Laboratory Trainer Theory

The Training Program for Health Institute Graduates has been
developed for the health institute graduates of the Kingdom of

Saudi Arabia. The Ministry of Health General Directorate of
Training and Scholarship, in collaboration with subject matter
experts from other Ministry of Health bodies that include, but
are not limited to: The General Directorate of Pharmaceutical
Affairs; The General Directorate of Nursing Affairs; The
General Directorate of Radiology and Applied Services; The
General Directorate of Lab and Blood Banks; Medical Records
Administration; and The Field Epidemiology Training Program
have produced the enclosed Training Program.

A special thank you to Teesside University and others for their
contribution to the development of the materials for the First
Edition of the Training Program for Health Institute Graduates

Kingdom of Saudi Arabia - Ministry of Health
General Directorate of Training and Scholarship
Training Program for Health Institute Graduates

Table of Contents
Program Outline ............................................................................................................................................................. 1
General Orientation (day 1) .................................................................................................................................. 10
General Orientation (day 2) .................................................................................................................................. 21
Collaboration and Teamwork in Health Care ...................................................................................... 21
Notes for trainer .............................................................................................................................. 21
Lecture ................................................................................................................................................. 29
Infection Prevention and Control............................................................................................................. 40
Lecture ................................................................................................................................................. 45
Workplace Safety and Injury Prevention .............................................................................................. 58
Lecture ................................................................................................................................................. 61
Bacteriology ................................................................................................................................................................... 68
Notes for trainer .............................................................................................................................................. 69
An Introduction to the Bacterial Cell, its Organization and Members ...................................... 71
Elements of Microbial Nutrition ............................................................................................................... 82
Sterilization and Disinfection .................................................................................................................... 90
Tools of the Laboratory ............................................................................................................................. 102
Antimicrobial Chemotherapy and Sensitivity Testing ................................................................. 114
The Cocci of Medical Importance .......................................................................................................... 122
Gram Negative Bacilli of Medical Importance.................................................................................. 134
Gram Positive Bacilli of Medical Importance ................................................................................... 145
Cerebrospinal Fluid (CSF) Culture ........................................................................................................ 156
Blood Culture ................................................................................................................................................. 161
Urine Culture ................................................................................................................................................. 166
Respiratory Tract Infections ................................................................................................................... 175
Parasitology ................................................................................................................................................................ 184
Notes for trainer ........................................................................................................................................... 185
Medical Parasitology Lab .......................................................................................................................... 186
The Parasites of Medical Importance - Protozoa ............................................................................ 212
The Parasites of Medical Importance - Helminth ........................................................................... 220
Clinical Chemistry .................................................................................................................................................... 226
Technology in Clinical Chemistry (Part I) ......................................................................................... 227
Technology in Clinical Chemistry (Part II) ........................................................................................ 231
Units and Calculation in Clinical Chemistry ...................................................................................... 236
Blood Glucose Tests .................................................................................................................................... 238
Kidney Function Tests ............................................................................................................................... 242
Electrolytes and Minerals ......................................................................................................................... 246
Clinical Enzymology.................................................................................................................................... 253
Liver Function Tests ................................................................................................................................... 259
Cardiac Biomarkers .................................................................................................................................... 264
Lipid Profile .................................................................................................................................................... 275
Special Tests in Clinical Chemistry ....................................................................................................... 281
Body Fluid Chemistry (Urine CSF- Other Body Fluids) ............................................................ 287
Blood Bank ................................................................................................................................................................ .. 295
Donor Selection ............................................................................................................................................ 299
Blood Collection ........................................................................................................................................... 305
Blood Donor Adverse Reactions ............................................................................................................ 307
Donor Recruitment and Retention ....................................................................................................... 313
Blood Component Preparation and Storage ..................................................................................... 328
Blood Component Quality Control ........................................................................................................ 333
Leukoreduced and Irradiated Blood Components ......................................................................... 336
Basic Immunology ....................................................................................................................................... 343
Blood Grouping Discrepancies ............................................................................................................... 348
Pretransfusion Guidelines and Neonatal Transfusion Policy .................................................... 356
Antibody Identification ............................................................................................................................. 360
Transfusion Transmitted Diseases ....................................................................................................... 372
Bacterial Contamination ........................................................................................................................... 382
Screening and Confirmatory ................................................................................................................... 393
NAT Types....................................................................................................................................................... 400
Hematology ................................................................................................................................................................ . 406
Introduction to Hematology .................................................................................................................... 408
Red Cells .......................................................................................................................................................... 412
Anemia ............................................................................................................................................................. 415
Abnormal Hemaglobins ............................................................................................................................ 418

White Cells ...................................................................................................................................................... 420
Lymphocytes and their Disorders ........................................................................................................ 422
Acute Leukemia ............................................................................................................................................ 424
Chronic Leukemia ........................................................................................................................................ 426
Investigation of Bleeding Disorders ..................................................................................................... 428
Blood Coagulation ....................................................................................................................................... 430
Thrombin Time (TT) Test ........................................................................................................................ 431
Automation .................................................................................................................................................... 432
Specimen Collection ............................................................................................................................................... 434
Lecture ............................................................................................................................................................. 436
Quality Control and Assurance ......................................................................................................................... 445
Lecture ............................................................................................................................................................. 446

Kingdom of Saudi Arabia Ministry of Health

Competency Based Training Flow Chart for Laboratory Technician

Trainee Laboratory
Technician
Orientation to program (2 days)
23 days teaching

MCQ on completion of theory block

16 weeks practical period

Pass
Fail
Extend the training for 2
months and repeat the
Technical Exam or MCQ
Assigned to availability
of vacant positions Dept.
needs
Pass
Fail
End the training
Period
English Course
6 Months
Fail

Pass

Repeat
2months
1

Course Title
Hospital-based Training for the Laboratory Technician

Introduction
The Ministry of Health believes in the value of their human resources. Every effort is made to
provide Saudi and non-Saudi health professionals with the tools and resources that are necessary for
success. An important first step in the process is an inclusive training program for new graduate
trainees who have not worked in their profession for more than one year. This is a 6-month program
which will provide them with the classroom and clinical experiences needed to embark on a
successful career in the laboratory field. The program has been designed as a transition program to
link the gap between the academic and the service settings and to prepare the target group to utilize
decision skills in the care of patients. The program's theoretical foundations are based on Kolbs
model of experiential learning (1984), Knowles' adult-learning principles (1970), and Kramer's
classic research on reality shock (1974).

Kolbs (1984) model for experiential learning
This 6-month training period supports the competency and professional development of novice
laboratory technicians. The program components include both theoretical knowledge and practical
learning experience.
Course Description
This Unit will provide students with a background of the analytical methods, skills and
instrumentation used in healthcare sciences. Core classes in this program study human diseases and
laboratory tests that identify them. Students learn to operate equipment in medical laboratories and
perform a wide range of procedures. Didactic and clinical instruction emphasize proper specimen
collection and handling, understanding test procedures, safety, quality control, acquisition of
technical skills, and troubleshooting techniques. Lectures and laboratory practical will cover several
key areas including clinical chemistry, haematology, blood bank, specimen collection, parasitology
and microbiology / bacteriology.
2

Program Goals
The goals of the program are the following:
To provide students with a body of knowledge and clinical training to develop entry-level
competencies in all routine areas of the clinical laboratory;
To produce graduates who demonstrate ethical behavior and professional attitudes;
To provide a quality program, which is assessed, evaluated, and revised as needed;
To provide graduates who will enrich the laboratories in which they are employed;
To provide a stimulating educational experience that encourages continuing education in both
students and participating laboratory staff.
Program Objectives
Theoretical Part
Upon successful completion of the program and initial employment, graduates should be able to
demonstrate entrance-level competencies in the following major areas of professional practice:

Knowledge:
1. Demonstrate a basic knowledge of bacteria, parasites and viruses
2. Demonstrate a basic knowledge of haematology and how this relates to abnormal results
3. Demonstrate a basic knowledge of Biochemistry
4. Demonstration of knowledge of the relationship of laboratory findings with common diseases
processes

Clinical part
Upon successful completion of the program and initial employment, graduates should be able to
demonstrate entrance-level competencies in the following major areas of professional practice:
Skills:
1. Collection, handling, preparation, and storage of biological specimens for laboratory analysis;
2. Performance of technical analyses on body fluids, cells, products, and organisms;
3. Recognition of factors that affect procedures and results and take appropriate action within
predetermined limits;
4. Ability to operate basic laboratory instrumentation;
5. Performance of quality control measures on instrumentation and technical analyses;
6. Recognition of and adherence to clinical laboratory safety policies;
7. Ability to troubleshoot instrumentation and technical analyses;
8. Ability to perform preventative and corrective maintenance on basic laboratory equipment
and instrumentation;
9. Ability to recognize when to refer instrumentation problems to the appropriate sources;
10. Demonstration of professional conduct with patients and health care workers both within and
outside the laboratory;
11. Demonstration of effective interpersonal communication skills
Practice Experience
Following theoretical training, trainees will go into clinical practice to enable them to practice and
consolidate skills under the supervision of a clinical preceptor. Trainees will rotate between different
areas of the laboratory. These areas include:
Clinical Chemistry
3

Hematology
Microbiology / Bacteriology
Blood Bank
Specimen Collection
Parasitology

Placements will be on a rotational basis and during each placement the clinical supervisor will offer
formative and summative feedback on the trainees progress and tick the relevant box when a
competency has been met. At the end of each placement a summative assessment will take place of
all competencies.

Methods of Teaching
The unit will be taught through a mixture of lecture and practical sessions. This will take the form of
6 hours of lectures and practicals per week. Competencies will be acquired in the following
coursework through didactic presentation and laboratory experience. The program coursework is
designed to show student progression of knowledge and skill.

Methods of Assessment
Theory Assessment: One MCQ assessing learning outcomes for knowledge areas 1 4
Practical Assessment:
Continuous evaluation of competencies at the end of each practical rotation according
to the form of evaluation available assessing outcomes S1-11.

Summative assessment
Summative assessment will take place at various points:
1. During the theoretical component trainees will be given a 2 hour MCQ examination of 60
questions at the end of the theory block.
2. By the end of trainees rotation in the relevant area, trainees will be assessed against the
relevant competencies. By the end of the practical component trainees should have been
assessed against all competencies. Competencies A-D are to be assessed at every point of
assessment in the practical component of the program.

The overall breakdown of marks awarded is as follows:
20% theory
70% practice
10% attendance (trainee will receive a 0% if he is absent for more than 10% of the total
duration of the course)

Trainees are to achieve a weighted average of 60% in order to pass the course

Should the trainee fail either part of the assessment then they will have 2 months to resit the
component. If at this point they are still unable to pass then the training program may be
discontinued.
Evaluation
As this program is new and developed for a specific purpose it is essential to gain a full evaluation to
inform future developments. Evaluation will be undertaken in several ways.
4

Evaluation from Trainee Laboratory Technicians
Ongoing feedback will be obtained from trainees through collection of qualitative data and a final
course evaluation questionnaire will be given on completion.

Evaluation from Laboratory Educators in clinical practice
A focus group will be facilitated in each of the delivery sites to bring together educators from clinical
practice. This feedback will identify if trainees are fit for practice and purpose on completion.
Qualitative data will be collected and experiences shared which will be fed back into the program
design.

Evaluation from speakers
Speakers will be given a short questionnaire to evaluate the delivery of their session. This will
include time to deliver content, student engagement, teaching methods and any future changes.
References
Monica Cheesbrough, (2006). District Laboratory Practice in Tropical Countries 2nd Edition.
Cambridge university press

Marshall, WJ and Bangert, SK. (2008). Clinical Biochemistry: Metabolic and Clinical Aspects 2
nd

Edition. Churchill Livingstone

5

Laboratory Trainee Technician Program Outline

Theoretical Component
Duration of
session
Topic Details of Session
2 days General Orientation Orientation to the program
Communication, Collaboration & Teamwork Delegation in
Health Care Setting
Infection Control in Health Care Setting
Workplace Safety & Injury Prevention
4 days Clinical Chemistry Technology in Clinical Chemistry (Part I)
Technology in Clinical Chemistry (Part II)
Units and Calculations in Clinical Chemistry

Objective is to identify:
Different methods (with their principles) applied in clinical
chemistry field.
Units Used in Clinical Chemistry & how calculate some
concentrations.
Blood Sugar Tests
Kidney Function Tests
Electrolytes and Minerals

Objective is to know:
Investigations done for blood glucose.
Parameters for evaluating the kidney function & how
calculate creatinine clearance.
Types of electrolytes, minerals of bone & iron profile
Clinical Enzymology
Liver Function Tests
Cardiac Biomarkers

Objective is to be aware of:
Enzymes of clinical significance and their role in diagnosis &
prognosis of some diseases.
Parameters for evaluating the liver function & their role in
diagnosis & prognosis of liver diseases.
Investigations for diagnosis of myocardial infarction, heart
failure & pulmonary embolism
Lipid Profile
Special Tests in Clinical Chemistry
Body Fluid Chemistry (Urine CSF Other Body Fluids)

To clarify:
Parameters of lipogram and how calculate LDL-cholesterol
from other parameters.
Types of non-routine investigations done in Clinical
Chemistry.
6

Chemical tests done on urine, CSF & other body fluids
4 days Haematology
Placement
Function of blood, haematopoisis
Collection of blood ,
Anticoagulants ,stains used in Hematology

Red cell function , morphology, PCV, and indices
Reticulocytic count
Haemoglobin function, structure, Introduction to Hb
abnormalities

Anaemia Classifications:
According to RBCs Indices
According to cause of defect:
o Membrane defect (Spherocytosis)
Quantitative Hemoglobinopathies (Thalathaimias)
Enzymopathies (G6PD)

White Blood Cells: Granulocytes, Monocytes and their
Benign Disorders

Lymphocytes and their disorders
Spleen
Acute Leukaemias

Chronic Leukaemias

Investigations of Bleeding Disorders:
Vascular endothelium damage
Platelet Number, Structure, Function
B.T Test
Blood Coagulation
External and Internal Pathways
PT, PTT tests introduction

Screening for a Circulating Anticoagulants
PT, APTT, TT test method

Fibrinolytic System FDP test
Information Provided by Platelet count , PT, APTT, TT
Automation role & principle
4 days Blood Bank
Placement
Criteria of donor selection and deferral
Blood donor sampling and collection
Management of donor adverse reaction
Donor recruitment and retained strategies

Objective is for trainees to be able to:
7

Prepare a donation program & deal with the factors affecting
motivation of volunteers to donate blood.
Determine if the donor is eligible to donate, temporally
deferred, or permanently deferred.
Counsel donors about the benefits and risk of donating
blood products.
Recognize adverse events related to blood donation and
manage them appropriately.
Blood component preparation and storage
Blood component quality control
Special blood component (irradiated leucoreduced
frozen)

Prepare, process, store, release and quality control for blood
components.
Manage and participate actively in all processes needed for
safe effective manufacturing and storage of blood
components.
Recognize plasma derivatives that are prepared
commercially.
Can explain the metabolic changes that occur during storage
Lymphocytes and their disorders
Principles of immunology
Blood grouping systems
Compatibility testing policy and procedure
Neonatal transfusion policy
Antibody screening and identification

Perform the identification and pre-transfusion testing of
patient/ unit, including ABO/Rh testing, RBC antibody
screen, and antibody identification.
Perform crossmatch: minor, major, Direct antiglobulin test
rapid spin and type & screen
BTTDs
Bacterial contamination of blood
Screening and confirmatory testing
Principles and different methods of NAT

Be able to perform various methodologies such as ELISA,
chemiluminescence and rapid assays used in screening of
TTDs.
Can perform the various methodologies such as western
blot, neutralization and reba testing used in confirmatory of
transfusion transmitted infection and interpret the results.
Can perform the NAT testing and interpret the results.
8

4 days Microbiology /
Bacteriology
Placement
Introduction to the bacterial cell, its Organization, and
Members
Elements of Microbial Nutrition, Ecology, and Growth
Sterilization & Disinfection
Tools of the Laboratory: The Methods for Studying
Microorganisms
Antimicrobial chemotherapy and sensitivity testing
The Cocci of Medical Importance
The Gram-Negative Bacilli of Medical Importance
The Gram-Positive Bacilli of Medical Importance
Cerebrospinal Fluid (CSF) Culture
Blood culture
Urine sample process
Respiratory sample culture
2 days Parasitology
Placement
Medical Parasitology Lab. Part1
Medical Parasitology Lab. Part2
The Parasites of Medical Importance Protozoa Part 1
The Parasites of Medical Importance Protozoa Part 2
The Parasites of Medical Importance Helminth Part 1
The Parasites of Medical Importance Helminth Part 2
1 day Specimen Collection
Placement
Receiving samples
Patient registration/data
Preparing patients
Handling of specimens
Blood specimen tubes
Blood withdrawal
Types of blood samples
Preparation of sample
Transport of samples
Sample retention
Sample rejection
1 day Quality Control and
Assurance
Placement
The policies and procedures associated with Quality Control
and Assurance
Good Laboratory Practice (GLP)
The principles of Good Clinical Practice (GCP)

9
MinistryofHealth
KingdomOfSaudiArabia
Training Program for Health
Institute Graduates
Day 1 General Orientation
LectureOutline
AbouttheTrainingProgramforHealthInstitute
Graduates
LaboratoryDomain CourseDescription
LaboratoryProgramOutline
RolesandResponsibilities Trainer
RolesandResponsibilities Trainee
PoliciesandProcedures
ImportantProgramDates
2
WelcometotheTrainingProgram
forHealthInstituteGraduates
3
Introduction
The Training Program for Health Institute Graduates has been undertaken by the
Ministry of Health.
The aim is to assist and promote the transition of technicians within the Kingdom of
Saudi Arabia (KSA) across various health care delivery settings
The Training Program for Health Institute Graduates design reflects a model of
delivery encompassing ten registered technician role domains. The ten specialty
area domains developed for training under the program are:
Pharmacy
Nursing
Radiology
Laboratory
MedicalRecords
Epidemiology,PublicHealth,andEnvironmentalHealth
Biomedical
AnesthesiaandRecovery
HealthandHospitalAdministration
OccupationalHealthandSafety
ThesuccessfulcompletionoftheTrainingProgrambytraineesofthedomainswill
fosterastrongerhumanresourcesdevelopmentstrategyandenhancethedeliveryof
competencybasedpracticestoKingdomofSaudiArabiacitizens.
4
HistoricalContext
His Royal Majesty, King Abdullah bin Abdulaziz Al Saud has
proactively identified the need to build capacity amongst its
youth population.
Thousands of Saudi youth have attended health institutes
across the Kingdom to advance their knowledge in their areas
of interest. However, the educational experience has created
a theorypractice gap for learners, thus posing difficulties for
graduates searching for employment in their field.
In response to Royal Decree A/29, dated 20/3/1432H, this
unique program provides health institute graduates with a
unique opportunity to participate in a Training Program for
Health Institute Graduates.
5
ProgramValues
UndertheKingdomofSaudiArabia,MinistryofHealth,Training
ProgramforHealthInstituteGraduates akeygoalofthe
programistoexperienceandbuildhumanresourcescapacity
reflectingthefollowingvalues:
Patientfirst
Justice
Professionalism
Quality
Honestyandtransparency
Teamwork
AcademicIntegrity
Initiativeandproductivity
Societalinvolvement
6
10
ProgramDescription
The Training Program for Health Institute Graduates was
envisioned for delivery in two important training / learning
phases.
7
Training Programfor HealthInstituteGraduates PhaseOne
Kingdomof Saudi Arabia,
Ministryof Health Trainee
selectionprocess
completed
6000+ Traineesparticipate
ina 6monthLanguage
StudyCourse toLearn
Englishfor applicationina
healthcare setting.
Traineesmustsuccessfully
complete the language
trainingtoprogressto
Phase Two.
Training Programfor HealthInstituteGraduates Phase
Two
Traineesparticipate inPhase Twoof
the TrainingProgram.
Thisentailscompletionof a 6month
Hospital/ HealthCare Agency based
course & placementsite practice
basedTrainingProgram.
The goal of thisphase of the
competency basedlearningprogram
asdeliveredineachone of the
domainspecific areas isa novice
tobeginner level trainedRegistered
Technicianwho canunderstandand
demonstrate safe andcompetent
scope of practice rolesand
responsibilities.
OutcomeEvaluation
All Traineeswill participate ina)
course basedlearningevaluation
andb) domainspecific trainee
placementpractice evaluation.
Thiswill include pretest/ post
testMCQ's, trainee self
assessmentwithmultisource
feedback, trainer assessment
andfeedback evaluationof both
global anddomainspecific
competenciesatvariouspoints
of the TrainingProgram.
ProgramDescription
Phase One
Trainees participation in a 6 month English language training course and its successful
completion is a prerequisite to entering the clinical learning phase of the Training Program
for Health Institute Graduates.
Phase Two
Requires the trainees participation in a 6 month domainspecific on the job training. This
involves a theoretical component, as well as a practical placement component
The theoretical component will allow the trainee to gain a fundamental understanding and
knowledge of the skills they will be acquiring. The practical placements will allow the
trainee to develop their professional practice role(s) and the delivery of practice based
competencies for use in the health care / hospital setting.
Evaluation of each trainees learning outcomes will be based on both global core
competencies and domain specific scope of practice competencies as a novice level
technician.
8
ProgramDesign
The Training Program has been developed from KSA Ministry of Health
mission, vision and values
The Program was designed with local and international professional input
from subject matter experts
The competencies developed for the program are designed in compliance
with the Saudi Commissions standards for health knowledge and skills
and/or best practices for the respective domains, while simultaneously
aligned with the MoHs commitment to high quality and safe healthcare
delivery across all of its health services.
The Program utilizes a participatory learning and competency guided
approach, from the novice to more advanced beginner level practice
9
ProgramDesign:KolbsModelforExperiential
Learning
10
LaboratoryTechnician
Introductionto Laboratorydomain
TheMinistryofHealthbelievesinthevalueoftheir
humanresources.Everyeffortismadetoprovide
theirhumanresourceswiththetoolsandresources
thatarenecessaryforsuccess.
Animportantfirststepintheprocessisaninclusive
trainingprogramfortechnicianswhohavenot
workedintheirdomainformorethanoneyear.
12
11
ProgramSynopsis
This Unit will provide students with a background of the
analytical methods, skills and instrumentation used in
healthcare sciences. Core classes in this program study
human diseases and laboratory tests that identify them.
Students learn to operate equipment in medical
laboratories and perform a wide range of procedures.
Didactic and clinical instruction emphasize proper
specimen collection and handling, understanding test
procedures, safety, quality control, acquisition of technical
skills, and troubleshooting techniques. Lectures and
laboratory practical will cover several key areas including
clinical chemistry, haematology, blood bank, sample
collection, parasitology and microbiology.
13
CourseDescription
CourseDescription
Toincludethefollowing:
Programobjectives
Chosenprogramrollout
Programoutline
Materialsneededfortheprogram
Teachinglearningprocess
Learningmethods
Methodsofassessment (intheoryandpractical
training)
15
CourseDescription:ProgramObjectives
(theory)
Uponsuccessfulcompletionoftheprogramandinitialemployment,
graduatesshouldbeabletodemonstrateentrancelevelcompetenciesinthe
followingmajorareasofprofessionalpractice:
Knowledge:
Demonstrateabasicknowledgeofbacteria,parasitesandviruses
Demonstrateabasicknowledgeofhaematologyandhowthisrelatesto
abnormalresults
DemonstrateabasicknowledgeofBiochemistry
Demonstrationofknowledgeoftherelationshipoflaboratoryfindings
withcommondiseasesprocesses
16
CourseDescription:ProgramObjectives
(practical)
Uponsuccessfulcompletionoftheprogramandinitialemployment,graduatesshouldbeableto
demonstrateentrancelevelcompetenciesinthefollowingmajorareasofprofessionalpractice:
Skills:
Collection,handling,preparation,andstorageofbiologicalspecimensforlaboratoryanalysis;
Performanceoftechnicalanalysesonbodyfluids,cells,products,andorganisms;
Recognitionoffactorsthataffectproceduresandresultsandtakeappropriateactionwithin
predeterminedlimits;
Abilitytooperatebasiclaboratoryinstrumentation;
Performanceofqualitycontrolmeasuresoninstrumentationandtechnicalanalyses;
Recognitionofandadherencetoclinicallaboratorysafetypolicies;
Abilitytotroubleshootinstrumentationandtechnicalanalyses;
Abilitytoperformpreventativeandcorrectivemaintenanceonbasiclaboratoryequipmentand
instrumentation;
Abilitytorecognizewhentoreferinstrumentationproblemstotheappropriatesources;
Demonstrationofprofessionalconductwithpatientsandhealthcareworkersbothwithinand
outsidethelaboratory;
Demonstrationofeffectiveinterpersonalcommunicationskills
17
CourseDescription:programrollout
ThesixmonthTrainingProgramhasatheoretical
componentandapracticalcomponent
Programrolloutwillbeasfollows:
TwodaysofGeneralOrientationtotheprogram
Onemonthoftheory
Fivemonthsofpractical
18
12
CourseDescription:ProgramOutline(theory)
Thefollowingareaswillbecoveredintheory:
Bacteriology
Parasitology
Clinicalchemistry
Haematology
Bloodbank
Specimencollection
Qualitycontrolandassurance
19
CourseDescription:Materials neededforthe
Program
Traineeswillreceive
Bookletfortheory
Toincludelectureslides
Toincludematerialsforclassroomactivities
Bookletforpractice
Toincludecompetenciestobemet
Toincludeweeklyselfevaluationtobecompletedat
theendofeveryweek
20
CourseDescription:TeachingLearningProcess
Supporting the Trainee
in the teaching
learning placement
experience(s) are:
Preceptor
Clinical Supervisor
Course Lecturer
Local Staff Teams
21
Trainee
Preceptor
Clinical
Supervisor
LocalStaff
Teams
Course
Lecturer&
Classes
CourseDescription:LearningMethods
The unit will be taught through a mixture of lecture
and practical sessions. Competencies will be
acquired in the following coursework through
didactic presentation and laboratory experience. The
program is designed to show student progression of
knowledge and skill.
22
CourseDescription MethodsofAssessment
Traineeswillbeassessedbasedonthefollowingbreakdown:
20%theory
Assessmenttoolstoinclude:MCQs
70%practical
Basedontheglobaland domainspecificcompetencies
Traineestocompleteweeklyselfevaluation(butwillnotreceivea
finalgradetowardsweeklyselfevaluation)
10%attendance
Traineestoachieveaweightedaveragescoreof
60%
23
TraineeEvaluationinPractice
The assessment of trainees in the Program against global and
domain specific standardized practice competencies will occur
in the following ways:
Trainee Selfassessmentofcompetencybasedpractice
Trainee Trainerassessment&feedbackofcompetencybased
practice
Trainee IndividualWeeklySelfEvaluation
24
13
CompetencyGradingScale
SelfAssessment
0=NoKnowledgeand/orexperiences(Nocompetency)
1=Limitedknowledgeand/orexperiences(Some
competency)
2=Knowledgeableandfeelsconfident(Complete
competency)
FinalSummativeAssessment
0=Notabletoperform(Nocompetency)
1=Limitedabilitytoperform(Somecompetency)
2=Isabletoperform(Completecompetency)
25
Trainee Assessment & Evaluation
Scope of Practice Competencies & Performance
26
Self
Assessment
Competencies
(The trainee will be able to )
Placement Placement
0 1 2 0 1 2 0 1 2
TraineeEvaluation GlobalPracticePerformance
Competencies(thetraineewillbeableto):
A.Professionalism,EthicalPractice&Teamwork
Demonstratesaprofessional,ethicalapproachtopractice&teamwork
B.MaintainingPractice&ProfessionalDevelopment
Takesresponsibilityforkeepingownknowledgeandskillsuptodatethroughcontinuing
professionaldevelopment
C.Communication/TherapeuticCommunicationinPractice
Demonstrateseffectivecommunicationandinterpersonalskillstodevelopandmaintain
effectiverelationshipswithpatientsandcolleagues
D.WorkplaceEnvironment&Safety
Practisessafelyanddemonstrateunderstandingofthecorrectuse,limitationsandhazardsof
theenvironment
WeeklySelfEvaluation
28
RolesandResponsibilities
Trainer
TrainersinTrainingProgram
For theory training:
Course Lecturer
For practical training:
Clinical Supervisor
Preceptor
30
14
RoleoftheCourseLecturer
The Course Lecturer member oversees the trainee
and is responsible for the delivery of classroom
sessions supporting the Training Program for Health
Institute Graduates.
The content presented and discussed in the
classroom sessions is then integrated by the trainee
into practice within the practical placement
experience environment.
31
ResponsibilitiesofCourseLecturer
Providesufficientinformationduringgeneralorientationtofacilitatetrainee
preparationfortheprogram;
Supportaconstructiveteaching/learningprocessintheclassroomsetting;
Setmeetingswiththetraineetodiscusstheprogressorlearningchallengesas
needed;
Encouragetraineestobuildontheirownexperience,personalknowledgeand
wisdom;
Promoteprofessionalgrowthoftrainees
Supporttraineesinunderstandingtheprovisionofsafecareintheplacement
setting;
Monitorandmediateinteractionsandconcernsbetweenotherstaffandtrainees
ifwarranted;
Assignaclassroomsessionfinalgradebasedonthetraineesperformanceon
classroomtests,examsandassignedclassroomworkforevaluationinthe
TrainingProgram
32
ResponsibilitiesoftheCourseLecturer
(continued)
Coordinate with the responsible administrative domainspecific representative for
batch of trainees regarding the following:
Register Trainee in the First Week
Report Attendance Per Month
Tests retrieve tests from representative and make necessary copies
Report Test Result per trainee
Submit incident report upon the occurrence of an incident, download
incident report and complete. Form to be sent to Ministry of Health General
Directorate of Training and Scholarship. The course lecturer is to indicate the
level or urgency of the incident
Extremely urgent for incidents that are detrimental to the patient and/or the trainee
Urgent for incidents that cause potential harm to the patient and/or the trainee
Moderate for incidents that are important and require the immediate attention of the
General Directorate of Training and Scholarship, but are not detrimental to the patient
and/or trainee
Not urgent for incidents that are to be brought to the attention of the General
Directorate of Training and Scholarship, but do not require immediate attention
**Trainer reserves the right to suspend trainee, but the ultimate decision to terminate a
trainee rests with the General Directorate of Training and Scholarship
33
ResponsibilitiesoftheClinicalSupervisor
Overallresponsibilityforthemanagementandadministrationoftraineeswithin
theclinicalenvironment.Keyrolesincludeorganizingtherotas toensurethatthe
learningoutcomesareachievedandthatadequatesupervisionisprovidedbythe
preceptors
Makeregularcontactwiththepreceptor
Providesupportandadvicetopreceptors
Speaktothepreceptorandthetraineeandreviewprogress,traineeformsand
workbooks
Signsoffontraineeweeklyselfevaluationandprovidessupporttotraineewhere
thereareareasforimprovement
Formativesigningoffofrelevantplacementcompetenciesoncecompetencyhas
beenachieved
34
ResponsibilitiesoftheClinicalSupervisor
(continued)
Coordinatewiththeresponsibleadministrativedomainspecific
representativeforbatchoftraineesregardingthefollowing:
ReportAttendance PerMonth
Submittraineecompetencyresult
Submitincidentreport upontheoccurrenceofanincident,
downloadincidentreportandcomplete.FormtoIndicatethelevel
orurgencyoftheincident
Extremelyurgent forincidentsthataredetrimentaltothepatientand/or
thetrainee
Urgent forincidentsthatcausepotential harmtothepatientand/orthe
trainee
Moderate forincidentsthatareimportantandrequiretheimmediate
attentionoftheGeneralDirectorateofTrainingandScholarship,butarenot
detrimentaltothepatientand/ortrainee
Noturgent forincidentsthataretobebroughttotheattentionofthe
GeneralDirectorateofTrainingandScholarship,butdonotrequire
immediateattention
35
TheroleofthePreceptor
Orientatethetraineetothedepartmentorunit,procedures,reference
manuals,andmembersofthehealthcareteam
Collaboratewiththetraineesclinicalsupervisoraboutprogress
throughoutthesemester;
Assistthetraineetoaccessresourcesandrelevantexperiences;
Supportthetraineetohelpincreasetheirlevelofcompetenceand
confidence;
Shareverbalandwrittenfeedbackwithboththetraineeandclinical
supervisor
Contribute(inwritingwherepossible)informationforthetraineesfinal
appraisal;
Immediatelyreportconcernsaboutunsafepracticetoboththetrainee
andclinicalsupervisor
Reviewtraineelearninggoalsandselectingappropriatelearning
experiencesfrompersonalassignmentstomeetthetraineeslearning
objectives
36
15
TheroleofthePreceptor(continued)
Takenoteofanytraineeabsencesandinformingimmediatesuperiors
(andclinicalsupervisor)andProgramleadsasrequired
Providesupervisionandlearningfacilitationtothetraineeinthepractical
area
Collaboratewiththetraineetoensurelearningneedsareidentified,and
thatsupervisionisappropriatetotraineesknowledgeandskillforeach
situation
Regularlyconsultwithotherstaffmembersbasedonindividualneeds
andtheneedsofthetraineetoobtainfeedbackonthetrainees
performance
Providefeedbacktothetraineethroughregularlyestablishedmeeting
timestoidentifynewlearningneeds,andtoplanthenextdaysactivities
**Theroledoesnotinvolvemarkingthewrittenassignmentsthatthe
traineecompletesaspartoftheirpracticeexperienceorassigningafinal
grade.
37
RolesandResponsibilities
Trainee
Traineerolesandresponsibilities
CompleteallpreparatoryTrainingProgram TraineePlacementPrerequisites
ReviewandcompletewithsignaturetheTrainingProgramagreements
Completeallpreparatoryclassroomandactivitiesinaselfdirectedlearningstyle
toenhancesuccessfullearningoutcomesandsafepracticedelivery
Completeallrequiredevaluationtools,asperdomainspecificrequirements
Understandthatattendanceismandatory
Actprofessionalandberesponsibleatalltimes
Orientateselftotheorganizational,departmentalandpracticeenvironment
Orientateselftorolesandresponsibilitiesofthevarioushealthcarestaff/team
members;
Orientateselftoavailablehumanandphysicalresources
Orientateselftocareandservicedeliveryanddocumentationprotocols
Buildonone'sownexperience,personalknowledgeandwisdom
39
Traineerolesandresponsibilities
Know(andoperateatalltimes)underthepoliciesandproceduresoftheagencyandthe
TrainingProgramforHealthInstituteGraduates
ConstantlychecktheProgramwebsiteforanyannouncements
Workcollaborativelyandinterdependentlywithothers
Bepreparedtosubmitrequiredevidenceofyourtraineepractice&learning
Maintainconfidentiality
Know/acknowledgethatalltraineeandevaluationdataincludingtraineelearning
outcomesdatabelongstotheTrainingProgramandwillbeusedinreportsandpublications
bytheMinistryofHealth
Understand/knowtheTrainingProgramconditionsoftraineegradingarebasedonthe
traineesuccessfullycompletingallTrainingProgramtests,assignmentsandactivitiesin
accordancewithprescribeddeadlinesafterwhichafinalgradewillbedeterminedbasedon
theweighteddistribution
Understand/knowthefinalgradeweighteddistribution
Understand/knowthattheTrainingProgramrequiresalltraineestoachieveatraineefinal
grade(60%)fromtheweightedaveragedistributioninordertoachieveagradestatusof
PASS
40
Traineerolesandresponsibilities(continued)
Arriveontheunitordepartmentandreporttothepreceptorforeachassigned
shiftofthepracticalexperience
Workundertheguidanceandsupervisionofthepreceptorandotherqualified
personnelasnecessary
Informthepreceptorpastexperienceinrelationtolearninggoals,scopeof
practicelimitationsandreliablyconveystheamountofguidanceandsupervision
required
Seekfeedbackdailyonpersonalperformanceandadjustslearninggoalsand
creatingremedialplansasnecessary
Participateasacollaborativeteammember,documentscare,demonstrate
knowledgeofallunit/facilitypoliciesandmeasuresforfireandother
emergencies
Notifyclinicalunitordepartmentandpreceptorofallabsencesinadvanceofa
plannedclinicalshift.
Reportanddocumentallcriticalincidentstothetrainerinpracticeimmediately
(medicationerrors,procedureortreatmenterrors,apatientorpersonalfallor
injury,needlestickinjuriesetc.)
Completetheweeklyselfevaluation
41
TraineePrerequisitesforEntrytoPlacement
Each trainee will meet the following prerequisites prior to
entering the placement site clinical environment:
Note: All prerequisites will be completed prior to entry to the
placement setting and will be completed / signed off to the
Ministry of Health by 26/7/1435H:
Trainee to complete the Placement Practice Agreement (PPA)
by 12/7/1435H
Trainee Health Examination by Physician current,
completed and deemed fit and able to fully participate in
writing
Trainee to present confirmation of TB Skin Testing
Identification of Allergies & Workplace Hazard Risks
medication, environmental sensitivities, latex allergy etc.
42
16
Policies&Procedures
Program&Professional CodeofConduct
Each Trainee acknowledges and will practice the following
elements supporting the values, attitudes and behaviors of the
program at all times:
Respect
TraineeResponsibilitiesforProfessionalService
DressCode
Integrity&Accountability
Confidentiality&ProtectionofPrivacy
Teamwork&Collaboration
Safety&CompetencyinPractice
Attendance&Punctuality
AcademicIntegrity
Incident&InjuryReporting
44
Safe&EthicalCareProvision
Asprofessionals,traineesareresponsibletoprotect
therightsandinterestsofthepatient/client.
Thereisagreatdealoftrustplacedinhealth
professionals,astheyareexpectedto:
Bequalified
Providesafe&competentcare
Respectbasichumanrightsofallindividuals
Betruthfulandactwithintegrityintheirconductand
interactionswithothers
45
TraineePractices&CodeofEthics
Trainees will reflect moral & ethical standards of practice.
Trainees, as members of a domain specific profession are required
to uphold the standards of practice and scope of competencies of
practice.
Trainees will seek guidance for decisionmaking in concerns
related to patient and service delivery ethical matters.
The trainees code of ethics supports the primary values of
ensuring patient / client health & well being, choice, dignity,
confidentiality, fairness, accountability, and the environment of
practice and its delivery are conducive to safe, competent and
ethical care.
46
TraineeDomain ScopeofPracticeBoundaries
It is the responsibility of each trainee to read,
understand and practice within their trainee role and
scope of practice competencies
It is the responsibility of the trainee to read, understand
and adhere to all policies and procedures
Practice under the supervision and direction of their
preceptor
Understand the limitations and boundaries of their role,
scope and related practices and seek guidance and
clarification if they are unsure prior to undertaking any
task or accepting responsibility for care and services.
47
TraineeRole&PracticeLiability
Theremaybeliabilityifactionscauseharm theassignment
ofliabilityisdependentupontheindividualsituation
Withinapreceptor traineesupervisedrelationshipthe
liabilityisusuallysharedbythetrainerinpractice,trainee&
hospital
Traineesareexpectedtoperformasaregisteredprofessional
andprovidesafepatient/clientcare
Traineesdonot practiceoutsideofrole/
domainjobdescriptionandpractice
scopeANDrequireongoingsupervision.
48
17
DressCodeinPlacementSetting
Trainees are required to comply with the dress code policies and procedures
AND all mandatory dress code health and safety requirements (example:
PPE personal protective equipment) to entre and practice in the placement
setting.
Domainspecific uniform may be required and if a trainee is no
appropriately attired or is unkempt, the trainee may be refused admission
to the agency
Footwear designated by site & domain specific work, and occupational
safety requirements.
*Note: all footwear must be closed toed
Photo ID name badges must be worn at all times
Adhere to scentfree environment request
Adhere to all health & safety and infection control dress code
requirements
49
Trainee PersonalHygiene&InfectionControl
TraineePersonalHygiene&InfectionPrevention
Restrainhair keepshairfromfallingforwardinfield
ofvision,hairmaybeavehicletotransport,carryor
dropmicroorganismsespeciallyifaseptic/sterile
techniqueisrequired
Keepfingernailsshort
Coveranyopenwoundswithanocclusivedressing
50
Trainee HandHygienePractices
When should hands be washed?
When visibly soiled
Before and after client contact
After contact with source of microorganisms (blood, body fluids,
mucus membranes, non intact skin or inanimate objects that might be
contaminated
Prior to performance of invasive procedures (IV catheters, indwelling
catheters)
Before and after removing gloves (wearing gloves does not remove
the need to wash hands)
At the beginning and end of every shift
51
PlacementSite&PracticeDelivery Safety
AllTraineesmust read,reviewandare
responsibleforknowingthefollowing
practice&workplacesafetyrequirements:
Confidentiality & privacy of personal patient and placement
organization information, practices, policies & procedures;
Documentation standards, policies & procedures;
Workplace Occupational Health & Safety requirements,
policies & procedures program and placement site specific
policies;
Environmental and Workplace Hazards practices, policies &
procedures, and guidelines.
52
PlacementSite&PracticeDelivery Safety
All Trainees will read, review and are responsible for knowing the
following practice & workplace safety requirements:
Infection Prevention and Control practices, policies & procedures
and guidelines;
Emergency Situations, Hazardous Threats, Fire Safety and Security
related practices, policies & procedures
Personal Protective Equipment & Hand Hygiene practices, policies
& procedures
Any specific domain related safety and quality assurance elements
that trainees need to be aware of / or needs to practice in the
domain specific department or in the delivery of their specific
practice role
Others specific to the Placement Site but not noted here.
53
HandbookReview&PreparingforPlacement
Trainees will complete the following:
Review of Training Program Handbook
Complete all prerequisites for entry into the placement
setting
Complete and sign off of all required forms /
acknowledgement agreements
Read, understand and comply with all policies and
procedures
Read, understand and comply with the code of conduct
and your scope of practice Trainee role
Practice with safety in mind, know your boundaries or
limitations in knowledge and scope
Comply with dress code and all safety regulations
54
18
TraineeEvaluationinProgram
The Training Program for Health Institute Graduates Trainee
evaluation will allow for the assignment of a final grade.
The trainees final grade at completion of the program will be
determined on the following basis:
Every Training Program trainee is aware and understands that all
Training Program conditions for determining a trainee final
grade are based on and subject to the trainee successfully
completing all Training Program tests, assignments and activities
in accordance with prescribed deadlines and reflective of
academic integrity requirements of the program.
55
TraineeEvaluationRequirements&Data
AsaconditionofunderstandingforentryintotheTrainingProgram
forHealthInstituteGraduates:
Every traineeisrequiredtocompleteallTrainingProgram~Trainee
EvaluationandProgramEvaluationrequirementsandPre/Post
ProgramFeedbackSurveysasrequestedbytheTrainingProgram
andMinistryofHealth
AND
Every traineeisawareandacknowledgeshisunderstandingthatall
TraineeandProgramEvaluationdataincludingtraineeslearning
outcomedataisthepropertyoftheTrainingProgramandmaybe
usedinreportsandpublicationsasdeterminedbytheMinistryof
Health.
56
TestsandExams:absences
Attendanceiscompulsoryforall scheduledtestsandexaminations.
WrittendocumentationISREQUIREDtosupportclaimsofextenuatingcircumstances,i.e.,illness
(MedicalCertificatemustbeprovided)ordeathofafamilymember.
Atraineemissinganytypeofevaluativemethodmustcontacttheirtrainerpriortothe
test/exam/assignmentorevaluationsessionduedateandtimeandclarifythereasonforabsence.
Failuretonotifythetrainerinadvance willresultinagradeofzeroforthatevaluativemethod.
**Alternatemakeupactivitiescouldbenegotiatedandcontractedwiththeinstructor.Ifthereasonsfor
missedtest/exam/assignment/evaluation areinkeepingwithexpectationsoftheTrainingProgrampolicy
ANDallappropriatedocumentationisprovidedasstatedabove,aninstructormaychoosetoofferatraineean
alternativeduedateandtimeforthetest/exam/assignmentorevaluationifthetraineemeetsthecriteria
warrantinganalternativetime&datetocompletetheevaluativemethod.
Itisthetraineesresponsibility tofollowupwiththetrainerregardingmissed,lateorabsentassignments,
testsorexams. ThetrainerinconsultationwiththeMinistryofHealthhavetherightandobligationtomake
thefinaldecisionaboutcourseevaluationactivities,notingthatcertainpoliciesmayneedtobeupheldin
supportoffairnesstoalltraineesintheprogram.
Pleasebeadvised:Recordsofabsencefromtests/examinationsmaybekeptintraineefiles
57
PolicyGuidelinesforTraineesScholarlyWork
Trainees are responsible for promoting academic integrity within their training
program and the development of their scholarly work.
If trainees feel the faculty have made a mistake in computing the assigned grade,
trainees have a responsibility to come to faculty as soon as possible prepared to
provide a rational re why they feel a mistake has been made.
Academic dishonesty includes any misrepresentation of any part or whole of the
process or product in preparation of academic scholarly work
Plagiarism specifically can be understood as, the act of copying, reproducing, or
paraphrasing significant portions of someone elses published or unpublished
material and representing someone elses thinking as ones own thinking by not
acknowledging the appropriate source or by the failure to use appropriate
quotation marks.
Plagiarism is one of many forms of academic misconduct (such as cheating,
misrepresentation, and submission of false information) that are described in the
Code of Academic Conduct. The minimum penalty is a mark of 0 for the work.
Trainees may fail the course or be expelled from the program (as per the
discretion of the General Directorate of Training and Scholarship)
58
StatementofAgreement/Acknowledgement
Eachtraineewillread,understandandagreestocomply
with:
TraineePlacementPracticeAgreement(PPA)(Form1)
TrainingProgramforHealthInstituteGraduates Trainee
ConfidentialityStatementofUnderstanding&
Agreement(Form2)
CodeofConductPolicy
PlacementSitespecificTraineeConfidentiality
AcknowledgementForm(ifapplicable)
*Note:Thisismandatorytoentertheplacementsite
59
ImportantProgramDates
19
Program GeneralTimetable&KeyDates
GeneralTimetable&KeyDates
Program
Begins
2/7/1435H
EnglishLanguageStudy Course ends
12/7/1435H
Day 1 TrainingProgramfor Health Institute Graduates General Orientation
13/7/1435H
Day 2 TrainingProgramfor Health Institute Graduates General Orientation
Eid AlFitr 25/9/1435H 4/10/1435H
EidAlFitr Holiday
Noclasses
National
Day
28/11/1435H
NationalDayHoliday
Noclasses
Eid AlAdha 2/12/1435H 17/12/1435H
EidAlFitr Holiday
Noclasses
Program
Ends
6/1/1436H
Training Programfor HealthInstituteGraduates ends
61
20

General Orientation Trainer

Communication Collaboration & Teamwork Delegation in Health Care Setting

- Slide 5 - Communication & Therapeutic Interaction
What does communication in the health care setting mean to you ?
[ Lecturer to explore this by asking trainees in the classroom setting the above question and
inviting a brief dialogue ]
Following the short dialogue with trainees continue the presentation by sharing the
following:
Health care workers utilize appropriate communication skills and personal interactions, in
order to effectively build and maintain relationships, and to facilitate optimal health care.
Relationships may be with patient / clients, families, or other health care professionals, for
example.
Each relationship can contribute something important to health and health decision making,
therefore learning about communication and therapeutic interaction is an important topic to
review today.
- Slide 6 - Communication and Human Interaction
Communication in some form allows expression and supports all human interaction
Verbal and non verbal
(Verbal communication refers to spoken language. Non verbal communication may include
facial expressions, posture, eye contact, body language, tone of voice, displayed emotion, etc.,
and often provides valuable information, for both conscious and unconscious individuals)
Written and unwritten
(Written information records and communicates important information, that may be vital to
care, for example. Unwritten information can include utilizing picture tools, or using computer
technology, for example.)
- Slide 7 - What other Factors Influence Communication ?
Several factors may influence the process of communication.
The Transactional Model of Communication is depicted here. This model shows:
1) The communicators There is the person who is sending outgoing information, and the person
who is receiving incoming information.
2) The message or messages This is the information being transmitted. Remember, this
information may be verbal or non verbal information, written and unwritten.
3) Noise This can be anything that interferes with effective communication. This could include
other conversations, phones ringing, or being called away to attend to something else.
Transactional refers to the fact that communication is a continuous and ongoing process.
Additionally, there are a variety of people with whom we are communicating with.
It is important to remember that our personal beliefs, experiences, attitudes, (and other
factors) impact the way we send information and also impact the way we interpret incoming
information. This can be helpful at times, but it may also impair communication or lead to
misinterpretation of information, for example.
Communication is considered developmental in the experience of human interaction.
As such recognizing or having a common language is an important aspect of our
communication.
When we speak or have a different language from one another, it can make it difficult to
establish communication with one another.
If you think back about your own development or the development of a baby to a child to a
young adult you may recall some developmental changes in the way the individual
communicated with their environment or with people around them.
21

Thus, how a baby communicates is different than that of a toddler or young child. Each is
progressing in their development of communication and language. This development will be
influenced by their own brain and bodys functioning ability but also by the influences of their
environment.
- Slide 9 - Communication Process Influencing Factors
When we think about the communication process
There are many influencing factors that can impact what we send and receive as messages in
the communication process.
These may include: (As above - read slide)
- Slide 10 - Communication in Health Care Setting
In the health care setting communication is essential in every aspect of the care we provide and
the meaning it has to our patients and families
Therefore, being able to support effective communication is of vital importance to our work and
care delivery in the health care setting.
As effective communication may be essential to: (As above - read slide)
- Slide 11 - The Communication Process
This slide depicts the communication process as a 2-way dialogue between the sender and
receiver.
Understanding how the communication process works is necessary to building effective
communication skills and abilities as a health care provider.
This includes your ability to identify and focus on understanding the other factors that may
influence the communication process or interactions as we engage with others.
This includes understanding the way an incoming message is interpreted/or decoded.
The Sender transmits information, in verbal and non verbal ways, each having the ability to
influence the way the message will be received and interpreted.
The Receiver (or listener) decodes the verbal and non verbal information together, and in
doing so, interprets the message.
Once a message is decoded, feedback occurs (a response), which is once again
communicated in verbal and non verbal ways.
This can be an ongoing process, providing both/all people are open to the communication.
Many other factors can influence the communication process.
Our interpersonal variables, such as personal experiences and attitudes, affect what
information is sent and how it is received. Additionally, environmental factors, such as a noisy
environment, a lack of privacy, etc., may also have an impact on the effectiveness of sending and
receiving the communication.
- Slide 12 - Communication & Gender
There can be communication differences related to ones gender.
(As above - read slide)
The differences may be due to growth and development variations by gender OR they may
be differences due to ones environment while growing up (such as differences in social, cultural
diversity or even educational / learning opportunity differences).
Therefore, how one communicates can be influenced by the norms, values and behaviors
which they have learned from their life experiences.
As a result, improving ones communication and its effectiveness can also be learned or
improved at any time in the life span.
- Slide 13 - The Meaning in Communication
Notice the differences between left and right sides of the brain.
The left side - we see logical thinking and analysis, a fondness for knowing facts, strength in
mathematics, and areas involved in understanding language and speaking.
22

We may keep this in mind, because it helps us understand differences in ways of thinking
and ways of communicating.
It also helps us to understand the variation in communication and the interpretation of
meaning from communication we may notice in others like our patients.
It is also worthy of noting:
The left side of the brain controls the right side of the body and the right side of the body
controls the left side of the body.
For example: damage to the left side of the brain may affect the patients ability to speak, or
understand language. Or it may cause weakness or paralysis of the right side of the body.
- Slide 14 - Right Brain Left Brain & Communication
The important message here is the following:
It is important to appreciate how differences in thinking, perception and therefore the
influence of the brain create meaning in the communication process and how this may vary
from person to person ~
It can even offer some explanation of the differences in how people receive and interpret
information in the communication process differently.
As a result of these differences it may impact your relationships with other people
including those you work with.
This is being presented to you to help you understand one possible explanation for the
diversity in the workplace we sometime experience as a result of communication.
- Slide 15 - Influences - Values, Perceptions & Behaviors
Our values, perceptions, and behaviors also influence the communication processes, by way
of how information is transmitted, and by how information is interpreted/perceived.
It is important to be mindful of each of these influences, and how they may impact things
like communication, health, decision making, and delivery of health care.
These differences may also impact your relationships with other people including those you
work with.
This is being presented to you to help you understand one possible explanation for the
diversity in the workplace that you may experience as a result of communication.
- Slide 16 - Types of Communication in Health Care
There are different types or ways that communication can occur in the health care setting.
Verbal communication is one type.
It is important in verbal communication to validate that the messages are being received
and interpreted accurately in the 2- way dialogue as a shared interaction.
Another type or way that communication can occur in the health care setting is written
communication.
As health care providers you will also be engaged in the written communication process
through your documentation of the patients shared information and messages that were being
shared with you.
Another type or way that communication can occur in the health care setting is non verbal
communication.
This can occur when the patient can not or is unable to speak verbally. Keep in mind a
patient may be able to communicate in other ways (eye contact, facial expressions, gestures,
writing, use of pictures etc.)
23

In non verbal communication it can be important to try and confirm or validate your
interpretation of messaging when ever possible as not every thing may have the same cultural
significance or is universally understood.
Another type or way that communication can occur in the health care setting is through
electronic and digital communication.
The use of electronic and digital communication is often guided or controlled by strict
policies and procedures to protect the privacy and confidentiality of information, who gets to
access it and where and who can share it.
Please refer to your placement organizations policies regarding your use of all electronic
and digital communication.
- Slide 20 - Personal Space & Communication
In understanding the communication process, it is important to recognize the influence of
ones personal space as measured between the sender and receiver during the communication
episode.
Personal space can actually change the perception of how the message is received and
interpreted by the receiver.
You will want to keep this in mind when dealing with patients and other health care
providers in the health care setting as the measure of personal space can influence how
others perceive your messages and the appropriateness of the message and any
corresponding actions that may follow.
The greater the congruency between what we say and how say it the clearer and more
accurate the message by the receiver. When congruency does not exists then misinterpretation
and misunderstanding results in the communication process.
The strength of the communication process for use in the workplace is essential in health
care settings.
Misinterpretation and misunderstandings can have very negative effects on collaboration,
teamwork, productivity and patient care outcomes.
- Slide 21 - Therapeutic Communication
Therapeutic communication simply refers to the way a health care provider communicates
and interacts with their patient and the patients family.
Therapeutic communication occurs in such a way that a trusting and respectful professional
relationship can be established.
Communicating and interacting in a compassionate, kind, confidential, and authentically
caring way, can help health care workers develop this relationship, which is essential if patients
are going to communicate their concerns and needs with us.
It is essential if we are also going to be able to develop a health care plan with the patient,
and establish how best to meet his/her health needs.
Establishing a therapeutic relationship may also help to create an optimal healing
environment for the patient.
- Slide 22 - Patient First (Person Centred Care)
Patient First = is also known as person centred care
In health care delivery settings, the patient is our priority. They are at the center of their
own care and within an ethical practice care delivery setting we are at all times advocating this
for the patient and their care.
This means that we need to get to know the patient. This sometimes also means we will get
to know the patients family or others who are important in their lives.
As health care providers, we need to gather information about what is important to the
patient, what their goals and hopes are for their health situation. Where do they live? Do they
24

live alone? Do they have friends and family nearby, who can help them with their physical,
emotional, or other health related needs?
We need to be compassionate, ask the right questions, show concern and respect, and really
listen to the information that the patient wishes to share with us.
This information will help ensure that an appropriate health care plan can be developed
with the patient and help ensure that the patients health needs can be best met.
- Slide 23 - Characteristics of Therapeutic Relationship
There are several important characteristics you will need to consider in establishing or building
the therapeutic relationship with a patient.
- Slide 24 - Communication & Patient Documentation
Another important component of communication and the patient care process is reflected in
documentation as in the patients chart or health record.
- Slide 25 - Types of Documentation
There can be several different types of documentation that occur specific to the patients care
process and the health care providers roles and responsibilities.
You are responsible for and will need to become familiar with the policies and procedures for
documentation in your trainee role while in the placement setting.
- Slide 26 - Dos & Donts of Documentation
Please keep in mind the following general aspects of documentation in the health care setting:
- Slide 27 - Communication & Collaborative Practice
Communication is important in building a therapeutic rapport with a patient and the
documentation of our communication is important in the ongoing sharing the needs, wishes,
goals and progress of the patients care as a process.
Communication and documentation are also critically important in our work delivery (as
care or services) both as and with other health care providers in the hospital or health care
setting.
Thus, our ability to communicate with and work with others on the health care team toward
the patients goals which we often refer to as collaborative care or collaborative practice can
be an important aspect of success as a registered technician in the health care setting.
In concluding this section of the presentation:
Hopefully, you have gained greater understanding of the concepts of communication and
therapeutic communication for use within the health care setting.
As you continue your experience in the Entry to Practice Training Program we invite you
to continue building your abilities to demonstrate and exhibit a strong therapeutic
communication style of behavior --- as this will be a very important component to your ongoing
success during the placement experience and as you continue your career path in health care.
- Slide 30 - Collaboration & Teamwork
During the next part of this lecture session on collaboration and teamwork, our learning
objective and outcomes will include building an:
Understanding of who is on your team
Increased understanding of the benefits/barriers to collaboration and teamwork
And
Understanding of delegation and how this may occur in the delivery of work in the health
care setting.
- Slide 31 - Communication & Collaborative Practice
In our previous presentation we talked about the importance of communication and
collaborative practice in the health care setting.
25

To begin our next session, let us begin by reviewing some of those definitions of the key
terminology
- Slide 32 - What does Teamwork Mean to You ?
Now lets take a moment and consider what teamwork means to you ?
So in reflecting on this photograph what does it say to you about teamwork ?
How does it fit with your own beliefs about teamwork ?
[With gentle encouragement invite the trainees to share their thoughts]
To summarize
Sometimes things can be easier to achieve when we have help.
Thinking of your own life we all may have certain responsibilities. Our parents, our
children, our spouse but when we distribute these responsibilities, with each person having
their own duties to perform, we are acting like a team, and as a result the entire family and
household may function properly because of this sharing. This is like teamwork.
Teamwork is necessary if we are to make health care function properly for the sake of the
patient.
- Slide 33 - The Patients Healthcare Team
[Invite the trainees to share their ideas]
- Slide 34 - Patients Interprofessional Healthcare Team
The patients interprofessional health care team can include the following
There may also be others not identified here who will or can also be invited to assist in the
patients care or its planning.
- Slide 35 - Why is teamwork - as the health care team important?
So why is teamwork as a health care team important ?
Can anyone think of an example to share with the group?
Perhaps drawing from personal experience?
[Invite the trainees to share their ideas from the perspective of the registered technician]
- Slide 36 - Importance of Teamwork
There are several important aspects to consider when thinking about teamwork in the
health care setting.
- Slide 37 - Benefits of Teamwork Approach
There are also some important benefits in using a teamwork approach which you should
keep in mind as you develop your role in the health care setting.
- Slide 38 - Barriers of Working on a Team
There
Role boundaries and limits can create barriers therefore team members must have clearly
defined roles and responsibilities, which are clearly communicated to all team / staff members.
Some overlap may occur, but major responsibilities are designated to appropriately qualified
professionals.
Expressing your needs and views - Every team member needs to feel comfortable in
expressing valid concerns, needs, information, and views, and each team member needs to have
the opportunity to do so.
26

Teams can be too flexible and avoid decisions Often teams can try to be flexible or
agreeable, instead of contributing important information, and this can hinder team effectiveness
and efficiency.
Power and decision making imbalance when an unequal or unfair distribution of power
and decision making abilities, this can affect team sharing/contributions and team decision
making.
Workload imbalances - can lead to anger, frustration, conflict, missed deadlines, as well as
physical and mental fatigue and even illness can occur which can all negatively impact team
functioning.
Disagreements and conflicts may occur, therefore utilizing appropriate conflict resolution
techniques are essential to handle conflict and support ongoing healthy team functioning.
- Slide 39 - What do you Share with the Team?
As a team member it is important to understand what it means to be a fellow professional
and what may or may not be appropriate and ethical to share within teams.
Generally, the following three areas of focus are a good guide to ensure your communication
of information on teams is appropriate.
- Slide 40 - Benefits of Teamwork in Health Care
In summary, this has been a brief overview of collaboration and teamwork to assist you in
the health care setting.
The benefits of teamwork are significant in this type of workplace environment.
Teamwork is also a critical aspect of the workplace values and workplace culture of the
hospital or health care setting.
The benefits of teamwork can
Your developing and being open to a strong collaborative - teamwork style of behavior will
be an important aspect of your success during this placement and learning experience.
In concluding this section of the presentation:
Hopefully, you have gained greater understanding of the concepts of collaboration and
teamwork for use within the health care setting.
to continue building your abilities to demonstrate and exhibit a strong collaboration and
teamwork style of behaviors --- as they will be very important components in your ongoing
success during the placement experience and as you continue your career path in health care.
- Slide 42 - Delegation & Technician Practice
In this next lecture session, we hope to learn about and understand the following objective:
To review the process of delegation and the benefits and barriers to its use for teamwork
and practice delivery in the health care setting.
- Slide 43 - Delegation of Work Tasks
The importance of delegation in work tasks is vital to the success of work in health care
settings.
More specifically,
- Slide 45 - Understanding the Terminology
It is important to have a common understanding of some key terms as it relates to
delegation in the workplace.
- Slide 54 - Supervision & Reporting on Teams
It is important that as a trainee in the role of a registered technician that you recognize that
supervision and reporting is an expectation of all staff who work on or with teams in the health
care setting.
27

Within your trainee role you will need to identify and clarify these expectations with your
preceptor / supervisor and clinical instructor especially as it relates specifically to your unique
placement setting
Thus
- Slide 59 - Teamwork Choices & Decisions
In team work and delegation, there are reasons why an individual may decline the
delegation of a task.
As a trainee, it is important that understand the limits of your role and practice and under
what conditions you should decline the delegation of a task or responsibility.
- Slide 61 - Quality of Care Impact
Participating in teamwork and more specifically the delegation process which supports
patient care and services provision in the health care setting - is an important concept that you
as trainees in the placement setting need to understand.
Hopefully, you have gained a greater understanding of the concept of delegation and
appreciate its impact on care and service delivery, collaborative practice and teamwork as used
in the health care setting.
You will need as a trainee, to understand more specifically how this is done in your
placement and departmental setting.
to continue building your abilities to demonstrate and exhibit a strong response to participate
in roles of delegation as a work style behavior ---
This is important --- as it will be a very important component to your ongoing success
during the placement experience and as you continue your career path in health care as a
registered technician.

28
MinistryofHealth
Institute Graduates
Day 2 Communication,
Collaboration and Teamwork in
Health Care
MinistryofHealth
KingdomofSaudiArabia
TrainingProgramforHealthInstitute
Graduates
Collaboration,Communication&
TeamworkinHealthCare
LectureOutline
3
1) Introducetheconceptsofcommunicationand
therapeuticcommunicationinthehealthcare
setting
2) Toreflectandreviewtheimportanceofteamwork
andcollaborationincareandservicedeliveryto
patients
3) Toreviewtheprocessofdelegationandthe
benefitsandbarrierstoitsuseforteamworkand
practicedelivery
TrainingProgramforHealth
InstituteGraduates
Communication&Therapeutic
Interactions
Communication&TherapeuticInteraction
5
CommunicationandHumanInteraction
What is communication & why is it important ?
Allows the exchange of information
Permits thoughts and ideas to be shared
Conveys feelings and desires
Encourages questions and exploration of new things
Promotes diversity
Communication in some form allows expression and
supports all human interaction
Verbal and non verbal
Written and unwritten
6
29
WhatotherFactorsInfluenceCommunication?
7
Language&CommunicationDevelopment
Language and communication skills develop through
stages across a lifespan
How a person communicates as Newborn Infant
Toddler Young Child Adolescent Adult Older
Adult changes
Language and communication can be expressed
through movement, play, drawing , reading, writing,
story telling , facial expressions / body language and
sound / vocalization / speech
8
CommunicationProcess InfluencingFactors
Attitudes and biases
Values and perceptions
Roles and relationships
Environment (level of noise, personal space proximity)
Gender
Life stages growth & development, emotional state
Social / Cultural characteristics
Proximity personal space / territoriality
9
CommunicationinHealthCareSetting
Effective communication is essential to:
Understand and identify and the patients health
status understand and identify illness /disease based
needs
Provide all aspects of safe, patient centered care
Collaborate with others in the delivery of care and
services
Creating meaning and learning through shared
experiences
10
TheCommunicationProcess
Sender as source encoder
Message what is actually said
/ written, body language, how
words are transmitted or
channelled
Receiver is listener decoder
perception of intention
Response Feedback
11
Communication&Gender
Communication differences are noted between males and
females from an early age:
Boys learn to establish independence and negotiate
status
Girls seek confirmation, belonging and acceptance
Challenges to successful communication, can arise from
cultural, personality, age, and gender differences. If you
can recognize them in the interaction, you may be better
able to deal with them ~ in the process of communication.
12
30
TheMeaninginCommunication
Good communication is the key to success in just about everything in life.
Understanding how the two hemispheres of the brain are responsible for
different ways of thinking can help. People use both hemispheres, but some have
a distinct preference for one over the other.
13
RightBrain LeftBrain&Communication
Research suggests, leftbrained subjects focus on logical
thinking, analysis, and accuracy.
Rightbrained subjects focus on aesthetics, feeling, and
creativity. Rightbrained people can remember details
better if they have a story quality.
Thus, appreciating how differences in thinking,
perception and therefore the influence of the brain
creating meaning in the communication process ~ could
impact your relationships with other people including
those you work with.
14
Influences Values,Perceptions&Behaviors
Values standards that influence behaviors
Perceptions personal views of an event
Unique personality traits and experiences
Validation of previous experiences
Role modeling the behaviors of others
Sociocultural characteristics such as culture,
education and socio economic level can impact
development of language and the communication
process
15
TypesofCommunicationinHealthCare
16
Verbal Communication:
Use of common language, requires comprehension and receivers
perception / value judgement of information shared for understanding of
its meaning in the specific situational context
Can range from sharing of simple to complex thought and ideas
A persons level of literacy (knowledge and ability to understand the
spoken and written word in that language) and the ability to actually
understand the language being used will influence their ability to process
the message and understand the information or data shared
Pace, intonation of voice, clarity of speech , noise level in the
environment, body language, perceived congruence of the information
(verbal and nonverbal messages match), perceived credibility of the
speaker etc. Can all impact what and how information is heard and
interpreted by the receiver
Effective Written Communication: (legible)
Can be similar to verbal communication, but it does not
convey nonverbal cues
It typically depicts appropriate use of language and
terminology (impacts credibility / believability of
information)
It typically depicts logical organization of thoughts /
ideas
It can reflect a more permanent recording of events or
interactions
17 18
31
Electronic and Digital Communication:
Electronic communication offers advantages due to speed of
transmission, efficiency and legibility however it has
disadvantages as it compromises patient / client
confidentiality , may not comply with policy guidelines and
may not be accessible to everyone due to variation in
socioeconomic status
Trainee need to recognize agency specific guidelines and will
need consent to use electronic communication related to
sharing of patient information and care.
19
PersonalSpace&Communication
Personal space is defined as the
distance people prefer in
interactions with others
Intimate distance often see
in direct care
Personal distance less
overwhelming
Social distance increased
eye contact & decreased
touch
Public distance farther
away
20
TherapeuticCommunication
At the core of health care and services are therapeutic
relationships based on caring, mutual respect and dignity.
Communication is the way that these helping healing
relationships are established and reflects an interactive
process between the health care provider and patient / client
Therapeutic communication between health care providers
and patients empowers and enables the patient to know
themselves and make choices for themselves especially in
times of stress
21
Patient First(PersonCentredCare)
22
CharacteristicsofTherapeuticRelationship
Focus on the patient / client, build rapport
Respect the patient as an individual
Respect patient confidentiality
Focus on patient / client wellbeing
Based on mutual trust, respect and acceptance
Is supported by empathy, listening and uses silence
Is non judgemental and avoids personal information
Educate and empower the patient / client
Maintain truth telling
Communication&PatientDocumentation
Communication related to documentation in the patient
record or chart is vital to the care delivery process
Communication as documentation can reflect discussion
(teams) recording (charting of care and related health /
illness information) and formally documenting (as legal
documents)
Only those formally engaged in the care of the patient /
client have access to the patients record or chart
24
32
TypesofDocumentation
Source oriented record (examples admission sheet, vital
signs graphic record, MAR, diagnostic reports etc)
Problemoriented record where the documentation is
arranged according to the patients problems which can
encourage collaboration and alert caregivers to patients
needs
Charting by exception and reflecting standards of care with
the aid of bedside flow sheets and forms
Trainees will need to review documentation policies and
procedures in their placement setting for adoption and
their compliance in care and service delivery requirements
25
Dos&DontsofDocumentation
Dos
Chart changes in condition of patient
Be timely, read prior notes
Be objective & factual
Properly correct errors
Write legibly and neatly
Date & time all entries
Adhere to professional standards
Follow agency policy and procedures,
ethical guidelines
Treat as legal document
26
Donts
Do not leave blank spaces
Do not chart in advance
Do not use vague terms
Do not chart for others
Never alter the record
Do not record assumptions
Do not remove pages or destroy
any part of the patient record /
chart
If chart is electronic do not leave
computer terminal unattended
Do not share personal password or
security codes
Communication&CollaborativePractice
Collaboration is a way of working together that recognizes
collective responsibility and the need for interdependency of
relationships to achieve outcomes
Collaborative practice is defined as working toward mutually
identified goals while valuing different perspectives and
accountabilities of team members
Communication skills are used to gather, analyze and transmit the
information and to accomplish the goal work at each step in the
process
Good communication and collaborative practices can impact a
healthy teamwork environment and delivery of patient care quality
(Potter&Perry,2009)
27
Questions/Discussion
28
InstituteGraduates
Collaboration&Teamwork
Collaboration&Teamwork
Learning Outcomes:
Understand who is on your team
Increase understanding of the benefits/barriers
Understand delegation
30
33
Communication&CollaborativePractice
Collaboration is a way of working together that recognizes
collective responsibility and the need for interdependency of
relationships to achieve outcomes
Collaborative practice is defined as working toward mutually
identified goals while valuing different perspectives and
accountabilities of team members
Communication skills are used to gather, analyze and transmit the
information and to accomplish the goal work at each step in the
process
Good communication and collaborative practices can impact a
healthy teamwork environment and delivery of patient care quality
(Perry&Potter,2009)
31
WhatdoesTeamworkMeantoYou?
ThePatientsHealthcareTeam
Who makes up the patients Healthcare Team?
PatientsInterprofessional HealthcareTeam
Patient/client
Family/significant others
Physician
Specialists
Registered Staff
Registered Nurses
Physiotherapists
Occupational therapist
Registered Technician Domain Specific
Others
Whyisteamwork asthehealthcareteam
important?
RegisteredTechnician
ImportanceofTeamwork
Provides patient first holistic approach to care
Provides a consistent approach in supporting the
patients health goals / outcomes
Promotes all aspects of patients life
Physical
Emotional
Social
Intellectual
Spiritual
We depend on each other to perform roles and
communicate effectively with each other
34
BenefitsofTeamworkApproach BarriersofWorkingonaTeam
Role boundaries and limits
Expressing your needs and views
Being too flexible and avoiding decisions
Power and decision making imbalance
Workload imbalances
Handling conflict
WhatdoyouSharewiththeTeam?
What was done for the patient
What needs to be done for the patient
How the patient is responding to care & treatment
BenefitsofTeamworkinHealthCare
Supports collaboration
Supports communication
Assist with learning & knowledge building
Can improve decision making
Can improve problem solving
Distribution of work can be cooperatively shared
Assists with access to expertise as needed in care and
services delivery
41
InstituteGraduates
Delegation&TechnicianPractice
DelegationofWorkTasks
Some professional disciplines that are registered
often exhibit a scope of practice which provides
them the authority to delegate a task (physicians,
registered nurses, radiologists, pharmacists, medical
laboratory directors, etc)
When a task is delegated
do not be offended or angry
if you are not allowed to
perform a task that you usually do
Delegation Defined
The process by which responsibility and authority
are transferred to another individual
Transferring the authority to perform a controlled
act procedure to a person
Delegation is a mutual transfer of responsibility &
authority that occurs on the basis of competence &
trust
44
UnderstandingtheTerminology
Responsibility
An obligation to accomplish a task
Accountability
The act of accepting ownership for the results or
lack thereof
Delegation is a learnable skill and involves good
communication techniques, the spirit of teamwork
and understanding of the principles of delegation
45
PrinciplesofDelegationinPractice
1. You can only delegate those tasks for which you are
responsible
2. You must transfer responsibility and authority for
the performance of an activity
3. You must know the task to be delegated
4. You must keep in mind the goal is to accomplish
the care or work safely
5. Registered staff may remain accountable for the
outcome
46
BenefitsofDelegation
Benefits to the Delegator
Time & efficiency
Development of new skills and abilities
Increase selfesteem & confidence
Job satisfaction
Increase teamwork
Increase goals and objective outcomes
Increase the quality of care
Decrease absenteeism and overtime
47
TheDelegationProcess
1. Define the task
What can and cant be delegated?
You can only delegate what you have responsibility
and authority over
You can only delegate effectively what you know
best
Need to define the aspects of the task
48
36
2. Determine who to delegate to
What skill and / or qualifications is required
Scope of practice
Who is available
Who is willing to accept the delegation
49
3. Clearly communicate and describe expectations
Clear and complete communication of expectations
Describe the task
Provide rationale for the task
Describe the outcome expected
Validate understanding of the person being
delegated the task
Be clear and reasonable about time frames
50
4. Mutual agreement
Seek agreement that he is willing to accept
responsibility and authority
51
5. Monitor performance and give feedback
Assessment and follow up evaluation
Be accessible
Give support
Praise and give recognition
Be public about accomplishments
52
Trainee AcceptingDelegation
Have a clear understanding of what is being asked of you
Be realistic and examine whether you have the skills and
abilities for the task
Do you have the time? If you do not be honest!
Ask questions and seek clarification if uncertain
Five Rights (5) of Delegation:
Right task, right circumstances, right person, right direction
& communication, right supervision & evaluation .
53
Supervision&ReportingonTeams
Registered Technicians are responsible to the patient / client
and coworkers in their service provision
Trainees will be accountable to their trainer in practice in the
placement setting however they will work collaboratively with
other Team members in the practice setting
As an employee the Registered Technician is accountable to
their supervisor and also within the chain of reporting /
decision making command to:
Department Head / Department Manager who may report to a
more senior executive leader
37
CommonEnvironmentalObstaclestoDelegation
Compliance with Standards
Job Descriptions which limit roles & responsibilities
Organizational policies which limit roles
Organizational structures which limit roles
Management styles
Challenging norms of practice
Resource limitations
55
ObstaclestoDelegation Delegator
Lackoftrust&
confidence
Believeothers
incapable
Believeself
indispensable
Fearofcompetition
Fearofcriticism
Fearofliability
Fearofblame
Fearoflossofcontrol
Fearofoverburdening
Fearofdecreasedjob
satisfaction
Insecurity
Inexperiencein
delegation
Inadequate
organizationalskills
56
ObstaclestoDelegationbyDelegatee
Inexperience
Fear of failure and reprisal
Lack of confidence
Overdependence on others
Avoidance of responsibility
57
WhyDelegationcanbeIneffective
Under Delegation
Major reason for under delegation is the need for control and
perfection
It occurs when full authority is not given
Delegator fails to provide proper equipment or instruction
Reverse Delegation
Delegator takes back responsibility
Over Delegation
Too much authority and/or responsibility is given
58
TeamworkChoices&Decisions
Trainees have right to say no to a delegated task when:
Beyond the practice scope or limits of your role
Not prepared to perform the task safely
Could harm the person
Condition of patient has changed
You do not know how to use
the supplies or equipment in the task
Do not ignore a delegated act
Must communicate concerns to delegator
Must have sound reason for your refusal
Trainee InDelegationwhatdoIreport
Completion of the task / responsibility, any issues or
concerns in delivering the task, anything that is
different from what was expected to happen
Report any changes in the patients presentation, their
abilities or presenting signs / symptoms
Need to develop trust & rapport with the staff member
delegator which emerges from you responsibility for
practice task and reliability of the information reported
38
QualityofCareImpact
By participating as a collaborative team member,
who values and embraces the spirit of teamwork as
well as, communicates clearly his scope of practice
roles and responsibilities a Trainee can have a
significant impact on quality patient care and
services delivery.
61
62
References
Potter, P.A., & Perry, A. G. (2009). Canadian fundamentals
of Nursing (4
th
ed.). Toronto, Canada: Elsevier Canada.
63
39

Infection Control in Health Care Setting

- Slide 3 - Trainee Orientation to Infection Control
Every health care worker has the responsibility to provide safe health care.
This responsibility includes knowledge of how to keep themselves safe from infection, and
knowledge of how to prevent the spread of infection to patients.
Health care workers need to:
Recognize patient and health care staff safety as an important responsibility in practice
delivery and build awareness of the role(s) of infection prevention and infection control
protocol systems.
Apply required knowledge in preventing and/or minimizing infection in daily practice.
Perform appropriate behaviors required to prevent health care associated infections.
Demonstrate required competence to provide patients with safe care and reduce / protect
others from the risk of hospital acquired infections.
- Slide 5 - Infection Control
There is an important need to control the spread of infection.
There may be many ways to help control the spread, and each strategy plays an important
part in infection control.
Each strategy can also protect both healthcare workers and patients, as well as everyone
else that each of us are near to or may come in contact with.
Thus, infection prevention and infection control is an important practice in the hospital
health care setting given the high risk of infectious illness and disease type agents that may be
found in the setting.
Infection control typically,
Includes all of the practices used to prevent the spread of microorganisms that could cause
disease in a person, and
Infection control practices help to protect clients and healthcare providers and families and
visitors from disease by reducing and/or eliminating sources of infection.
- Slide 6 - What are Nosocomial Infections ?
What are nosocomial infections?
Nosocomial infections are also known as hospital acquired infections.
They result from delivery of health services in a healthcare setting, and clients are at an
increased risk of catching these infections.
Unfortunately, HAIs (Hospital Acquired Infections) may lead to extended hospital length of
stay, healthcare costs and potential mortality.
This is another reason why we need to learn how to prevent the spread of infection.
- Slide 7 - Health Care - Hospital Associated Infections
Here are interesting facts about hospital associated infections or HAIs.
It is easy to see how costly HAIs can be.
Costly to the health of loved ones and costly to treat.
This is another reason why it is important for health care workers to learn how they can
help prevent the spread of infection.
- Slide 11 - What is the Chain of Infection ?
These factors involved in the transmission of infection and are considered part of the Chain
of Infection (as seen in the diagram).
From the diagram we can see that the Chain of Infection involves:
the Causative agent (the micro-organism that has the ability to cause infection or disease)
the Reservoir (the place where this organism can live, reproduce, thrive. It may be a person,
or a table top)
40

the Portal of Exit (the place where this organism may exit or escape from, such as a nose
or mouth during a cough, or the bowel as feces, are examples),
the Mode of Transmission (the method by which the organism is transferred from one place
to the next. For example, from the hands of a healthcare worker to another person, through the
air, or through sharing another contaminated object)
the Portal of Entry (the place where the organism enters a host, such as through broken skin
or mucous membranes.)
the Susceptible Host (the person who cannot resist the micro-organism from invading their
body and causing infection or disease. Someone who has a chronic illness, or who is elderly, are
more susceptible than healthier individuals, for example.)
Healthcare workers need to be aware of the chain of infection, as well as the opportunities
to break the chain.
In doing so, workers protect themselves and their patients from the spread of infectious
disease.
The chain of infection, and ways of breaking this chain, will be discussed a little later in this
presentation.
- Slide 15 - Breaking the Chain of Infection Transmission
In this diagram we can see the Chain of Infection depicted,
We also see ways of breaking that chain of infection identified.
This list outlines several ways of reducing transmission of infectious organisms, and
therefore they are strategies which we can utilize to prevent spread of infection.
The chain of infection, and the strategies listed above, will be discussed further, in
subsequent slides.
- Slide 16 - Chain of Infection
Understanding some of the key terminology used in the Chain of Infection, that we are
discussing will be helpful in facilitating a better understanding of infection prevention and
infection control practices that will now begin to explore in this presentation.
- Slide 17 - Chain of Infection
Airborne refers to infectious agents/organisms which are carried long distances by air
currents. They are carried through very small airborne particles, and are not seen with the eye.

Example of infectious disease, which can be transmitted through the air, include Measles
and Tuberculosis.
Contact refers to infectious agents which are spread through touch.
For example, touch/contact of an infected person of the infectious organism coming in
contact with things like furniture, or equipment may allow for transmission of the infectious
agent / organism from one person to another
Droplet refers to infectious agents which are transmitted by large respiratory droplets,
that may travel up to 1-2 meters from the patient.
- Slide 18 - Chain of Infection & Mode of Transmission
Understanding the mode of transmission can be helpful in understanding how to break or
interrupt the chain of infection and therefore reduce the transmission or spread of infection.
- Slide 21 - Early Recognition & Planned Precautions
An important key in the prevention of infection begins with the health care worker
recognizing and identifying that there may be a potential source of infection and therefore
reason for concern.
- Slide 22 - Epidemiology and Surveillance Outbreak
This slide contains common terminology to be aware of, regarding the study of population
based infection, illness and disease patterns and trends.
41

Each healthcare worker has the responsibility of helping to break the chain of infection.
Additionally, health care providers should remember that this may involve educating the
patient and the patients family / visitors about ways to also prevent the spread of infectious
organisms or agents.
This may include ways of isolating the pathogen and eliminating its mode of transmission
thereby breaking the chain of infection.
Breaking any potential area in the chain (as in the diagram) can reduce the risk of further
transmission.
- Slide 29 - Infection Prevention & Control Practices
In your hospital health care placement setting there are resources to help educate and
guide your practice, regarding infection prevention and infection control strategies.
This includes the organizations key policies and procedures related to safe practices.
As trainees, you will need to identify and understand the key policies, procedures and
practices related to infection prevention and infection control that will be required to safely
fulfill your registered technician role and responsibilities.
- Slide 31 - Infection Control Know the Precautions
Infection Control Know the Precautions:
Standard Precautions
Standard precautions should be applied for ALL patients.
Standard precautions include the need to wash your hands between any contact with or
before and after any direct contact with a patient.
This includes for example, contact with blood, other body fluids or excretions, non-intact
skin, and wound dressings.
You should also wash your hands after touching anything in the patients area and after
removing gloves.
You should also wear personal protective equipment (like gloves or gowns) if you anticipate
contact with bodily fluids,
You should discard sharp instruments and needles in a puncture resistant container, and
clean shared equipment between patient use,
You should teach patients to cover mouth when coughing, and teach patients to wash their
hands.
Standard precautions are used for all patients regardless of their diagnoses to ensure
protection of the patient, the health care worker and any other patients being cared for in the
same location.
For certain highly transmissible or epidemiologically important pathogens, other
transmission-based precautions are used in addition to standard precautions.
Transmission-Based Precautions to Isolate Infectious Agents
Contact
Droplet
Airborne
Contact, droplet, and airborne precautions are meant to block the different routes or modes
of transmission.
- Slide 35 - Contact Precautions
Contact precautions offer a simple prevention strategy:
In hospital health care setting it is helpful to post signs called practice precautions
(sometimes also called isolation precautions) at the entrance/doors of the rooms containing
patients with infectious illness or disease.
The signs/precautions are an important way of informing staff and visitors that infection
has been identified and that certain protective measures may be require before entering the
room.
This is an important step in preventing the further spread of infection.
42

Essentially the signs alert healthcare staff, family and visitors to the presence of infection,
and advise them of how to protect themselves before entering the room.
Notice the sign on top for contact precautions in the diagram.
It informs people the patient is isolated for contact precautions.
It also informs people to clean their hands, wear gloves and a gown before entering, and to
disinfect equipment before using with other patients.
Your will need to check the precautions required for use in your own placement setting.
- Slide 38 - Droplet / Contact Precautions
This is an example of a Droplet/Contact Precautionary sign (as a prevention strategy).
It contains instructions for visitors and healthcare staff, including the personal protective
equipment that is required before entering the patients room.
Notice it advises people to wear a mask (example: an N95 mask, which filters out up to
95% of airborne agents, if fitted properly).
It also advises gowns, gloves, hand washing, and eye protection.
Family and visitors need to be educated about these precautionary requirements.
- Slide 41 - Airborne Precautions
This is an example of an Airborne Precaution sign (a prevention strategy).
It contains instructions for family, visitors and healthcare staff, including the personal
protective equipment that is required before entering the room.
Notice this sign informs us that respiratory protection, such as a special N95 mask is
required, hand washing and the need to keep the patients room door closed.
- Slide 43 - Prevent Infectious Disease Transmission by:
In summary, this diagram illustrates some of the ways that infection is spread or
transmitted in the hospital health care setting environment.
For example, this may include transmission through the use of shared contaminated
equipment and furniture, being transported on the hands of healthcare workers, family
members and visitors.
The infectious organism / agent is then carried to the next susceptible patient - as a host.
Also important in the diagram is the depiction of ways to help break or prevent the spread
of infection.
The precautionary measures for health care workers to follow regarding droplet
transmission of infection are:
- Slide 47 - PPE for Standard Precautions
PPE as Standard Precautions include:
This is true, unless additional precautions are necessary for airborne, droplet, or contact
concerns.
In that case, we have additional precautions (as discussed in earlier slides, such as N95
mask etc), that are utilized in addition to those of the above standard precautions.
You will notice here that standard precautions are used with all patients.
- Slide 48 - Bloodborne Pathogens
In considering the necessary precautions for health care workers to be protected from
bloodborne pathogens, the following approach should be utilized.
It is important to understand, that bloodborne pathogens are the infectious organisms
found and transmitted within the blood.
- Slide 50 - Bloodborne Universal Precautions
This is an example of a precaution sign that depicts the universal precautions taken to
protect the health care worker and other patients from the risk of transmission of bloodborne
infections.
- Slide 52 - Personal Protective Equipment (PPE)
43

PPE is an important strategy in the prevention of the spread of infection.
We have been discussing the use of personal protective equipment as a mandatory
requirement of all health care workers in order to support the prevention and control of
infection as being spread in the health care setting from person to person.
In summary , there are some important things to remember.
- Slide 54 - Personal Protective Equipment PPE
Personal protective equipment is shown here.
It includes gown, gloves, shoe covers, mask, eye shield, and hair net/cover.
It may also be worn by a member of a surgical team wishing to support surgical asepsis.
- Slide 60 - Effective Hand Washing Technique
Ensuring an effective hand washing technique also includes the following:
Tip to remember:
Keep in mind dirty hands turned the tap/water on. Therefore, the handles/tap are dirty.
You may use the paper towel to turn tap/water off.
The paper towel can also be used to open the door and then throw the paper towel away.
- Slide 64 - Aseptic Technique Types
The word aseptic refers to being free from germs that cause disease or infection or being
free from contamination of infection-causing organisms.
Aseptic techniques are ways by which we prevent the spread of these germs, or ways we try
to minimize contamination.
The two types mentioned here are:
- Slide 74 - Summary of Infection Control Practices
Participating in infection prevention and supporting infection control in patient care and
services provision in the health care setting - are important concepts that you as trainees in the
placement setting need to understand.
Hopefully, you have gained a greater understanding of the concept of infection prevention
and infection control and appreciate its impact on care and service delivery and practice
precautions as used in the health care setting.
You will need as a trainee, to understand more specifically how this is done in your
to continue building your abilities to demonstrate and exhibit a strong response to participate
in roles of infection prevention and control as your work behavior ---
This is important --- as it will be a very important component to your ongoing success

44
MinistryofHealth
Training Program for Health Institute
Graduates
Day 2 - Infection Prevention & Control
LectureOutline
2
1) DescribeHealthcareAssociatedInfection(HAI)
2) Describethechainofinfection&standard/
universalprecautions
3) Beawareandunderstandtherole(s)ofisolation
precautionsinpreventingthespreadof
transmissionforcertaininfections
4) Describeeachtypeofisolationprecautionusedto
reducethespreadofcontact,droplet,airborne
infectiousorganismsintheplacementsetting
5) Reviewhandhygieneanditsrequireduse
TraineeOrientationtoInfectionControl
Recognizepatientandhealthcarestaffsafetyasan
importantresponsibilityinpracticedeliveryandbuild
awarenessoftherole(s)ofinfectionpreventionand
infectioncontrolprotocolsystems.
Applyrequiredknowledgeinpreventingand/or
minimizinginfectionindailypractice.
Performappropriatebehaviorsrequiredtoprevent
healthcareassociatedinfections.
Demonstraterequiredcompetencetoprovidepatients
withsafecareandreduce/protectothersfromtherisk
ofhospitalacquiredinfections.
3
Infection
Aninvasionofpathogensormicroorganismsintothe
bodythatarecapableofproducingdisease.
Theinvasionandreproductionofmicroorganismsin
abodytissuethatcanresultinalocalorsystemic
clinicalresponsesuchascellulitis,feveretc.
4
InfectionControl
Includesallofthepracticesusedtopreventthe
spreadofmicroorganismsthatcouldcausedisease
inaperson.
Infectioncontrolpracticeshelptoprotectclientsand
healthcareprovidersandfamiliesandvisitorsfrom
diseasebyreducingand/oreliminatingsourcesof
infection.
5
WhatareNosocomialInfections?
Resultfromdeliveryofhealthservicesina
healthcaresetting,clientsareatincreasedrisk.
Unfortunately,HAIsmayleadtoextendedhospital
lengthofstay,healthcarecostsandpotential
mortality.
45
HealthCare HospitalAssociatedInfections
AccordingtotheWorldHealthOrganization(2005):
Onaverage,8.7%ofhospitalpatientssufferhealthcare
hospitalassociatedinfections(HAI)
DevelopedcountriesHAIisapproximately510%
WhileindevelopingcountriestheriskofHAIis220
timeshigherandmayaffectmorethan25%ofpatients
Atanyonetime,over1.4millionpeopleworldwidesuffer
frominfectionsacquiredwhileinhospital.
7
HealthCare HospitalAssociatedInfection
WHOdefinesaHAIas:
Aninfectionacquiredinhospitalbyapatientwho
wasadmittedforareasonotherthanthatinfection.
Aninfectionoccurringinapatientinahospitalor
otherhealthcarefacilityinwhomtheinfectionwas
notpresentorincubatingatthetimeofadmission.
Thisincludesinfectionsacquiredinthehospitalbut
appearingafterdischarge,andalsooccupational
infectionsamongstaffofthefacility.
8
MainSourcesofInfection
Healthcareassociatedinfections:
Arecausedbybacteria,fungiorvirusesenteringthebody
throughoneormoreofthefollowingroutes:
Personpersonviahandsofhealthcareproviders,
patientsandvisitors;
Personalequipment(e.g.stethoscopes,computers)and
clothing;
Environmentalcontamination;
Airbornetransmission;
Carriersonthehospitalstaff;
Commonsourceoutbreaks
9
ImpactofHealthCareAssociatedInfection
HAIcan:
Increasepatientssuffering.
Leadtopermanentdisability.
Leadtodeath.
Prolonghospitalstay.
Increaseneedforahigherlevelofcare.
Increasethecoststopatientsandhospitals.
10
WhatistheChainofInfection?
There are several factors
which influence the
opportunity and risk of
acquiring an infection where
transmission occurs from one
person to another in a health
care setting.
11
TransmissionofNosocomial Infections
Mostnosocomialinfectionsaretransmittedby
healthcareworkersandclientsasaresultofdirect
contact.
We,mustpayparticularattentiontowashinghands
aftercontactwithclientsorequipment.
12
InfectionPrevention&ControlPractices
GuidelinesforInfectionPrevention&ControlPractices
SaudiMinistryofhealthreportingpolicies
GCCinfectioncontrolmanual
WHOguidelines
CentreforDiseaseControlGuidelines
LocalHealthCareSetting/HospitalPolicies&Procedures
DepartmentalProcedures
InfectionControl
ManagementofPrescribedContagiousConditions
FactSheets/SafetyAlerts
Forpatients,visitorsandstaff
13
PreventionofInfections
Requireshealthcareproviderswhohave:
Knowledgeofcommoninfectionsandmodesoftransmission
Knowledgeoftheextentoftheproblem;
Knowledgeofthestepsandstrategiestoprotectselfandothers
Anattitudeofcooperationandcommitment
Beinganeffectiveteamplayer
CommitmenttopreventingHAIs
Knowledgeofappropriatereportingprocedure
Knowingwhenandwhotoaskforhelp
14
BreakingtheChainofInfection Transmission
InfectionControl
Practices
Cleaningand
Disinfecting
InfectiousWaste
ContagiousConditions
Immunization
15
ChainofInfection
InfectiousAgent microorganism(bacteria,viruses)
Resident normallyresideontheskininstable
numbers
Transient attachlooselytotheskinbycontactwith
another easilyremovedbyhandwashing
Reservoir orsourceofpathogen.
Pathogensurvivesherebutmayormaynotmultiply.
PortalofExit
Fromthereservoir,exitthroughtheskin,respiratorytract,
orblood.Sitewheremicroorganismleaves.
16
ChainofInfection
ModeofTransmission ormeans ofspread:
Airborne
Contact
Droplet
PortalofEntry(tothehost)
Entersthesamewaytheyexitthehost(example:open
wound,breathein)
17
ChainofInfection&ModeofTransmission
18
For transmission of infection, there must be sufficient quantities of the pathogen,
AND the pathogen must be virulent enough to cause disease. The pathogen moves
through a route of transmission, and reaches a portal of entry, such as eyes, nose,
mouth, or puncture wound, to enter the susceptible host.
Disease transmission can be prevented by breaking one or more of the links in this
chain of transmission
Most pathogens have a preferred portal of entry, one that gives ready access to an
immediate environment suitable for the establishment of growth, or one that allows
the pathogens to reach their target tissues or organs.
Common portals of entry include the respiratory tract, Gastro intestinal tract, mucosa
(e.g., conjunctiva, nose, mouth), genitourinary tract, breach of skin integrity, or
mosquito bite
Next, need to review the different routes of transmission from an infected patient to
a susceptible host.
47
SusceptibilityofHost
Hostmustbesusceptibletothestrengthand
numbersofthemicroorganisms.
Toreducesusceptibility
provideadequatenutrition&
rest,promotebodydefenses
againstinfection&provide
immunization
19
PatientRisksforDevelopingInfections
1. Lowerresistancetoinfectiousmicroorganisms(dueto
illnessordisease).
2. Exposuretoanincreasednumberofandmoretypesof
diseasecausingorganisms.(Hospitalharborsahigh
populationofvirulentstrainsofmicroorganismsthat
areresistanttoantibiotics)MRSA,VRE superbugs.
3. Theperformanceofinvasiveprocedures.(IVcathetars
etc..Anythingthatcrossesprotectivebarriers)
20
EarlyRecognition&PlannedPrecautions
EarlyRecognition
Health care facility staff must quickly identify and separate
potential sources of infection from susceptible hosts
Since the infecting agent often is not known at the time of
admission to a health care facility, transmissionbased
precautions are used empirically, according to the clinical
syndrome and the likely etiologic agent at time, and then
modified when the pathogen is identified or a transmissible
infectious disease etiology is ruled out.
These infection control principles are also used for
laboratory and procedurespecific safety.
21
EpidemiologyandSurveillanceOutbreak
Outbreak is a term used in epidemiology to describe an
occurrence of disease greater than would otherwise be
expected in a particular time and place. It may be small
and localized group or impact upon thousands of people
across an entire continent.
Two linked cases of a rare infectious disease may be
sufficient to constitute an outbreak. Outbreaks may also
refer to epidemics, which affect a region in a country or
a group of countries, or pandemics, which describe
global disease outbreaks.
22
RequiredSkillstoPreventInfections
Applystandard/universalprecautions*
Usepersonalprotectionequipmentmethods
Knowwhattodoifyoubecomeexposed
Encourageotherstouseuniversalprecautions
Reportbreaksintechniquethatincreasepatient
risks
Observepatientsforsignsandsymptomsof
infectionandreportforisolationandoutbreakrisk
management
23
InfectionControlPractices
TraineesRoleinPrevention
and
InfectionControl
24
48
PrinciplesofInfectionControl
1. Microorganisms move through space on air
currents avoid shaking or tossing linen.
2. Microorganisms are transferred from one surface
to another whenever objects touch, a clean item
touching a less clean item becomes dirty keep
hands away from face, keep linens away from
uniforms, an item dropped on the floor is
considered dirty.
25
3. Microorganisms are transferred by gravity when
one item is held above another, avoid passing dirty
items over clean items, eg. Clean items on upper
shelves dirty items on lower shelves (bedpan).
4. Microorganisms are released into the air on
droplet nuclei whenever a person breathes or
speaks avoid breathing directly in someones
face; when someone coughs/sneezes, cover mouth
with tissue, discard, wash hands.
26
5. Microorganisms move slowly on dry surfaces, but
very quickly through moisture use paper towel to
turn facets off, dry bath basin before returning to
bedside table.
6. Proper handwashing removes many of the
microorganisms that would be transferred by the
hands from one item to another always wash
hands between patients.
27
BreakingtheChainofInfection
Various types of
infection prevention &
control strategies by
isolation of the
pathogen and
transmission mode can
reduce the risk(s) of
person to person
spread of hospital
acquired infections
28
InfectionPrevention&ControlPractices
GuidelinesforInfectionPrevention&ControlPractices
SaudiMinistryofhealthreportingpolicies
GCCinfectioncontrolmanual
WHOguidelines
LocalHealthCareSetting/HospitalPolicies&Procedures
DepartmentalProcedures
InfectionControl
ManagementofPrescribedContagiousConditions
FactSheets/SafetyAlerts
Forpatients,visitorsandstaff
29
PracticePrecautionsforRouteofTransmission
Contact:Infectionsspreadbydirectorindirect
contactwithpatientsorthepatientcare
environment
Droplet:Infectionsspreadbylargedroplets
generatedbycoughs,sneezes,etc.
Airborne (dropletnuclei):Infectionsspreadby
particlesthatremaininfectiouswhilesuspendedin
theair
30
49
InfectionControl KnowthePrecautions
StandardPrecautions
ShouldbeappliedforALLpatients
Standardprecautionsareusedforallpatientsregardlessoftheirdiagnoses
toensureprotectionofthehealthcareworkerandthepatient.
Forcertainhighlytransmissibleorepidemiologicallyimportantpathogens,
transmissionbasedprecautionsareusedinadditiontostandardprecautions.
TransmissionBasedPrecautionstoIsolateInfectiousAgents
Contact
Droplet
Airborne
Contact,droplet,andairborneprecautionsaremeanttoblockthedifferent
routesoftransmissionthatwediscussedearlier.
31
TransmissionbyDirect&IndirectContact
Some pathogens are spread through direct contact, while
others are spread through indirect contact.
Examples of direct contact include persontoperson
mechanisms such as kissing, skintoskin contact and sexual
contact.
As well there can be direct contact with animals, soil or
vegetation.
Indirect contact occurs when an agent is carried from a
reservoir (the source of infection) to a susceptible host
without direct contact with the source. For example, by
touching a contaminated surface a person can become
infected.
32
ContactTransmission Precautions
Trainees should avoid contact with their face, eyes or mouth when
their non gloved or gloved hands may be contaminated
Limit patient movement outside of their designated room
Limiting patient contact with other well patients
Contact Transmission
Persons infected with some common respiratory pathogens can
spread their disease by either contaminating their own hands, the
hands of a healthcare worker or an environmental surface.
Hands can transmit these diseases when they have contact with
contaminated surfaces followed by contact with either another body
surface such as the conjunctiva or nasal mucosa or an intermediate
object.
33
ContactTransmission Precautions
Usedforprotectionagainstcontact(i.e.,handcontaminationorself
contact)withlargedroplets
ThekeyelementsofContactPrecautionsforpatientssuspectedor
confirmedofhavingadiseasespreadbydropletsorsomecommon
respiratorypathogensare:
Usingclean,nonsterileglovesforallepisodesofdirectpatient
contact
Changingtheglovesaftereachpatientcontact
Useagown(disposableorrewashable)foreachpatientcontact
anddisposingofitaseitherwasteorlaundrydependingonits
type,aftereachepisode
Usededicatedspecificequipment(preferablesingleuse)fora
singlepatientandcleananddisinfectsharedequipmentbetween
patientuses.
34
ContactPrecautions
35
DropletTransmission
Droplet transmission involves contact of the conjunctivae (the mucous
membrane that lines the inner surface of the eyelid and the exposed surface
of the eyeball) or the mucous membranes of the nose or mouth of a
susceptible person with largeparticle droplets containing microorganisms
generated from a person who is infected with the microorganism.
Droplets are generated primarily during coughing, sneezing, or talking and
during the performance of certain procedures such as suctioning and
bronchoscopy.
Transmission via largeparticle droplets is associated with close contact
between people, probably because large droplets typically do not travel
beyond about 1 meter.
Some examples of diseases that spread via large droplets are pharyngeal
diphtheria, pertussis, and meningococcal disease.
36
50
DropletTransmission Precautions
Droplet Precautions are used in addition to Standard Precautions
to provide protection to clinicians and others protection from
infections spread by large droplets generated by coughs and
sneezes
Additional protection measures critical under Droplet Precautions are:
Use of a medical/ procedural mask (by Trainees) when within a
meter of a patient
Physically maintaining distance between the infected patient and
other persons by a distance of at least one meter from all other
persons,
Limit patient movement. If a patient has to leave the area, the
patient should wear a medical mask, if tolerated, for the duration
of their time away.
37
Droplet/ContactPrecautions
38
AirborneTransmission
Airbornetransmissionoccursby
disseminationofinhalable
infectiousmaterial.
Microorganisms carried in this manner can be dispersed widely
by air currents and those that remain infectious while in the air
may cause infection when inhaled or deposited on a susceptible
hosts respiratory tract, potentially over a long distances from
the source patient, depending on the environmental factors.
Certain therapeutic procedures (e.g., endotracheal intubation,
bronchoscopy) are associated with the generation of aerosols.
39
AirborneTransmission Precautions
Airborneprecautionsareusedforprotectionagainstinhalationoftinyinfectious
dropletnuclei
InadditiontoStandardPrecautions:
Useaparticulaterespirator(example:N95Mask)whenenteringthepatient
isolationroom. Performamask sealcheckbeforeeachuse.
Placethepatientinadequatelyventilatedroom(12airchangesper
hour)
Limitpatientmovementandensurethatthepatientwearsamedicalmaskif
outsidetheirroom.
Airborneprecautionsshouldalsobeperformedduringtheperformanceofany
aerosolgeneratingproceduresassociatedwithpathogentransmission(e.g.,
endotrachealintubation,bronchoscopy)
40
AirbornePrecautions
41
DrugResistantOrganisms&Carriers
Epidemiologicalevidencesuggeststhatmultidrug
resistantorganismsarecarriedfrompersontoperson
byhealthcareprofessionals.
Carriersareindividualswhoharbordiseaseorganismsin
theirbodywithoutvisiblesymptomsandmaypassthe
infectiontoanotherperson.
Itispossibletocarryanorganismwithoutbeingaware
ofitforexample,TyphoidMaryawomanwhocarried
thetyphoidbacillusandunknowinglystartedan
epidemicintheUSinthe1880s.
42
51
PreventInfectiousDiseaseTransmissionby:
SourceControlMeasures
Coughetiquette,cleaningand
disinfectionofenvironment
ModesofTransmission
Contact:handhygiene
Droplet:distancefrom
sourcelessthan12meters
Airborne:isolationrooms
andventilation
PortalofEntryintotheHost
Addingbarriers,e.g.,PPE
Host
Strengthenhostdefences,e.g.,
vaccination,immunization
43
RequiredPerformanceofTrainee
Traineesneedto:
Apply standard/universalprecautions
Consider immunizedagainstHepatitisB
Usepersonalprotectionequipment(PPE)methods
Knowwhattodoifexposedtoblood/bodilyfluids
Encourage otherstouseuniversalprecautions
Traineesneedtomakeeveryefforttominimizethe
spreadofinfectionandtoencouragepatientsandother
healthcareworkerstoactivelyengageinpracticesthat
minimizethespreadofinfectioninthehospital.
44
BasedontheconceptthatbodilyfluidsfromANYperson
canbeinfectious
Therefore,standardprecautionsshouldbeusedon
everypatient
All necessary PPE for protection use by Trainees is
mandatory and should comply with the Centre for Disease
Control Guidelines which requires that the use of category
specific isolation for infection in addition to the use of all
standard precautions be used when a patient is known or
suspected to have an infection.
45
StandardPracticePrecautions
Traineesshouldapproacheverysituation
ashavingthepotentialtoinfectapatient
orahealthcareworkerorthemselves.
Infectionsarepreventablewhenhealth
careworkersusetherighttechniquesand
remainonthelookoutforuncleanand
unsafesituations.
Guidelinesforpreventingexposetoblood,
bodyfluids,secretions,excretions,broken
skinormucousmembranesshouldbe
followed.
46
PPEforStandardPrecautions
IF direct contact with blood & body fluids,
secretions, excretions, mucous membranes, non
intact skin
Gloves
Gown
IF there is the risk of spills onto the body and/or face
Gloves
Gown
Face protection (mask plus eye protection goggle
or visor; face shield)
47
Bloodborne Pathogens
Do not touch or try to clean up any bodily fluids such as
blood, urine, stool or vomit unless you have been
trained in the use of proper PPE
Trained staff with latex gloves (and safety glasses) are
authorized to handle this type of hazardous waste
material and its proper disposal
If you are exposed to any bodily fluids notify your
supervisor immediately (example: needle stick injury,
contact through an open area of the body or mucous
membrane etc.)
48
52
Ifexposedtoblood/bodyfluids
Guidelines if exposed to blood or body fluids
If puncture type wound (needle stick injury) occurs it should
be washed immediately and should be caused to bleed.
If skin contamination should occur, wash the area
immediately
If splashes to the nose or mouth occur it should be flushed
with water
Eye slashed require irrigation with clean water or saline
Report incident to supervisor / preceptor for follow up
protocol instructions, check with occupational health or
physician as the situation warrants
Trainers must complete an Incident Report Form
49
Bloodborne UniversalPrecautions
50
UniversalPrecautions
Universal precautions: "Universal precautions," as
defined by CDC, are a set of precautions designed to
prevent transmission of human immunodeficiency
virus (HIV), hepatitis B virus (HBV), and other
bloodborne pathogens when providing first aid or
health care. Under universal precautions, blood and
certain body fluids of all patients are considered
potentially infectious for HIV, HBV and other
bloodborne pathogens. Retrieved from CDC web
site
http://www.cdc.gov/ncidod/dhqp/bp_universal_precautions.html
51
PersonalProtectiveEquipment(PPE)
Personal protective equipment includes the use of
gowns, gloves, aprons, eye protection and face masks.
The use of these equipment is usually based on
assessment of the risk of microorganism transmission to
the patient or to the carrier as well as the risk of
contamination of the healthcare practitioners clothing
and skin by the patients blood, bodily fluids, secretions
or excretions.
Health care workers should wear a face mask, eye
protection and a gown if there is the potential for blood
or other bodily fluids to splash.
52
PPE &ContactwithInfectiousAgent
All of the PPE listed here prevent contact with the infectious
agent, or body fluid that may contain the infectious agent, by
creating a barrier between the worker and the infectious material.
Gloves, protect the hands,
Gowns or aprons protect the skin and/or clothing,
Masks and respirators protect the mouth and nose, goggles protect
the eyes, and face shields protect the entire face. The respirator,
has been designed to also protect the respiratory tract from
airborne transmission of infectious agents. Well discuss this in
more detail later.
Goggles protect eyes
Face shields protect face, mouth, nose, and eyes
53
PersonalProtectiveEquipment PPE
54
53
InfectionPreventionthroughHandWashing
Hand washing is the single most important infection
control intervention strategy for use in the health
care hospital setting
Traineesrequireknowledge&skill:
Howtocleanhands
Rationaleforchoiceofcleanhandpractice
Techniquesforhandhygiene
Protecthandsfromcontaminants
Promoteadherencetoplacementsitehandhygiene
guidelines
55
HandWashing HandHygiene
Hand washing is the single most important intervention to be
performed it is done before and after patient contact.
Also preform hand hygiene immediately before donning and
after removing PPE
Every healthcare worker is required to act responsibly and
without fail to apply the techniques for hand washing at
every patient encounter. They also should advise patients and
families of the importance of hand washing and give them
permission to remind the staff.
Decontamination refers to the process for physical removal of
blood, bodily fluids and the removal or destruction of micro
organisms from the hands.
56
FiveMomentsofHandHygiene
Before patient contact
Before an aseptic task
After body fluid exposure
even if wearing gloves!
After patient contact
After contact with patient
surroundings or equipment
57
FiveMomentsofHandHygiene
58
WhenShouldHandsbeWashed
When visibly soiled.
Before and after client contact.
After contact with a source of microorganisms (blood,
body fluids, mucus membranes, non intact skin or
inanimate objects that might be contaminated.
Prior to performance of invasive procedures (IV
catheters, indwelling catheters).
Before and after removing gloves (wearing gloves does
not remove the need to wash hands).
At the beginning and end of every shift.
59
EffectiveHandWashingTechnique
Remove all wrist and hand jewelry.
Cover cuts and abrasions with waterproof dressings.
Keep fingernails short, clean, and free from nail polish.
Wet hands under tepid running water
Apply soap or antimicrobial preparation
solution must have contact with whole surface area of
hands
vigorous rubbing of hands for 2030 seconds
especially tips of fingers, thumbs and areas between fingers
Rinse completely
Dry hands with good quality paper towel.
60
54
UseofalcoholbasedHandSanitizer
Apply a 12 squirts of product in your
cupped hand
Rub hands palm to palm
Right palm over left hand with
interlaced fingers
Palm to palm with fingers interlaced
Backs of fingers to opposing palms with
fingers interlocked
Rub between thumb and forefinger
Rotational rubbing, backwards and
forwards with clasped fingers of right
hand in left palm and vice versa
Once dry, your hands are cleaned
61
PersonalProtection&SkinIntegrity
Frequent hand washing dries skin. Skin can
breakdown and crack, breaking our skin barrier
protection.
Use hand moisturizer frequently.
Protection of the client is priority, however, we must
also protect ourselves we are at risk for contact
with infectious materials or exposure to a
communicable disease.
62
BeforeAsepticContactwithEachPatient
A trainee should clean hands before an aseptic task.
It is essential that trainees clean their hands immediately before
any aseptic task. This is necessary to protect the patient against
harmful microorganisms, including the patients own micro
organisms, entering his or her body. Trainees must protect against
transmission through contact with mucous membrane: oral/dental
care, giving eye drops, secretion aspiration.
Often trainees will be treating patients who have open wounds and
any contact with nonintact skin: skin lesion care, wound dressing,
any type of injection is an opportunity for transmission.
Medical devices are well known for harbouring potentially harmful
microorganisms and contact with devices such as a catheter
insertion, opening a vascular access system or a draining system
must be done with careful preparation. Trainees should also be
diligent in preparation of food, medications and dressing sets.
63
AsepticTechnique Types
MedicalAsepsis Cleantechnique;proceduresused
toreduce&preventspreadofmicroorganisms
**Handwashing**
SurgicalAsepsis Steriletechnique;procedures
usedtoeliminatemicroorganisms**Sterilization**
64
EquipmentNeeds Masks&Gloves
Masks should be worn:
If aseptic or sterile technique is required
To protect an immune compromised patient
Masks or respirator mask (N 95) should be worn
If airborne infection is suspected or confirmed
Gloves must be worn for:
All invasive procedures
Contact with sterile sites
Contact with nonintact skin or mucous membranes
All activities assessed as having a risk of exposure to blood, bodily fluids,
secretions and excretions, and handling sharps or contaminated
instruments.
Hands should be washed before and after gloving
65
HandlingofSharps&InjectionEquipment
Trainees should be aware of the significant problem for
healthcare workers caused by needle stick injuries,
which are as prevalent as injuries from falls and handling
and exposure to hazardous substances.
If puncture type wound (needle stick injury) occurs it
should be washed immediately and should be caused to
bleed.
Report incident to supervisor / preceptor for follow up
protocol instructions, check with occupational health or
physician as the situation warrants
66
55
SafeUseandDisposalofSharps
Keephandlingofsharps/needlestoaminimum
Donotrecapneedles,bendorbreakafteruse
Discardeachneedleintoasharpscontaineratthe
pointofuse
Donotoverloadabinifitisfull
Donotleaveasharpbininthereachofchildren
67
EnvironmentalDecontamination
CleaningMUSTprecededecontamination
Disinfectantineffectiveiforganicmatteris
present
Usemechanicalforce
Scrubbing
Brushing
Flushwithwater
68
EnvironmentalCleaning&Wastes
EnvironmentalCleaning:
Useappropriateproceduresfortheroutinecleaninganddisinfection
ofenvironmentalandotherfrequentlytouchedsurfaces
WasteDisposal:
Treatwastecontaminatedwithblood,bodilyfluids,secretionsand
excretionsasclinicalwaste,inaccordancewithlocalregulations,
disposalprotocolsandstorageinproperlymarkedcontainers
69
ToMinimizetheSpreadofInfection
REMEMBER
Beforecontactwitheachandeverypatient:
cleanhandsbeforetouchingapatient
cleanhandsbeforeanaseptictask
cleanhandsaftertouchingapatient
cleanequipmentusedwithapatient
cleanhandswhenleavingpatientsurroundings
70
TraineePracticeResponsibilities
Review and understand the key guidelines, policies and
procedures for infection control and safety within your
health care placement site and clinical domain
environments.
Demonstrate responsibility for minimizing infection
transmission in your practice delivery.
Ask questions if you are unsure of a patients infection
control mode of transmission precautions
Let staff know if supplies are inadequate or depleted.
Educate others about clean hands and infection
transmission protocols / etiquette.
71
TraineeRole&Liability
Liable if actions cause harm usually shared by
instructor, trainee & hospital
Expected to perform as registered professional
safe client care
As a trainee you do not practice outside of role /
domain job description and you require supervision
72
56
SummaryofInfectionControlPractices
Earlyrecognitionandreporting
Infectioncontrolprecautions
Handhygiene:alcoholbasedhandrub,hand
washing
PPE:gloves,gowns,masks/respirators,eye
protection
Patientaccommodation isolationroom
Environmentalcleaningandwastedisposal
Occupationalhealthmanagement
73
SummaryofInfectionControlPractices
Maintaincleanlinessofthehospital
Cleananddisinfecthospital&patientcareequipment
Personalattentiontohandwashingbeforeandafter
everycontactwithapatientorobject
Usepersonalprotectiveequipmentwheneverindicated
Useanddisposeofsharpssafely
74
75
References
World Health Organization. (2010). WHO Patient
Safety Curriculum Guide for Medical Schools.
World Health Organization. (2010). Topic 1: What is
patient safety?
World Health Organization. (2010). Topic 9: Minimizing
infection through improved infection control.
76
57

Workplace Safety & Injury Prevention

- Slide 3 - Trainee Safety in the Health Care Setting
Work related injuries not only cause time away from the workplace activities but can also
result in time away from activities with your family & friends
Therefore it is very important for those working in health care settings to have a good
understanding of safety measures and work place policies and procedures promoting both
patient, staff / trainee and visitor safety.
In order to do this, as a trainee you will need to have a better understanding of the meaning
of working safely in the hospital / health care setting.
- Slide 4 - Definition of Patient Safety
It is also important to understand that patient safety is everyones responsibility.
In the workplace, we may have different roles and responsibilities and even different titles
related to our work but as health care professionals
We all have an equal responsibility to ensure the safety of each other and this is especially
important in how we promote the prevention of accidents and injury for patients.
- Slide 5 - Adverse Events & Workplace Safety
In the health care setting - an adverse event can cause harm to another person be it a patient
or health care provider.
When we refer to an adverse event for a patient --- it is typically the result of an error, lapse
or breach in safety or system of safety measures or protocols.
This means that as a health care worker we can be a contributor to the cause of an
adverse event and the event could have otherwise been prevented.
As a basic example, consider hand washing and your contact with a patient.
By not properly completing your hand washing or not hand washing before working directly
with each patient - you become a risk to a patients safety as you may transmit or carry the
potential for transmission of infection from one patient to another patient by not complying
with the safe hand washing protocol.
- Slide 6 - Emergency Situations & Response
Many unanticipated situations can arise in a hospital or other health care settings.
This can include Emergency situations that require all personnel to respond in accordance
with organizational planned safety responses.
- Slide 7 - Emergency Evacuation
Anticipating what to do in the event of an emergency situation is critical to saving lives in a
real emergency situation.
Therefore, hospital health care settings have established policies and procedures with a
defined plan of action to guide staff on the proper way to respond in these situations.
As trainees, it is important that you familiarize yourself with the emergency and safety
related policies, protocols and procedures of the organization in which you are completing your
placement.
An example of an unanticipated emergency situation might be that of an emergency
evacuation situation. It is not unusual for teams to practice their safety responses to build their
preparation and sense of readiness in the event of an actual emergency.
- Slide 8 - Fire Safety
Trainees understanding of fire safety in the hospital health care setting is also critical, as
time and knowing what to do can save lives in a real fire situation.
- Slide 9 - Fire Extinguishers & Training
Knowledge and understanding of fire safety is important as is how to utilize fire safety
equipment.
58

This may be very important as there can be higher risks when unsafe work practices are
undertaken by people in the workplace for example - when flammable substances are not
stored properly and safely which can then lead to a fire situation.
In thinking about fire safety and your training keep in mind
Each trainee needs to review the fire safety manual, be familiar with the fire safety protocols
of their department and the location of fire equipment in the event of an actual fire emergency.
- Slide 12 - Workplace Adverse Events & Safety
Every job has some risk to the worker if they do not comply with work related training or
they do not follow safety guidelines and protocols as established in the workplace setting.
Therefore, as trainees you will need to become familiar with all the safety precautions
related to your role and responsibilities while in the placement setting.
- Slide 15 - Trainees have a duty to
As a trainee it is especially important to recognize what you know and to acknowledge
what you do not know and find out
Not being truthful about your knowledge, gaps in your knowledge or scope of practice &
role limitations can hurt you and hurt others in the workplace. Professionalism and ethics
requires you to share this information.
- Slide 17 - Your Use of Personal Protective Equipment (PPE)
In the hospital health care setting the need for your use of personal protective equipment
or PPE as it is called is prescribed as a mandatory requirement according to the policies and
procedures of the placement organization.
By properly using PPE you help to ensure the safety of the patients, other health care
workers, the organization and the general public who enters the organization.
- Slide 18 - Personal Protective Equipment PPE
By building a better understanding of transmission and spread of infection and its
prevention / control through the proper use of PPE, you are promoting the safety of others and
yourself as a health care provider.
By complying with the guidelines and protocols for infection prevention and infection
control as we have briefly covered in todays session, you will also be protecting yourself in
the work environment.
- Slide 19 - Body Mechanics and Movement
A basic understanding of proper body mechanics and movement is important in the
prevention of accidents and injury and future health problems for the health care worker.
- Slide 27 - Lifts & Transfers
When asked to assist in lifting or transferring a patient - keep in mind you may become
trained in using other types of special equipment to assist in such lifts and transfers.
For the safety of the patient and your fellow team members, if you are not familiar with or
have not been trained in the use of such equipment, you will need to disclose this prior to
engaging in any patient transfer or lift and allow for another staff member to assist in the
procedure.
Once you have been trained in the safe use of the equipment and lift / transfer procedures
you are encouraged to assist others as needed.
- Slide 30 - Material Safety Data Sheets MSDS
To assist all staff in identifying safety information for the workplace setting, MSDS or
material safety data sheets should be available to staff / employees and individuals working in
the organization.
Speak with your department preceptor / supervisor regarding where they can be found in
your placement settings department.
59

- Slide 31 - Bloodborne Pathogens
As a trainee in the hospital health care setting it is very important for you to understand
the precautions and risks associated with bloodborne pathogens and their contact precautions.
This includes understanding the PPE needs in accordance with organizational and work task
related guidelines and policies.
- Slide 33 - Safe Use and Disposal of Sharps
To avoid accidents and injuries to yourself and others in the workplace related to blood and
body fluids transmission of infection -- you will need to practice the safe use and you must
dispose of all sharps in the appropriate disposal / biohazard marked containers.
Your compliance with this practice is mandatory
- Slide 36 - Injuries & Accidents Trainee Reporting
It is essential that should you, as a trainee experience an accident or injury in the placement
setting that this is reported in accordance with the Entry to Practice Training Program policies
and guidelines.
It is also essential that you report all injuries and accidents to your supervisor / preceptor in
the placement setting
immediately
- Slide 37 - Breaching Safety Practice & Disciplinary Action
The consequence of disregarding or not practicing required occupational health and safety
practice guidelines in the work place setting can result in your dismissal from the placement
setting and the program.
- Slide 38 - Key Messages Safety & Injury Prevention
In concluding this lecture session you should have gained a better understanding of the
following :
You recognize patient safety as an important responsibility in your practice and as a
healthcare delivery system
You may apply the required knowledge in preventing and/or minimizing workplace injury
and accidents
You will support performance of appropriate behaviors required to prevent health care
associated injuries, and
You will demonstrate the required competence to provide patients with safe care and to
avoid injuries.
As a trainee, you will need to understand more specifically how this applies to you in your
Thus, as you continue your experience in the Entry to Practice Training Program we invite
you to continue building your abilities to demonstrate and exhibit a strong response to
participate in roles of safe practice delivery and the prevention of accidents & injuries in the
work place as a valued work attitude and behavior.
This is also important --- as it will be a very important component to your ongoing success
60
MinistryofHealth
Graduates
Day 2 - Workplace Safety & Injury
Prevention
LectureOutline
2
1. Recognize patient safety as an important
responsibility in your practice and as a healthcare
delivery system.
2. Apply required knowledge in preventing and/or
minimizing workplace injury and accidents.
3. Perform appropriate behaviors required to prevent
health care associated injuries.
4. Demonstrate required competence to provide
patients with safe care and to avoid injuries.
TraineeSafetyintheHealthCareSetting
Work related injuries not only cause time away from the
workplace activities but can also result in time away from
activities with your family & friends
What is the meaning of working safely?
Completing every task the correct way and not taking
hazardous shortcuts
Understanding organizational policies & safety procedures
Wearing required personal protective equipment (PPE)
Being rested, alert and paying attention to the task you are
undertaking
Asking for instruction and guidance prior to completing
unfamiliar tasks
3
DefinitionofPatientSafety
Patient safety is a discipline in the health care sector
that applies safety science methods toward the goal of
achieving a trustworthy system of health care delivery.
Patient safety is also an attribute of health care
systems; it minimizes the incidence and impact of, and
maximizes recovery from, adverse events.
According to Institute of Medicine, safety is defined as
freedom of accidental injury.
(Emanuel, et al, 2008)
4
AdverseEvents&WorkplaceSafety
Adverse events are widespread and preventable
(Emanuel et al., 2008)
Much unnecessary harm is caused by health-care
errors, safety lapses and system failures.
Example: Hospital acquired infections (HAI) from poor hand-washing.
Example: Complications from errors in
medication orders or administering
the wrong medication.
Example: Work related accidents and injuries.
5
EmergencySituations&Response
In Emergency Situations in the health care setting
you must know what to do:
KNOW the Emergency Response Codes and
Responses for your Placement setting by reviewing
the policies and procedures and discussing what
your trainee role will be with your supervisor /
preceptor
6
EmergencyEvacuation
Evacuation of a building may be required if an
emergency situation threatens the life and safety of
patients, visitors and employees
Situations that may require evacuation may include:
Fire or smoke
Chemical spills
Power failure
Bomb threat
Violence or terrorist attacks
7
FireSafety
If you find a small fire smaller then a small trash can you
should call for help and then you may try to put it out.
If the fire is anything larger, you should sound the fire
alarm, calling the emergency response number with the
location of the fire and then following the fire protocols
of the health care placement setting
This may include removing any person in the immediate
area of the fire to an area of safety and
Reporting / assembling in your designated
area with coworkers to await other
instructions
8
FireExtinguishers&Training
Fire extinguishers only have a minute of retardant in
each extinguisher
So you will only be able to put out a fire the size of a
small trash can with it
To use a fire extinguisher REMEMBER
PASS
Pull the pin
Aim at the base of the fire
Squeeze the handle
Sweep the base of the fire
9
ElectricalSafety
Only trained maintenance employees are authorized
to investigate and conduct electrical repairs
Do not attempt any maintenance activities you are
not trained or authorized to conduct
Never use a damaged extension cord or any other
piece of damaged equipment
Never use electrical equipment in a damp or wet
area
10
WorkplaceSafetyinHealthCareSetting
Patient safety is a discipline in the health care
sector that applies safety science methods
toward the goal of achieving a trustworthy
system of health care delivery.
Patient safety is also an attribute of health
care systems; it minimizes the incidence and
impact of, and maximizes recovery from,
adverse events
(Emanuel et al., 2008)
11
WorkplaceAdverseEvents&Safety
Workplace safety can assist to decrease the
risk of adverse events that can occur in the
workplace environment
Unnecessary harm can be caused by health-
care worker fatigue, errors, taking shortcuts,
lack of attention to environmental safety and
poor planning for emergency or system failures.
Many accidents and injuries that occur in the
workplace are preventable
12
62
HowCanSomeonebeHurtatWork
Youhavearoletoplayinstayingsafeby:
Learningtorecognizehazardsandthe
potentialforfallrisks
Lookingforhazardseverywhere
Thinkingofallfourhazardcategories
Physical
Chemical
Biological
Ergonomic
Reporthazardstoyourpreceptor/trainerin
practiceforappropriateaction
13
Patient&EnvironmentalSafety
Prevent and/or minimize the adverse events and
eliminate preventable harm in the health care setting
As all health care professionals, you are responsible for
ensuring patient and environmental safety
Help ensure a safe workplace protects your health
Safety is everyones responsibility - if everyone does
their part it can make a real difference
Practicing health & safety on the job lowers the rate
of accidents and injuries in the workplace
14
Traineeshaveadutyto
Do your trainee role / job safely and in the way you
have been trained, using the right safety devices and
personal protective equipment
Comply and meet the prerequisites for entry into the
health care setting work environment
Know and comply with the health care setting work
place policies and procedures
Ask questions and obtain clarification of the things you
do not know or understand
15
Health&SafetyisYourResponsibility
Your are encouraged to report any safety problems
to your supervisor / preceptor
Before taking on any task consider the necessary
safety measures and check policy / procedures
LISTEN, ask questions and talk to one another
REPORT immediately any accident, injury or health
& safety problem to your supervisor / preceptor
16
YourUseofPersonalProtectiveEquipment(PPE)
Safetyglasses/faceshields
Hearingprotection
Gloves
Respirators/Masks
Gowns
Protectivefootwear(enclosedshoes)
Xray/radiationshields
TheseareconsideredpartofyourrequiredUNIFORM
17
PersonalProtectiveEquipment PPE
18
63
BodyMechanicsandMovement
Body Mechanics are the coordinated efforts of the
musculoskeletal and nervous systems as the person
moves, lifts, bends, stands, sits and completes daily
activities
(Thibbeau & Patton, 2007)
19
DefinitionofBodyAlignment
Body alignment refers to the relationship of one
body part to another body part along a horizontal of
vertical line.
It is the condition of joints, tendons, ligaments and
muscles in various body positions where correct
alignment reduces strain on musculoskeletal
structures , maintains adequate muscle tone and
contributes to balance.
Typically when at rest and where the correct
alignment is supported it does not cause pain
20
DefinitionofBodyBalance
Body Balance is achieved when a relatively low centre of
gravity is balanced over a wide, stable base of support is
present, and t a line falls from the centre of gravity
vertically through the base of support. The body loses
balance when the line from the centre of gravity does
not fall vertically through the base of support
Body Balance is also enhanced by proper posture and
body positions that most favours the functions of
movement and requires the least amount of muscular
work / strain on muscles, ligaments and bones
21
LiftingTechnique&ProperBodyMechanics
Maintaining balance, proper body alignment and posture
during work activity can be supported by doing the following:
1) Maintain a broad base of support by separating your feet to
a comfortable distance while standing
2) To increase your balance bring your centre of gravity
closer to your base of support by bending your knees
slightly, flexing your hips and keeping your trunk erect in
proper back alignment
3) Bring the load (item you are carrying / lifting ) close to body
to help maintain your balance
22
KNOWYourBackSafetyGuidelines
More than 50% of all back pain in health care
workers is associated with manual lifting tasks
The most common back injury is strain on the
muscle group around the lumbar vertebrae
Injury in this area affects the ability to bend
forward, backward, and from side to side
It can also decrease your ability to rotate the
hips and lower back
KNOW your health care facilitys safety
information regarding transfer, positioning
and lifting patients and use the recommended
back safety guidelines to prevent injury
23
ObjectAssessmentPriortoLifting
Always check the weight of the object prior to lifting it
person and determine if assistance is needed and
resources are available
If moving a patient, use safe patienthandling equipment
and engage other knowledgeable staff as your lift /
transfer team member supports
Manual lifting of the patient should be the last resort
and should only be used when it does not involve lifting
most or all of the patients weight
24
64
BodyMechanics&LiftingObjects
Improperversus
properobjectlifting
technique
Properlifting&
standing
technique
25
LiftingTechniqueInstructions
If the object seems heavy, get help from another person or
more
Plan the path of travel before the lift and ensure the path is
clear, allows adequate space for movement and is safe to
move the object freely
Determine who will lead / give instructions for lifting
Always lift with your legs / knees and keep your back straight
Never twist while carrying a load / object, move your feet in
the direction you wish to move
26
Lifts&Transfers
27
EnvironmentalAwareness&SafetyforOthers
It is important to maintain a high level of environmental
cleanliness throughout the health care setting for the safety
of others
Falls and accidents from tripping hazards
Water / Spills cause slip hazards
Clean up or immediately notify your supervisor of any
environmentally unsafe conditions
Call bells within reach for all patients
Bedrails / stretcher railings up for patients
28
HazardousMaterials
All chemicals and substances in the health care setting should be
labeled with the name of the chemical and manufacturer
Bulk chemicals and chemicals with a hazard must be labeled with a
Hazard Management Information System Label
This label may use symbols or a number rating the higher the
number the more hazardous the chemical (dangerous vapor,
ignites, flammable or reactivity with contact etc.)
Note: hazardous materials are typically stored requiring special safety
conditions.
If your trainee work area houses hazardous
materials you must KNOW & review the
related policies & procedures
29
MaterialSafetyDataSheets MSDS
Health care settings often use MSDS or material safety data sheets to
assist in identifying important safety information to staff / employees
The MSDS provides indepth information on health hazards, reactivity,
flammability, chemical properties, guidelines on usage and storage
Typically, MSDS for all products used at a facility are in binders within
department areas where the materials are being used
Identify with your Supervisor / Preceptor where the MSDS binder is
located for your reference & review any special precautions for the
department work delivery
EXAMPLE: Eye wash stations do exist for washing of hazardous materials
from eyes, then seek medical attention
30
65
Bloodborne Pathogens
Do not touch or try to clean up any bodily fluids such as
blood, urine, stool or vomit unless you have been
trained in the use of proper PPE
Trained staff with latex gloves (and safety glasses) are
authorized to handle this type of hazardous waste
material and its proper disposal
If you are exposed to any bodily fluids notify your
supervisor immediately (example: needle stick injury,
contact through an open area of the body or mucous
membrane etc.)
31
IfExposedtoBlood/BodyFluids
Follow healthcare guidelines if exposed to blood or body fluids
If puncture type wound (needle stick injury) occurs it should be
washed immediately and should be caused to bleed.
If skin contamination should occur, wash the area immediately
If splashes to the nose or mouth occur it should be flushed with
water
Eye splashed require irrigation with clean water or saline
Report incident to trainer in practice immediately for follow up
protocol instructions, and for the need of immediate visit to the
occupational health physician for the assessment for the need of
post exposure prophylaxis.
Trainer in practice will be requested to complete an Incident
Report Form
32
SafeUseandDisposalofSharps
Keep handling of sharps / needles to
a minimum
Do not recap needles, bend or break
after use
Discard each needle into a sharps
container at the point of use
Do not overload a bin if it is full
Do not leave a sharp bin in the reach
of children
33
HandlingPatientCareEquipment
Handle patient care equipment (soiled with blood or
other body fluid secretions or excretions) in a way (PPE)
that prevents contact with skin and mucous membranes
Handle patient care equipment in a way that prevents
contamination of clothing and the spread of
microorganisms to other patients
Appropriately dispose of single use equipment
Clean and disinfect reusable equipment after each
patient episode of use. Do not share equipment with
other patients without properly cleaning it first
34
SeriousAccidentorMedicalEmergency
If a trainee has a serious accident or injury or needs
Emergency medical care follow the guidelines below:
Summon a first responder to the injured person and call
for help
Inform the Trainees supervisor / preceptor
Transport the trainee to receive medical attention
(example: to Emergency Department)
Notify the Entry to Practice Training Program Ministry
of Health Staff and complete required documentation
forms as required
Trainee requires a physicians note indicating he is fit
and able to return to placement and perform duties
35
Injuries&Accidents TraineeReporting
All injuries and accidents must be reported to your
supervisor / preceptor immediately
This includes first aid injuries and close calls.
Accidents and injuries resulting in medical treatment
must be documented on an Incident Report Form
Disciplinary Action:
Disregarding occupational health & safety rules or
established safety practice in the health care setting
can result in dismissal from the placement
36
66
BreachingSafetyPractice&DisciplinaryAction
Disciplinary Action:
Disregarding occupational health & safety rules or
established safety practice in the health care setting can
result in dismissal from the placement
Example of Violations:
Not wearing required PPE
Not immediately reporting an injury or damage
Committing an unsafe act such as tampering with
equipment, removing safety warnings etc.
Operating a piece of equipment you are not authorize to
operate
37
KeyMessages Safety&InjuryPrevention
Workplace safety and injury prevention is the
responsibility of every professional health care
provider
It is critical to become knowledgeable on the plan of
action, policies / procedures , and key team member
roles essential for response ~ in advance of an
emergency situation
All Trainees must review the Emergency
Preparedness Policies & Procedures for their
assigned Placement Site
39
References
Emanuel,L.,Berwick,D.,Conway,J.,Combes,J.,Hatlie,
M.,Leape,L.,Reason,J.,Schyve,P.,Vincent,C.,&
Walton,M.(2008).Whatexactlyispatientsafety?
AdvancesinPatientSafety,Vol.1:Assessment.Retrieved
from
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ap
s2v1&part=advancesemanuelberwick_110
Thibbeau,G.A.,&Patton,K.T.,(2007).Anatomyand
physiology(6
th
ed.).,St.Louis,MO:Mosby.
http://www.slideshare.net/gtwaddell/generalsafety
presentation
40
67
Ministry of Health
Kingdom Of Saudi Arabia
Bacteriology
68

Bacteriology

Sterilization and Disinfection
- Slide 24 Moist Heat Methods
Most effective when air is removed initially and replaced by super heated steam. This vacuum
allows steam to get to places not normally reached as pockets of air would normally insulate
that area and kill temperature would not be reached.
- Slide 52 Alcohols
70% alcohol gives maximum kill for bacteria and viruses.
- Slide 55 Aldehydes
Not used these days routinely for disinfection due to their toxicity.

Tools of the Laboratory
- Slide 33
Can also use Eosin as counterstain (brighter than Safranin)

The Cocci of Medical Importance
- Slide 20 Prevention of Staphylococcal Infections
Universal now known as standard precautions
- Slide 71 Other Gram-negative Cocci and Coccobacilli
Now known as Moraxella catarrhalis

The Gram-Negative Bacilli of Medical Importance
- Slide 12 Burkholderia
Used to be known as Pseudomonas pseudomallei
- Slide 13 - Acinetobacter and Stenotrophomonas
Used to known as Pseudomonas maltophilia

The Gram-Positive Bacilli of Medical Importance
- Slide 56 Diagnosis
Now available to differentiate between BCG vaccination and true TB is Quantiferon Gold

Cerebrospinal Fluid (CSF) Culture
- Slide 13 Microscopic Examination
Care needs to be taken when staining as the dried sediment can easily come off the slide and
you are only looking at stain deposit. Take extreme care in the staining process. Preferable to do
manual staining.

Urine Culture
- Slide 23 Specimen Transport
This is one option. Not necessarily preferred option. Usually when delay in transport is
envisaged, but not routinely
- Slide 27 Laboratory Examination of Urine
This is not normally classed as abnormal result if urates seen in urine.
- Slide 29 Examine the specimens microscopically
- Slide 44 Materials
MacConkey plate can be used instead of CLED plate as well.

Respiratory Tract Infections
69

- Slide 6 Flora of respiratory tract
Host defense mechanisms normally remove any microbes which invade the bronchial tract or
lung alveoli in healthy people. These mechanisms keep LRT sterile
- Slide 7 Types of specimens received
Blood cultures should not always be collected for a LRT. Maybe sometimes if warranted but not
every time.
- Slide 18 Endotracheal and Tracheostomy secretions
A tracheostomy is a hole made in the front of the neck, opens through trachea. breathing tube
can be placed into the windpipe.
- Slide 21 - *Important
Delays in processing of sputums can give false results. Yeasts especially grow well in sputum
- Slide 29 Rejection criteria for tracheal aspirates
This could also be indicative of TB and should be checked to rule this out.

70
MinistryofHealth
Graduates
Laboratory Technician
AnIntroductiontothebacterialCell
ItsOrganization,andMembers
CharacteristicsofCellsandLife
All living things (single and multicellular) are made of cells that share some
common characteristics:
Basicshape spherical,cubical,cylindrical
Internal content cytoplasm, surrounded by a membrane
DNA chromosome(s), ribosomes, metabolic capabilities
Two basic cell types: eukaryotic and prokaryotic
2
CharacteristicsofCells
Eukaryotic cells: animals, plants, fungi, and protists
Contain membranebound organelles that compartmentalize the
cytoplasm and perform specific functions
Contain doublemembrane bound nucleus with DNA chromosomes
Prokaryotic cells: bacteria and archaea
No nucleus or other membranebound organelles
3
CharacteristicsofLife
Reproduction and heredity genome composed of DNA packed in
chromosomes; produce offspring sexually or asexually
Growth and development
Metabolism chemical and physical life processes
Movement and/or irritability respond to internal/external stimuli; self
propulsion of many organisms
Cell support, protection, and storage mechanisms cell walls, vacuoles,
granules and inclusions
Transport of nutrients and waste
4
ProkaryoticProfiles
5
ProkaryoticProfiles
Structures that are essential to the functions of all prokaryotic cells are a
cell membrane, cytoplasm, ribosomes, and one (or a few) chromosomes
6
Structureofabacterialcell
7
ExternalStructures
Appendages
Two major groups of appendages:
Motility flagella and axial filaments (periplasmic flagella)
Attachment or channels fimbriae and pili
Glycocalyx surface coating
8
Flagella
3 parts:
Filament long, thin, helical structure composed of protein Flagellin
Hook curved sheath
Basal body stack of rings firmly anchored in cell wall
Rotates 360
o
Number and arrangement of flagella varies:
Monotrichous, lophotrichous, amphitrichous, peritrichous
Functions in motility of cell through environment
9
Flagella
10
FlagellarArrangements
1. Monotrichous single flagellum at one end
2. Lophotrichous small bunches emerging from the same site
3. Amphitrichous flagella at both ends of cell
4. Peritrichous flagella dispersed over surface of cell; slowest
11
Electronmicrographsofflagellararrangements
12
72
FlagellarResponses
Guide bacteria in a direction in response to external stimulus:
Chemical stimuli chemotaxis; positive and negative
Light stimuli phototaxis
Signal sets flagella into rotary motion clockwise or counterclockwise:
Counterclockwise results in smooth linear direction run
Clockwise tumbles
13
Theoperationofflagella
14
Chemotaxisinbacteria
15
PeriplasmicFlagella
Internal flagella, enclosed in the space between the outer sheath and the
cell wall peptidoglycan
Produce cellular motility by contracting and imparting twisting or flexing
motion
16
Periplasmicflagella
17
Fimbriae
Fine, proteinaceous, hairlike bristles emerging from the cell surface
Function in adhesion to other cells and surfaces
18
73
Pili
19
Rigidtubularstructuremadeofpilin protein
Foundonlyingramnegativecells
FunctiontojoinbacterialcellsforpartialDNAtransfercalled
conjugation
Glycocalyx
Coating of molecules external to the cell wall, made of sugars and/or
proteins
Two types:
1. Slime layer loosely organized and attached
2. Capsule highly organized, tightly attached
Functions:
Protect cells from dehydration and nutrient loss
Inhibit killing by white blood cells by phagocytosis, contributing to
pathogenicity
Attachment formation of biofilms
20
21 22
Biofilmonacatheter
23
TheCellEnvelope
External covering outside the cytoplasm
Composed of two basic layers:
Cell wall and cell membrane
Maintains cell integrity
Two different groups of bacteria demonstrated by Gram stain:
Grampositive bacteria: thick cell wall composed primarily of
peptidoglycan and cell membrane
Gramnegative bacteria: outer cell membrane, thin peptidoglycan
layer, and cell membrane
24
25
StructureofCellWalls
Determines cell shape, prevents lysis (bursting) or collapsing due to
changing osmotic pressures
Peptidoglycan is primary component:
Unique macromolecule composed of a repeating framework of long
glycan chains crosslinked by short peptide fragments
26
Peptidoglycan
27
GramPositiveCellWall
Thick, homogeneous sheath of peptidoglycan
2080 nm thick
Includes teichoic acid and lipoteichoic acid: function in cell wall
maintenance and enlargement during cell division; move cations
across the cell envelope; stimulate a specific immune response
Some cells have a periplasmic space, between the cell membrane
and cell wall
28
29
GramNegativeCellWall
Composed of an outer membrane and a thin peptidoglycan layer
Outer membrane is similar to cell membrane bilayer structure
Outermost layer contains lipopolysaccharides and lipoproteins (LPS)
Lipid portion (endotoxin) may become toxic when released during
infections
May function as receptors and blocking immune response
Contain porin proteins in upper layer regulate molecules entering and
leaving cell
Bottom layer is a thin sheet of peptidoglycan
Periplasmic space above and below peptidoglycan
30
75
31
ComparisonofGramPositiveandGram
Negative
32
TheGramStain
Differential stain that distinguishes cells with a grampositive cell wall
from those with a gramnegative cell wall
Grampositive retain crystal violet and stain purple
Gramnegative lose crystal violet and stain red from safranin counterstain
Important basis of bacterial classification and identification
Practical aid in diagnosing infection and guiding drug treatment
33 34
NontypicalCellWalls
Some bacterial groups lack typical cell wall structure, i.e., Mycobacterium
and Nocardia
Grampositive cell wall structure with lipid mycolic acid (cord factor)
Pathogenicity and high degree of resistance to certain chemicals and
dyes
Basis for acidfast stain used for diagnosis of infections caused by these
microorganisms
Some have no cell wall, i.e., Mycoplasma
Cell wall is stabilized by sterols
Pleomorphic
35
ExtremevariationinshapeofMycoplasma
pneumoniae
36
76
CellMembraneStructure
Phospholipid bilayer with embedded proteins fluid mosaic model
Functions in:
Providing site for energy reactions, nutrient processing, and synthesis
Passage of nutrients into the cell and the discharge of wastes
Cell membrane is selectively permeable
37
Cellmembranestructure
38
BacterialInternalStructures
Cell cytoplasm:
Dense gelatinous solution of sugars, amino acids, and salts
7080% water
Serves as solvent for materials used in all cell functions
39
Chromosome
Single, circular, doublestranded DNA molecule that contains all the
genetic information required by a cell
Aggregated in a dense area called the nucleoid
DNA is tightly coiled
40
Chromosomestructure
41
Plasmids
Small circular, doublestranded DNA
Free or integrated into the chromosome
Duplicated and passed on to offspring
Not essential to bacterial growth and metabolism
May encode antibiotic resistance, tolerance to toxic metals, enzymes, and
toxins
Used in genetic engineering readily manipulated and transferred from cell to
cell
42
Ribosomes
Made of 60% ribosomal RNA and 40% protein
Consist of two subunits: large and small
Prokaryotic differ from eukaryotic ribosomes in size and number of proteins
Site of protein synthesis
Present in all cells
43
Prokaryoticribosome
44
Inclusions and granules
Intracellular storage bodies
Vary in size, number, and content
Bacterial cell can use them when environmental sources are depleted
Examples: glycogen, poly hydroxybutyrate, gas vesicles for floating, sulfur
and phosphate granules (metachromatic granules), particles of iron oxide
45
Bacterialinclusionbodies
46
Cytoskeleton
Manybacteriapossessaninternal
networkofproteinpolymersthatis
closelyassociatedwiththecellwall
47
Endospores
Inert, resting, cells produced by some G+ genera: Clostridium, Bacillus, and
Sporosarcina
Have a 2phase life cycle:
Vegetative cell metabolically active and growing
Endospore when exposed to adverse environmental conditions;
capable of high resistance and very longterm survival
Sporulation formation of endospores
Hardiest of all life forms
Withstands extremes in heat, drying, freezing, radiation, and chemicals
Not a means of reproduction
Germination return to vegetative growth
48
78
Sporulation cycle
49
Endospores
Resistance linked to high levels of calcium and dipicolinic acid
Dehydrated, metabolically inactive
Thick coat
Longevity verges on immortality, 250 million years
Resistant to ordinary cleaning methods and boiling
Pressurized steam at 120
o
C for 2030 minutes will destroy
50
BacterialShapes,Arrangements,
andSizes
Vary in shape, size, and arrangement but typically described by one
of three basic shapes:
Coccus spherical
Bacillus rod
Coccobacillus very short and plump
Vibrio gently curved
Spirillum helical, comma, twisted rod,
Spirochete springlike
51
Commonbacterialshapes
52
ComparisonofSpiralShapedBacteria
53
BacterialShapes,Arrangements,andSizes
Arrangement of cells is dependent on pattern of division and how cells
remain attached after division:
Cocci:
Singles
Diplococci in pairs
Tetrads groups of four
Irregular clusters
Chains
Cubical packets (sarcina)
Bacilli:
Diplobacilli
Chains
Palisades
54
79
Arrangementofcocci
55
Thedimensionsofbacteria
56
ClassificationSystemsintheProkaryotae
1. Microscopic morphology
2. Macroscopic morphology colony appearance
3. Bacterial physiology
4. Serological analysis
5. Genetic and molecular analysis
57
DiagnosticSchemeforMedicalUse
Uses phenotypic qualities in identification
Restricted to bacterial disease agents
Divides bacteria based on cell wall structure, shape, arrangement,
and physiological traits
58
59
SpeciesandSubspecies
Species a collection of bacterial cells which share an overall similar
pattern of traits in contrast to other bacteria whose pattern differs
significantly
Strain or variety a culture derived from a single parent that differs in
structure or metabolism from other cultures of that species (biovars,
morphovars)
Type a subspecies that can show differences in antigenic makeup
(serotype or serovar), susceptibility to bacterial viruses (phage type) and
in pathogenicity (pathotype)
80
UnusualFormsofMedicallySignificant
Bacteria
Obligate intracellular parasites
Rickettsias
Very tiny, gramnegative bacteria
Most are pathogens that alternate between mammals and bloodsucking
arthropods
Obligate intracellular pathogens
Cannot survive or multiply outside of a host cell
Cannot carry out metabolism on their own
Rickettsia rickettisii Rocky Mountain spotted fever
Rickettsia typhi endemic typhus
61
UnusualFormsof
MedicallySignificantBacteria
Chlamydias
Tiny
Obligate intracellular parasites
Not transmitted by arthropods
Chlamydia trachomatis severe eye infection and one of the most
common sexually transmitted diseases
Chlamydia pneumoniae lung infections
62
ComparisonofThreeCellularDomains
63
81
MinistryofHealth
Graduates
ElementsofMicrobialNutrition,Ecology,andGrowth
MicrobialNutrition
Nutrition process by which chemical substances (nutrients) are acquired
from the environment and used for cellular activities
Essential nutrients must be provided to an organism
Two categories of essential nutrients:
Macronutrients required in large quantities; play principal roles in cell
structure and metabolism
proteins, carbohydrates
Micronutrients or trace elements required in small amounts; involved in
enzyme function and maintenance of protein structure
manganese, zinc, nickel
2
Nutrients
Inorganic nutrients atom or molecule that contains a
combination of atoms other than carbon and hydrogen
metals and their salts (magnesium sulfate, ferric nitrate, sodium
phosphate), gases (oxygen, carbon dioxide) and water
Organic nutrients contain carbon and hydrogen atoms and are
usually the products of living things
methane (CH
4
), carbohydrates, lipids, proteins, and nucleic acids
3
ChemicalAnalysisofMicrobial
Cytoplasm
70% water
Proteins
96% of cell is composed of 6 elements:
Carbon
Hydrogen
Oxygen
Phosphorous
Sulfur
Nitrogen
4
SourcesofEssentialNutrients
Carbon sources
Heterotroph must obtain carbon in an organic form such
as proteins, carbohydrates, lipids and nucleic acids, made
by other living organisms
Autotroph an organism that uses CO
2
, an inorganic gas as
its carbon source
not nutritionally dependent on other living things
5
Nitrogen Sources:
Main reservoir is nitrogen gas (N
2
); 79% of earths atmosphere is N
2.
Nitrogen is part of the structure of proteins, DNA, RNA & ATP these
are the primary source of N for heterotrophs.
Some bacteria & algae use inorganic N nutrients (NO
3
, NO
2
, or NH
3
).
Some bacteria can fix N
2.
Regardless of how N enters the cell, it must be converted to NH
3
, the
only form that can be combined with carbon to synthesis amino acids,
etc.
6
82
Oxygen Sources
Major component of carbohydrates, lipids, nucleic acids, and
proteins
Plays an important role in structural and enzymatic functions of
cell
Component of inorganic salts (sulfates, phosphates, nitrates) and
water
O
2
makes up 20% of atmosphere
Essential to metabolism of many organisms
7
Hydrogen Sources
Major element in all organic compounds and several inorganic
ones (water, salts and gases)
Gases are produced and used by microbes.
Roles of hydrogen:
maintaining pH
forming H bonds between molecules
serving as the source of free energy in oxidationreduction reactions of
respiration
8
Phosphorous (Phosphate Sources)
Main inorganic source is phosphate (PO
4
3
) derived from
phosphoric acid (H
3
PO
4
) found in rocks and oceanic mineral
deposits
Key component of nucleic acids, essential to genetics
Serves in energy transfers (ATP)
9
Sulfur Sources
Widely distributed in environment, rocks; sediments contain
sulfate, sulfides, hydrogensulfidegasandsulfur
Essential component of some vitamins and the amino acids:
methionineandcysteine
Contributestostabilityof proteinsbyformingdisulfidebonds
10
OtherNutrientsImportantin
MicrobialMetabolism
Potassium essential to protein synthesis and membrane function
Sodium important to some types of cell transport
Calcium cell wall and endospore stabilizer
Magnesium component of chlorophyll; membrane and ribosome
stabilizer
Iron component of proteins of cell respiration
Zinc, copper, nickel, manganese, etc.
11
GrowthFactors:
EssentialOrganicNutrients
Organic compounds that cannot be synthesized by an
organism because they lack the genetic and metabolic
mechanisms to synthesize them
Must be provided as a nutrient
essential amino acids, vitamins
12
NutritionalTypes
Main determinants of nutritional type are:
Carbon source heterotroph, autotroph
Energy source
chemotroph gain energy from chemical compounds
phototrophs gain energy through photosynthesis
13 14
Transport:MovementofChemicals
AcrosstheCellMembrane
Passive transport does not require energy; substances exist in a
gradient and move from areas of higher concentration towards areas of
lower concentration
diffusion
osmosis diffusion of water
facilitated diffusion requires a carrier
Active transport requires energy and carrier proteins; gradient
independent
active transport
group translocation transported molecule chemically altered
bulk transport endocytosis, exocytosis, pinocytosis 15 16
17 18
19 20
EnvironmentalFactorsThatInfluence
Microbes
Environmental factors fundamentally affect the function of metabolic
enzymes.
Factors include:
temperature
oxygen requirements
pH
electromagnetic radiation
barometric pressure
21
3CardinalTemperatures
Minimum temperature lowest temperature that permits
a microbes growth and metabolism
Maximum temperature highest temperature that permits
a microbes growth and metabolism
Optimum temperature promotes the fastest rate of
growth and metabolism
22
3TemperatureAdaptationGroups
1. Psychrophiles optimum temperature below 15
o
C; capable of
growth at 0
o
C
2. Mesophiles optimum temperature 20
o
40
o
C; most human
pathogens
3. Thermophiles optimum temperature greater than 45
o
C
23 24
85
GasRequirements
Oxygen
As oxygen is utilized it is transformed into several toxic products:
singlet oxygen (O
2
), superoxide ion (O
2
), peroxide (H
2
O
2
), and
hydroxyl radicals (OH
)
Most cells have developed enzymes that neutralize these chemicals:
superoxide dismutase, catalase
If a microbe is not capable of dealing with toxic oxygen, it is forced to
live in oxygen free habitats.
25
CategoriesofOxygenRequirement
Aerobe utilizes oxygen and can detoxify it
Obligate aerobe cannot grow without oxygen
Facultative anaerobe utilizes oxygen but can also grow in its absence
Microaerophilic requires only a small amount of oxygen
26
CategoriesofOxygenRequirement
Anaerobe does not utilize oxygen
Obligate anaerobe lacks the enzymes to detoxify oxygen so cannot
survive in an oxygen environment
Aerotolerant anaerobes do no utilize oxygen but can survive and
grow in its presence
27
CarbonDioxideRequirement
All microbes require some carbon dioxide in their metabolism.
Capnophile grows best at higher CO
2
tensions than normally present
in the atmosphere
28
29
EffectsofpH
Majority of microorganisms grow at a pH between 6 and 8
Obligate acidophiles grow at extreme acid pH
Alkalinophiles grow at extreme alkaline pH
30
86
OsmoticPressure
Most microbes exist under hypotonic or isotonic conditions
Halophiles require a high concentration of salt
Osmotolerant do not require high concentration of solute
but can tolerate it when it occurs
31
OtherEnvironmentalFactors
Barophiles can survive under extreme pressure and will
rupture if exposed to normal atmospheric pressure
32
EcologicalAssociationsAmong
Microorganisms
Symbiotic organisms live in close nutritional relationships; required by
one or both members
mutualism obligatory, dependent; both members benefit
commensalism commensal member benefits, other member not
harmed
parasitism parasite is dependent and benefits; host is harmed
33
EcologicalAssociationsAmong
Microorganisms
Nonsymbiotic organisms are freeliving; relationships not
required for survival
synergism members cooperate and share nutrients
antagonism some member are inhibited or destroyed
by others
34
InterrelationshipsBetweenMicrobes
andHumans
Human body is a rich habitat for symbiotic bacteria, fungi,
and a few protozoa normal microbial flora
Commensal, parasitic, and synergistic
35
MicrobialBiofilms
Biofilms result when organisms attach to a substrate by
some form of extracellular matrix that binds them together
in complex organized layers
Dominate the structure of most natural environments on
earth
Communicate and cooperate in the formation and function
of biofilms quorum sensing
36
87
TheStudyofMicrobialGrowth
Microbial growth occurs at two levels: growth at a cellular
level with increase in size, and increase in population
Division of bacterial cells occurs mainly through binary
fission (transverse)
parent cell enlarges, duplicates its chromosome, and
forms a central transverse septum dividing the cell into
two daughter cells
37 38
RateofPopulationGrowth
Time required for a complete fission cycle is called the
generation, or doubling time
Each new fission cycle increases the population by a factor
of 2 exponential or logarithmic growth.
Generation times vary from minutes to days.
39 40
RateofPopulationGrowth
Equation for calculating population size over time:
N
= (Ni)2
n
N
is total number of cells in the population.

Ni is starting number of cells.
Exponent n denotes generation time.
2
n
number of cells in that generation
41
ThePopulationGrowthCurve
In laboratory studies, populations typically display a predictable pattern over
time growth curve.
Stages in the normal growth curve:
1. Lag phase flat period of adjustment, enlargement; little growth
2. Exponential growth phase a period of maximum growth will continue as
long as cells have adequate nutrients and a favorable environment
3. Stationary phase rate of cell growth equals rate of cell death caused by
depleted nutrients and O
2
, excretion of organic acids and pollutants
4. Death phase as limiting factors intensify, cells die exponentially in their
own wastes
42
43
MethodsofAnalyzingPopulation
Growth
Turbidometry most simple
Degree of cloudiness, turbidity, reflects the relative
population size
Enumeration of bacteria:
viable colony count
direct cell count count all cells present; automated or
manual
44
45 46
47
89
MinistryofHealth
Graduates
Sterilization and Disinfection
Physical,chemical,andmechanicalmethodstodestroyorreduce
undesirablemicrobesinagivenarea(decontamination)
Primarytargetsaremicroorganismscapableofcausinginfectionor
spoilage:
Vegetativebacterialcellsandendospores
Fungalhyphae andspores,yeast
Protozoantrophozoites andcysts
Worms
Viruses
Prions
2
ControllingMicroorganisms
3
Highestresistance
Prions,bacterialendospores
Moderateresistance
Pseudomonassp.
Mycobacteriumtuberculosis
Staphylococcusaureus
Protozoancysts
Leastresistance
Mostbacterialvegetativecells
Fungalsporesandhyphae,yeast
Envelopedviruses
Protozoantrophozoites
4
RelativeResistanceofMicrobes
5
Sterilization aprocessthatdestroysallviablemicrobes,including
viruses,prions andendospores
Disinfection aprocesstodestroyvegetativepathogens,not
endospores;inanimateobjects
Antiseptic disinfectantsapplieddirectlytoexposedbodysurfaces
Sanitization anycleansingtechniquethatmechanicallyremoves
microbes
Degermation reducesthenumberofmicrobesthroughmechanical
means
cide=tokill,bactericide,viricide,etc
Statis/static standstill bacteriostatic
6
TerminologyandMethodsofControl
7
Permanentlossofreproductivecapability,evenunderoptimumgrowth
conditions
Hardtodetect,microbesoftenrevealnoconspicuousvitalsignstobegin
with
8
MicrobialDeath
Theeffectivenessofaparticularagentisgovernedbyseveralfactors:
Numberofmicrobes
Natureofmicrobesinthepopulation
TemperatureandpHofenvironment
Concentrationordosageofagent
Modeofactionoftheagent
Presenceofsolvents,organicmatter,orinhibitors
Lengthofexposuretotheagent
9
FactorsThatAffectDeathRate
10
Numberofmicrobes
11
Natureofmicrobes
12
Modeofactionoftheagent
13
Lengthofexposuretotheagent
Selectionofmethodofcontroldependsoncircumstances:
Doestheapplicationrequiresterilization?
Istheitemtobereused?
Cantheitemwithstandheat,pressure,radiation,orchemicals?
Isthemethodsuitable?
Willtheagentpenetratetothenecessaryextent?
Isthemethodcost andlaborefficientandisitsafe?
14
PracticalConcernsinMicrobialControl
Cellulartargetsofphysicalandchemicalagents:
1. Thecellwall cellwallbecomesfragileandcelllyses;someantimicrobial
drugs,detergents,andalcohol
2. Thecellmembrane losesintegrity;detergentsurfactants
3. Proteinandnucleicacidsynthesis preventionofreplication,transcription,
translation,peptidebondformation,proteinsynthesis;chloramphenicol,
ultravioletradiation,formaldehyde
4. Proteins disruptordenatureproteins;alcohols,phenols,acids,heat
15
AntimicrobialAgentsModesofAction
16
17
1. Heat moistanddry
2. Coldtemperatures
3. Desiccation
4. Radiation
5. Filtration
18
MethodsofPhysicalControl
Moistheat lowertemperaturesandshorterexposuretime;
coagulationanddenaturationofproteins
Dryheat moderatetohightemperatures;dehydration,altersprotein
structure;incineration
19
ModeofActionandRelative
EffectivenessofHeat
20
Bacterialendosporesmostresistant usuallyrequire
temperaturesaboveboiling
21
HeatResistanceandThermalDeath
Thermaldeathtime(TDT) shortestlengthoftimerequired
tokillalltestmicrobesataspecifiedtemperature
Thermaldeathpoint(TDP) lowesttemperaturerequiredto
killallmicrobesinasamplein10minutes
22
ThermalDeathMeasurements
23
Steamunderpressure sterilization
Autoclave15psi/121oC/1040min
Steammustreachsurfaceofitembeingsterilized
Itemmustnotbeheatormoisturesensitive
Modeofaction denaturationofproteins,destructionof
membranesandDNA
24
MoistHeatMethods
93
25
Tyndallization intermittentsterilizationforsubstancesthat
cannotwithstandautoclaving
Itemsexposedtofreeflowingsteamfor3060minutes,
incubatedfor2324hoursandthensubjectedtosteamagain
Repeatcyclefor3days
Usedforsomecannedfoodsandlaboratorymedia
Disinfectant
26
NonpressurizedSteam
Boilingat100Cfor30minutestodestroynonsporeforming
pathogens
Disinfection
27
BoilingWater Pasteurization
Pasteurization heatisappliedtokillpotentialagentsofinfectionand
spoilagewithoutdestroyingthefoodflavororvalue
63C66Cfor30minutes(batchmethod)
71.6Cfor15seconds(flashmethod)
Notsterilization killsnonsporeformingpathogensandlowersoverall
microbecount;doesnotkillendosporesormanynonpathogenic
microbes
28
Dryheatusinghighertemperaturesthanmoistheat
Incineration flameorelectricheatingcoil
Ignitesandreducesmicrobesandothersubstances
Dryovens 150180oC coagulateproteins
29
DryHeat
30
Microbiostatic slowsthegrowthofmicrobes
Refrigeration015oCandfreezing<0oC
Usedtopreservefood,media,andcultures
31
Cold
Gradualremovalofwaterfromcells,leadstometabolic
inhibition
Noteffectivemicrobialcontrol manycellsretainabilityto
growwhenwaterisreintroduced
Lyophilization freezedrying;preservation
32
Desiccation
IonizingRadiation deeppenetratingpowerthathassufficientenergyto
causeelectronstoleavetheirorbit,breaksDNA
Gammarays,Xrays,cathoderays
Cold(lowtemperature)sterilization
Usedtosterilizemedicalsuppliesandfoodproducts
33
Radiation
34
35
Nonionizingradiation littlepenetratingpower mustbe
directlyexposed
UVlightcreatespyrimidinedimers,whichinterferewith
replication
36
Radiation
37
Physicalremovalofmicrobesbypassingagasorliquid
throughfilter
Usedtosterilizeheatsensitiveliquidsandairinhospital
isolationunitsandindustrialcleanrooms
38
Filtration
39
Heat moistvs.dry
Moist steam(pressureornone),boilingwater,pasteurization
Dry incineration,hotair
Thermaldeathtime(TDT)
Thermaldeathpoint(TDP)
Coldanddessication
lyophilization
Radiation
Filtration
40
Summary:MethodsofPhysicalControl
Disinfectants,antiseptics,sterilants,degermers,andpreservatives
Somedesirablequalitiesofchemicals:
Rapidactioninlowconcentration
Solubilityinwateroralcohol,stable
Broadspectrum,lowtoxicity
Penetrating
Noncorrosiveandnonstaining
Affordableandreadilyavailable
41
ChemicalAgentsinMicrobialControl
Highlevelgermicides killendospores;maybesterilants
Devicesthatarenotheatsterilizable andintendedtobeusedinsterile
environments(bodytissue)
Intermediatelevel killfungalspores(notendospores),tuberclebacillus,
andviruses
Usedtodisinfectdevicesthatwillcomeincontactwithmucous
membranesbutarenotinvasive
Lowlevel eliminateonlyvegetativebacteria,vegetativefungalcells,
andsomeviruses
Cleansurfacesthattouchskinbutnotmucousmembranes
42
LevelsofChemicalDecontamination
Natureofthematerialbeingtreated
Degreeofcontamination
Timeofexposure
Strengthandchemicalactionofthegermicide
43
FactorsthatAffectGermicidalActivity
ofChemicals
44
1. Halogens
2. Phenolics
3. Chlorhexidine
4. Alcohols
5. Hydrogenperoxide
6. Detergents&soaps
7. Heavymetals
8. Aldehydes
9. Gases
10. Dyes
45
GermicidalCategories
Chlorine Cl2,hypochlorites (chlorinebleach),chloramines
Denaturate proteinsbydisruptingdisulfidebonds
Intermediatelevel
Unstableinsunlight,inactivatedbyorganicmatter
Water,sewage,wastewater,inanimateobjects
Iodine I2,iodophors (betadine)
Interfereswithdisulfidebondsofproteins
Intermediatelevel
Mildermedicalanddentaldegerming agents,disinfectants,ointments
46
Halogens
47
Disruptcellwallsandmembranesandprecipitateproteins
Lowtointermediatelevel bactericidal,fungicidal,virucidal,notsporicidal
Lysol
Triclosan antibacterialadditivetosoaps
48
Phenolics
49
Asurfactantandproteindenaturantwithbroadmicrobicidal properties
Lowtointermediatelevel
Hibiclens,Hibitane
Usedasskindegerming agentsforpreoperativescrubs,skincleaning,and
burns
50
Chlorhexidine
51
Ethyl,isopropylinsolutionsof5095%
Actassurfactantsdissolvingmembranelipidsandcoagulating
proteinsofvegetativebacterialcellsandfungi
Intermediatelevel
52
Alcohols
Producehighlyreactivehydroxylfreeradicalsthatdamageproteinand
DNAwhilealsodecomposingtoO2gas toxictoanaerobes
Antisepticatlowconcentrations;strongsolutionsaresporicidal
53
HydrogenPeroxide
54
98
Glutaraldehyde andformaldehydekillbyalkylatingproteinandDNA
Glutaraldehyde in2%solution(Cidex)usedassterilant forheatsensitive
instruments
Highlevel
Formaldehyde disinfectant,preservative,toxicitylimitsuse
Formalin 37%aqueoussolution
Intermediatetohighlevel
55
Aldehydes
56
Ethyleneoxide,propyleneoxide
Strongalkylatingagents
Highlevel
Sterilizeanddisinfectplasticsandprepackageddevices,foods
57
GasesandAerosols
58
59
Quaternaryammoniacompounds(quats)actassurfactantsthatalter
membranepermeabilityofsomebacteriaandfungi
Verylowlevel
Soaps mechanicallyremovesoilandgreasecontainingmicrobes
60
DetergentsandSoaps
99
61 62
63
Solutionsofsilverandmercurykillvegetativecellsinlow
concentrationsbyinactivatingproteins
Oligodynamic action
Lowlevel
Merthiolate,silvernitrate,silver
64
HeavyMetals
65 66
100
Anilinedyesareveryactiveagainstgrampositivespeciesofbacteriaand
variousfungi
Sometimesusedforantisepsisandwoundtreatment
Lowlevel,narrowspectrumofactivity
67
DyesasAntimicrobialAgents
Lowlevelofactivity
Organicacidspreventsporegerminationandbacterialandfungalgrowth
Aceticacidinhibitsbacterialgrowth
Propionicacidretardsmolds
Lacticacidpreventsanaerobicbacterialgrowth
Benzoicandsorbic acidinhibityeast
68
AcidsandAlkalis
69
101
MinistryofHealth
Graduates
Laboratories
Tools of the Laboratory:
The Methods for Studying Microorganisms
2
Discovery of Microorganisms
Antonyvan
Leeuwenhoek(1632
1723)
firstpersontoobserve
anddescribemicro
organismsaccurately
3
magnification abilitytoenlargeobjects
resolvingpower abilitytoshowdetail
4
Lenses and the Bending of Light
lightisrefracted(bent)whenpassingfromone
mediumtoanother
refractiveindex
ameasureofhowgreatlyasubstanceslowsthevelocity
oflight
directionandmagnitudeofbendingisdetermined
bytherefractiveindexesofthetwomediaforming
theinterface
5
Lenses
focuslightraysataspecificplacecalledthefocal
point
distancebetweencenteroflensandfocalpointis
thefocallength
strengthoflensrelatedtofocallength
shortfocallengthmoremagnification
6
102
7 8
9
WorkingDistance
distancebetweenthefrontsurfaceoflensandsurfaceofcoverglass
orspecimen
10
11
Microscope Resolution
Abilityofalenstoseparateordistinguishsmall
objectsthatareclosetogether
Wavelengthoflightusedismajorfactorin
resolution
Shorterwavelengthgreaterresolution
12
103
13
The Light Microscope
Manytypes
Brightfieldmicroscope
Darkfieldmicroscope
Phasecontrastmicroscope
Fluorescencemicroscopes
Arecompoundmicroscopes
Imageformedbyactionof2lenses
14
The Bright-Field Microscope
Producesadarkimageagainstabrighter
background
Hasseveralobjectivelenses
Parfocal microscopesremaininfocuswhenobjectives
arechanged
Totalmagnification
Productofthemagnificationsoftheocularlensand
theobjectivelens
15
The Dark-Field Microscope
Producesabrightimageoftheobjectagainstadark
background
Usedtoobserveliving,unstainedpreparations
16
17
The Phase-Contrast Microscope
Enhancesthecontrastbetweenintracellular
structureshavingslightdifferencesinrefractive
index
Excellentwaytoobservelivingcells
18
104
The Differential Interference Contrast
Microscope
Createsimagebydetectingdifferencesinrefractive
indicesandthicknessofdifferentpartsofspecimen
Excellentwaytoobservelivingcells
19
The Fluorescence Microscope
Exposesspecimentoultraviolet,violet,orbluelight
Specimensusuallystainedwithfluorochromes
Showsabrightimageoftheobjectresultingfrom
thefluorescentlightemittedbythespecimen
20
21 22
Specimens Preparation
Wetmounts&hangingdropmounts allow
examinationofcharacteristicsoflivecells:motility,
shape,&arrangement
Fixedmounts aremadebydrying&heatingafilmof
specimen.Thissmear isstainedusingdyestopermit
visualizationofcellsorcellparts.
23
Preparation and Staining of
Specimens
Increasesvisibilityofspecimen
Accentuatesspecificmorphologicalfeatures
Preservesspecimens
24
105
Fixation
Processbywhichinternalandexternalstructures
arepreservedandfixedinposition
Processbywhichorganismiskilledandfirmly
attachedtomicroscopeslide
Heatfixing
Preservesoverallmorphologybutnotinternal
structures
Chemicalfixing
Protectsfinecellularsubstructureandmorphologyof
larger,moredelicateorganisms
25
Dyes and Simple Staining
Dyes
Makeinternalandexternalstructuresofcellmore
visiblebyincreasingcontrastwithbackground
Havetwocommonfeatures
Chromophore groups
Chemicalgroupswithconjugateddoublebonds
Givedyeitscolor
Abilitytobindcells
26
Dyes
cationicdyes basic,withpositivechargesonthe
chromophore
anionicdyes acidic,withnegativechargesonthe
chromophore
surfacesofmicrobesarenegativelychargedandattract
basicdyes positivestaining.
negativestaining microberepelsdye&itstainsthe
background
27 28
Simplestaining
Asinglestainingagentisused
Basicdyesarefrequentlyused
Dyeswithpositivecharges
E.G.,Crystalviolet
29
Differential Staining
Dividesmicroorganismsintogroupsbasedontheir
stainingproperties
E.G.,Gramstain
E.G.,Acidfaststain
30
106
Gram staining
Mostwidelyuseddifferentialstainingprocedure
Dividesbacteriaintotwogroupsbasedon
differencesincellwallstructure
31
primarystain
mordant
decolorization
counterstain
Positive
negative
32
Escherichiacoli agramnegativerod 33
Acid-fast staining
Particularlyusefulforstainingmembersofthegenus
mycobacterium
E.G.,Mycobacteriumtuberculosis causestuberculosis
E.G.,Mycobacteriumleprae causesleprosy
Highlipidcontentincellwallsisresponsiblefortheir
stainingcharacteristics
34
Staining Specific Structures
Negativestaining
Oftenusedtovisualizecapsulessurroundingbacteria
Capsulesarecolorlessagainstastainedbackground
35
Staining Specific Structures
Sporestaining
Doublestainingtechnique
Bacterialendosporeisonecolorandvegetativecellisa
differentcolor
Flagellastaining
Mordantappliedtoincreasethicknessofflagella
36
107
Typesofstains
37
ElectronMicroscopy
Beamsofelectronsare
usedtoproduceimages
Wavelengthofelectron
beamismuchshorter
thanlight,resultingin
muchhigherresolution
38
TheTransmissionElectronMicroscope
Electronsscatterwhentheypassthroughthin
sectionsofaspecimen
Transmittedelectrons(thosethatdonotscatter)are
usedtoproduceimage
Denserregionsinspecimen,scattermoreelectrons
andappeardarker
39 40
SpecimenPreparation
Analogoustoproceduresusedforlightmicroscopy
Fortransmissionelectronmicroscopy,specimens
mustbecutverythin
Specimensarechemicallyfixedandstainedwith
electrondensematerial
41
Otherpreparationmethods
Shadowing
Coatingspecimenwithathinfilmofaheavymetal
Freezeetching
Freezespecimenthenfracturealonglinesofgreatest
weakness(e.G.,Membranes)
42
108
43
Ebola
44
Flyhead
45
TheScanningElectronMicroscope
Useselectronsreflectedfromthesurfaceofa
specimentocreateimage
Producesa3dimensionalimageofspecimens
surfacefeatures
46
47
NewerTechniquesinMicroscopy
Confocalmicroscopyand
scanningprobe
microscopy
Haveextremelyhigh
resolution
Canbeusedtoobserve
individualatoms
48
109
The5Isofculturingmicrobes
1. Inoculation introductionofasampleintoa
containerofmedia
2. Incubation underconditionsthatallowgrowth
3. Isolationseparatingonespeciesfromanother
4. Inspection
5. Identification
49
Isolation
Ifanindividualbacterialcellisseparatedfromother
cells&hasspaceonanutrientsurface,itwillgrow
intoamoundofcells acolony
Acolonyconsistsofonespecies
50
Isolationtechnique
51 52
Media providingnutrientsin
thelaboratory
Mostcommonlyused:
Nutrientbroth liquidmediumcontainingbeefextract&
peptone
Nutrientagar solidmediacontainingbeefextract,
peptone&agar
Agar isacomplexpolysaccharideisolatedfromred
algae
Solidatroomtemp,liquefiesatboiling(100
o
c),doesnot
resolidify untilitcoolsto42
o
c
Providesframeworktoholdmoisture&nutrients
Notdigestibleformostmicrobes
53
Typesofmedia
Synthetic containspureorganic&inorganic
compoundsinanexactchemicalformula
Complexornonsynthetic containsatleastone
ingredientthatisnotchemicallydefinable
Generalpurposemedia growsabroadrangeof
microbes,usuallynonsynthetic
Enrichedmedia containscomplexorganic
substancessuchasblood,serum,hemoglobinor
specialgrowthfactorsrequiredbyfastidious
microbes
54
110
Enrichedmedia
55
Selectivemedia containsoneormoreagentsthat
inhibitgrowthofsomemicrobesandencourage
growthofthedesiredmicrobes
Differentialmedia allowsgrowthofseveraltypes
ofmicrobesanddisplaysvisibledifferencesamong
desiredandundesiredmicrobes
56
Selective&DifferentialMedia
57
Selectivemedia
58
Differentialmedia
59
TypesofAgar BloodAgar
DefiningFeature:
Contains5%bloodcellsfrom
ananimal,usuallysheep.
WhatWillGrow:
Mostbacteriawillgrowon
this.
60
111
TypesofAgar ChocolateAgar
DefiningFeatures:
Madeoflysed redblood
cells,typicallysheep.
Nutrientmediumusedto
culturefastidious
organisms.
DOESNOTgive
haemolysis data
61
TypesofAgar ChocolateAgar
WhatWillGrow:Most
bacteriawillgrowonthis.
Haemophilus growsdue
toX&Vfactorsfromthe
sheepblood cannot
differentiatebetweenthe
speciesthough!
Neisseria growsdueto
nutrientrich
environment.
62
TypesofAgar MacConkey Agar
DefiningFeature:
E.coli differentiatesby
growingintoredcolonies
duetosugarfermentation.
Canbemixedwithor
withoutsugarlactose.
WhatWillGrow:
WillonlygrowGram
Negativebacteria.
63
TypesofAgar ThayerMartinAgar
DefiningFeature:
Designedtoisolate
Neisseria gonorrhoeae.
WhatWillGrow:
Specificallydesignedfor
Neisseria gonorrhoeae.
64
TypesofAgar Xylose LysineDeoxychocolate Agar
DefiningFeature:
Usedforculturingstool
samples.
Containstwodifferent
indicators.
Coloniesoforganismthat
fermentlactosewillappear
yellow.
WhatWillGrow:
FormulatedtoinhibitGram
positivebacteria.
FormulatedtoencourageGram
negativebacilli.
65
TypesofAgar Sabouraud Agar
DefiningFeature:
Usedtogrowfungi.
HasalowpH(killsmost
bacteria).
Containsgentamicin
antibiotic(particularly
usefulforGramNegative
bacteria).
WhatWillGrow:
Growsfungi.
66
112
Miscellaneousmedia
reducingmedium containsasubstancethat
absorbsoxygenorslowspenetrationofoxygeninto
medium;usedforgrowinganaerobicbacteria
carbohydratefermentationmedium contains
sugarsthatcanbefermented,convertedtoacids,
andapHindicatortoshowthereaction;basisfor
identifyingbacteriaandfungi
67
Carbohydratefermentationmedia
68
113
MinistryofHealth
Graduates
Antimicrobialchemotherapyandsensitivitytesting
PrinciplesofAntimicrobialTherapy
Administertoaninfectedpersonadrugthatdestroysthe
infectiveagentwithoutharmingthehostscells.
Antimicrobialdrugsareproducednaturallyorsynthetically.
2
3 4
OriginsofAntibioticDrugs
Antibioticsarecommonmetabolicproductsofaerobicspore
formingbacteriaandfungi.
bacteriaingeneraStreptomycesand Bacillus
moldsingeneraPenicillium andCephalosporium
Byinhibitingtheothermicrobesinthesamehabitat,
antibioticproducershavelesscompetitionfornutrientsand
space.
5
InteractionsBetweenDrugand
Microbe
Antimicrobialdrugsshouldbeselectivelytoxic drugsshould
killorinhibitmicrobialcellswithoutsimultaneouslydamaging
hosttissues.
Asthecharacteristicsoftheinfectiousagentbecomemore
similartothevertebratehostcell,completeselectivetoxicity
becomesmoredifficulttoachieveandmoresideeffectsare
seen
6
114
MechanismsofDrugAction
1. Inhibitionofcellwallsynthesis
2. Disruptionofcellmembranestructureorfunction
3. Inhibitionofnucleicacidsynthesis,structureorfunction
4. Inhibitionofproteinsynthesis
5. Blocksonkeymetabolicpathways
7 8
TheSpectrumofanAntimicrobicDrug
Spectrum rangeofactivityofadrug
Narrowspectrum effectiveonasmallrangeofmicrobes
Targetaspecificcellcomponentthatisfoundonlyin
certainmicrobes
Broadspectrum greatestrangeofactivity
Targetcellcomponentscommontomostpathogens
9
AntimicrobialDrugsThatAffectthe
BacterialCellWall
Mostbacterialcellwallscontainpeptidoglycan.
Penicillins andcephalosporins blocksynthesisof
peptidoglycan,causingthecellwalltolyse.
Activeonyoung,growingcells
Penicillins donotpenetratetheoutermembraneandareless
effectiveagainstGramnegativebacteria.
Broadspectrumpenicillins andcephalosporins cancrossthe
cellwallsofGramnegativebacteria.
10
11
AntimicrobialDrugsThatDisruptCell
MembraneFunction
Acellwithadamagedmembranediesfromdisruptionin
metabolismorlysis.
Thesedrugshavespecificityforaparticularmicrobialgroup,based
ondifferencesintypesoflipidsintheircellmembranes.
Polymyxins interactwithphospholipidsandcauseleakage,
particularlyingramnegativebacteria.
Amphotericinbandnystatin formcomplexeswithsterolsonfungal
membraneswhichcausesleakage.
12
115
13
DrugsThatInhibitNucleicAcid
Synthesis
Mayblocksynthesisofnucleotides,inhibitreplication,orstop
transcription
Chloroquine bindsandcrosslinksthedoublehelix;
quinolonesinhibitDNAhelicases.
Antiviraldrugsthatareanalogsofpurinesandpyrimidines
insertinviralnucleicacid,preventingreplication.
14
DrugsThatBlockProteinSynthesis
Ribosomesofeucaryotesdifferinsizeandstructurefrom
procaryotes;antimicrobicsusuallyhaveaselectiveaction
againstprocaryotes;canalsodamagetheeucaryotic
mitochondria
Aminoglycosides(streptomycin,gentamycin)insertonsites
onthe30SsubunitandcausemisreadingofmRNA.
TetracyclinesblockattachmentoftRNAontheAacceptorsite
andstopfurthersynthesis.
15 16
DrugsthatAffectMetabolicPathways
Sulfonamidesandtrimethoprimblockenzymesrequiredfor
tetrahydrofolate synthesisneededforDNAandRNA
synthesis.
Competitiveinhibition drugcompeteswithnormal
substrateforenzymesactivesite
Synergisticeffect anadditiveeffect,achievedbymultiple
drugsworkingtogether,requiringalowerdoseofeach
17 18
SurveyofMajorAntimicrobial
DrugGroups
Antibacterialdrugs
antibiotics
syntheticdrugs
Antifungaldrugs
Antiprotozoan drugs
Antiviraldrugs
About260differentantimicrobialdrugsareclassifiedin20drug
families.
19
PenicillinandItsRelatives
Largediversegroupofcompounds
Couldbesynthesizedinthelaboratory
Moreeconomicaltoobtainnaturalpenicillinthrough
microbialfermentationandmodifyittosemisyntheticforms
Penicillium chrysogenum majorsource
Allconsistof3parts:
Thiazolidine ring
Betalactamring
Variablesidechaindictatingmicrobialactivity
20
21
Betalactamantimicrobials allcontainahighlyreactive3
carbon,1nitrogenring
Primarymodeofactionistointerferewithcellwallsynthesis.
Greaterthanofallantimicrobic drugsarebetalactams.
Penicillins andcephalosporins mostprominentbetalactams
22
23
SubgroupandUsesofPenicillins
Penicillins GandVmostimportantnaturalforms
PenicillinisthedrugofchoiceforGrampositivecocci (streptococci)and
someGramnegativebacteria(meningococci andsyphilisspirochete).
Semisynthetic penicillins ampicillin,carbenicillin andamoxicillinhave
broaderspectra Gramnegativeentericrods
Penicillinaseresistant methicillin,nafcillin,cloxacillin
Primaryproblems allergiesandresistantstrainsofbacteria
24
117
Cephalosporins
Accountformajorityofallantibioticsadministered
IsolatedfromCephalosporium acremonium mold
Syntheticallyalteredbetalactam structure
Relativelybroadspectrum,resistanttomostpenicillinases,&
causefewerallergicreactions
Somearegivenorally;manymustbeadministered
parenterally.
Genericnameshaveroot cef,ceph,orkef.
25
Cephalosporins
4Generationsexist:eachgroupmoreeffectiveagainstGramnegatives
thantheonebeforewithimproveddosingscheduleandfewerside
effects
Firstgeneration cephalothin,cefazolin mosteffectiveagainstgram
positivecocci andfewgramnegative
Secondgeneration cefaclor,cefonacid moreeffectiveagainstgram
negativebacteria
Thirdgeneration cephalexin,ceftriaxone broadspectrumactivityagainst
entericbacteriawithbetalactamases
Fourthgeneration cefepime widestrange;bothgram negativeandgram
positive
26
27
TheAcquisitionofDrugResistance
Adaptiveresponseinwhichmicroorganismsbegintotolerate
anamountofdrugthatwouldordinarilybeinhibitory;dueto
geneticversatilityorvariation;intrinsicandacquired
Acquiredresistance:
Spontaneousmutationsincriticalchromosomalgenes
Acquisitionofnewgenesorsetsofgenesviatransferfromanother
species
Originatesfromresistancefactors(plasmids)encodedwithdrug
resistance,transposons
28
29
MechanismsofDrugResistance
Druginactivationbyacquiredenzymaticactivity
penicillinases
Decreasedpermeabilitytodrugorincreasedeliminationof
drugfromcell acquiredormutation
Changeindrugreceptors mutationoracquisition
Changeinmetabolicpatterns mutationoforiginalenzyme
30
118
31 32
ConsiderationsinSelectinganAntimicrobial
Drug
Identifythemicroorganismcausingtheinfection.
Testthemicroorganismssusceptibility(sensitivity)tovarious
drugsinvitro whenindicated.
Theoverallmedicalconditionofthepatient
33
IdentifyingtheAgent
Identificationofinfectiousagentshouldbeattemptedas
soonaspossible.
Specimensshouldbetakenbeforeantimicrobialsare
initiated.
34
TestingforDrugSusceptibility
Minimuminhibitoryconcentration[MIC]
Thesmallestconcentrationofantibioticthatinhibitsthegrowthof
organism
Liquidmedia(dilution)allowsMICestimation
Solidmedia(diffusion)
Diskdiffusion(KirbyBauer)
Etests
AllowsMICestimation
Betalactamaseproduction:quickscreeningmethod
35 36
119
Dilutioninliquidbroth
Tubescontainingincreasingantibioticconcentrations
Incubationduring18hrat37C
Tedious
37
Bacterial growth Inhibition
0 (Control) 0,25 0,50 1 2 4 8 mg/l
KirbyBauerdisctesting
38
Antibioticimpregnateddiscsplacedonanagarplateatthe
interfacebetweentestorganismandsusceptiblecontrolorganism
Resultingzonesofinhibitioncompared,useofcontrols
Susceptibilityisinferred(standardtables)
Etest
39
Plasticstripswithapredefined
gradientof
Oneantibiotic
Oneantifungal
Onlyonemanufacturer
Onestripperantibiotic
Widerangeofantibiotics
Easytouse
Storageat20C
Shortshelflife,expensive
ReadingEtests
40
Susceptible < 1
Resistant > 4 ug/ml
Ciprofloxacin for Yersinia pestis
Intermediate 1-4 ug/ml
Upper reading
Interpretation
41
Themainconceptistheclinicalcategorisation"
StrainsaresortedaccordingtolevelofMinimalInhibitory
Concentration(MIC)versusreferencebreakpoints
c andC aretheminorandmajorbreakpoints
Susceptible Intermediate Resistant
MIC < c MIC
<
C MIC
Criticalpointsinqualityassurance
1. Culturemedia:MullerHinton
2. Reagents:disks
3. Sizeoftheinoculums
4. Incubationcondition
5. Controlwithreferencestrains
6. Readinginhibitiondiameters(accuratemeasurement)
7. Knowledgeofstaff
42
120
Commoninterpretationproblems
Resultsdependsonthetechniqueused
Manyfactorsinfluenceresults
Lackofstandardizationoftheinoculums
Thicknessandqualityoftheculturemedia
Qualityandconservationofthedisks
Qualitycontrolwithstandardizedstrains
Conditionanddurationofincubation
43
44
An agar gel that is too thick leads to smaller zones
Problemwiththesizeof
theinoculums
Solution:
UseMcFarland0.5
photometer
Scale>sametubes
45
Contaminationwith
anotherorganism
46
47
Badmanipulation
InoculationoftheMullerHinton
Swabbing
Notbyflooding
48
ProblemswithEtestreading
121
MinistryofHealth
Graduates
TheCocci ofMedicalImportance
GeneralCharacteristicsoftheStaphylococci
Common inhabitant of the skin and mucous membranes
Spherical cells arranged in irregular clusters
Grampositive
Lack spores and flagella
May have capsules
31 species
2
3
Grows in large, round, opaque colonies
Optimum temperature of 37
o
C
Facultative anaerobe
Withstands high salt, extremes in pH, and high temperatures
Carried in nasopharynx and skin
Produces many virulence factors
4
5
VirulencefactorsofS.aureus
Enzymes:
Coagulase coagulates plasma and blood; produced by 97%
of human isolates; diagnostic
Hyaluronidase digests connective tissue
Staphylokinase digests blood clots
DNase digests DNA
Lipases digest oils; enhances colonization on skin
Penicillinase inactivates penicillin
6
122
VirulencefactorsofS.aureus
Toxins:
Hemolysins (, , , ) lyse red blood cells
Leukocidin lyses neutrophils and macrophages
Enterotoxin induce gastrointestinal distress
Exfoliative toxin separates the epidermis from the dermis
Toxic shock syndrome toxin (TSST) induces fever, vomiting,
shock, systemic organ damage
7 8
EpidemiologyandPathogenesis
Present in most environments frequented by humans
Readily isolated from fomites
Carriage rate for healthy adults is 2060%.
Carriage is mostly in anterior nares, skin, nasopharynx, intestine.
Predisposition to infection include: poor hygiene and nutrition, tissue
injury, preexisting primary infection, diabetes, immunodeficiency.
Increase in community acquired methicillin resistance MRSA
9
StaphylococcalDisease
Range from localized to systemic
Localized cutaneous infections invade skin through wounds, follicles, or
glands
Folliculitis superficial inflammation of hair follicle; usually resolved with no
complications but can progress
Furuncle boil; inflammation of hair follicle or sebaceous gland progresses
into abscess or pustule
Carbuncle larger and deeper lesion created by aggregation and
interconnection of a cluster of furuncles
Impetigo bubblelike swellings that can break and peel away; most common
in newborns
10
11
Systemic infections
Osteomyelitis infection is established in the metaphysis;
abscess forms
Bacteremia primary origin is bacteria from another
infected site or medical devices; endocarditis possible
12
13
Toxigenic disease
Food intoxication ingestion of heat stable enterotoxins;
gastrointestinal distress
Staphylococcal scalded skin syndrome toxin induces
bright red flush, blisters, then desquamation of the
epidermis
Toxic shock syndrome toxemia leading to shock and
organ failure
14
15
OtherStaphylococci
Coagulasenegative staphylococcus; frequently involved in nosocomial and
opportunistic infections
S. epidermidis lives on skin and mucous membranes; endocarditis,
bacteremia, UTI
S. hominis lives around apocrine sweat glands
S. capitis live on scalp, face, external ear
All 3 may cause wound infections by penetrating through broken skin.
S. saprophyticus infrequently lives on skin, intestine, vagina; UTI
16
IdentificationofStaphylococcusin
Samples
Frequently isolated from pus, tissue exudates, sputum, urine, and blood
Cultivation, catalase, biochemical testing, coagulase
17 18
ClinicalConcernsandTreatment
95% have penicillinase and are resistant to penicillin and ampicillin.
MRSA methicillinresistant S. aureus carry multiple resistance
Abscesses have to be surgically perforated.
Systemic infections require intensive lengthy therapy.
19
PreventionofStaphylococcalInfections
Universal precautions by healthcare providers to prevent
nosocomial infections
Hygiene and cleansing
20
GeneralCharacteristicsofStreptococci
Grampositive spherical/ovoid cocci arranged in long chains; commonly in
pairs
Nonsporeforming, nonmotile
Can form capsules and slime layers
Facultative anaerobes
Do not form catalase, but have a peroxidase system
Most parasitic forms are fastidious and require enriched media.
Small, nonpigmented colonies
Sensitive to drying, heat and disinfectants
25 species
21 22
Streptococci
Lancefield classification system based on cell wall Ag 17
groups (A,B,C,.)
Another classification system is based on hemolysis reactions.
hemolysis A,B,C,G and some D strains
hemolysis S. pneumoniae and others collectively called
viridans
23 24
HumanStreptococcalPathogens
S. pyogenes
S. agalactiae
Viridans streptococci
S. pneumoniae
Enterococcus faecalis
25
hemolyticS.pyogenes
Most serious streptococcal pathogen
Strict parasite
Inhabits throat, nasopharynx, occasionally skin
26
VirulenceFactorsofhemolytic
S.pyogenes
Produces surface antigens:
Ccarbohydrates protect against lysozyme
Fimbriae adherence
Mprotein contributes to resistance to phagocytosis
Hyaluronic acid capsule provokes no immune response
27 28
S.pyogenes
Extracellular toxins:
Streptolysins hemolysins; streptolysin O (SLO) and
streptolysin S (SLS) both cause cell and tissue injury
Pyogenic toxin (erythrogenic) induces fever and typical red
rash
Superantigens strong monocyte and lymphocyte
stimulants; cause the release of tissue necrotic factor
29
S.pyogenes
Extracellular enzymes
Streptokinase digests fibrin clots
Hyaluronidase breaks down connective tissue
DNase hydrolyzes DNA
30
Humans only reservoir
Inapparent carriers
Transmission contact, droplets, food, fomites
Portal of entry generally skin or pharynx
Children predominant group affected for cutaneous and throat infections
Systemic infections and progressive sequelae possible if untreated
31
ScopeofClinicalDisease
Skin infections
Impetigo (pyoderma) superficial lesions that break and form highly
contagious crust; often occurs in epidemics in school children; also
associated with insect bites, poor hygiene, and crowded living conditions
Erysipelas pathogen enters through a break in the skin and eventually
spreads to the dermis and subcutaneous tissues; can remain superficial
or become systemic
Throat infections
Streptococcal pharyngitis strep throat
32
33 34
ScopeofClinicalDisease
Systemic infections
Scarlet fever strain of S. pyogenes carrying a prophage that codes for
pyrogenic toxin; can lead to sequelae
Septicemia
Pneumonia
Streptococcal toxic shock syndrome
35
LongTermComplicationsof
GroupAInfections
Rheumatic fever follows overt or subclinical pharyngitis in children;
carditis with extensive valve damage possible, arthritis, chorea, fever
Acute glomerulonephritis nephritis, increased blood pressure,
occasionally heart failure; can become chronic leading to kidney failure
36
GroupB:Streptococcusagalactiae
Regularly resides in human vagina, pharynx and large intestine
Can be transferred to infant during delivery and cause severe infection
most prevalent cause of neonatal pneumonia, sepsis, and meningitis
15,000 infections and 5,000 deaths in US
Pregnant women should be screened and treated.
Wound and skin infections and endocarditis in debilitated people
37
GroupDEnterococciand
GroupsCandGStreptococci
Group D:
Enterococcus faecalis, E. faecium, E. durans
normal colonists of human large intestine
cause opportunistic urinary, wound, and skin infections,
particularly in debilitated persons
Groups C and G:
common animal flora, frequently isolated from upper
respiratory; pharyngitis, glomerulonephritis, bacteremia
38
Identification
Cultivation and diagnosis ensure proper treatment to prevent possible
complications.
Rapid diagnostic tests based on monoclonal antibodies that react with C
carbohydrates
Culture using bacitracin disc test, CAMP test
39 40
41
TreatmentandPrevention
Groups A and B are treated with penicillin.
Sensitivity testing needed for enterococci
No vaccines available
42
HemolyticStreptococci:
ViridansGroup
Large complex group
Streptococcus mutans, S. oralis, S. salivarus, S. sanguis, S. milleri, S. mitis
Most numerous and widespread residents of the gums and teeth, oral
cavity and also found in nasopharynx, genital tract, skin
Not very invasive; dental or surgical procedures facilitate entrance
43
ViridansGroup
Bacteremia, meningitis, abdominal infection, tooth abscesses
Most serious infection subacute endocarditis bloodborne bacteria
settle and grow on heart lining or valves
Persons with preexisting heart disease are at high risk.
Colonization of heart by forming biofilms
44
ViridansGroup
S. mutans produce slime layers that adhere to teeth, basis for plaque.
Involved in dental caries
Persons with preexisting heart conditions should receive prophylactic
antibiotics before surgery or dental procedures.
45
Streptococcuspneumoniae:The
Pneumococcus
Causes 6070% of all bacterial pneumonias
Small, lancetshaped cells arranged in pairs and short chains
Culture requires blood or chocolate agar.
Growth improved by 510% CO
2
Lack catalase and peroxidases cultures die in O
2
46
47
S.pneumoniae
All pathogenic strains form large capsules major virulence factor.
Specific soluble substance (SSS) varies among types.
84 capsular types have been identified
Causes pneumonia and otitis media
48
EpidemiologyandPathology
550% of all people carry it as normal flora in the nasopharynx; infections
are usually endogenous.
Very delicate, does not survive long outside of its habitat
Young children, elderly, immune compromised, those with other lung
diseases or viral infections, persons living in close quarters are
predisposed to pneumonia
Pneumonia occurs when cells are aspirated into the lungs of susceptible
individuals.
Pneumococci multiply and induce an overwhelming inflammatory
response.
Gains access to middle ear by way of eustachian tube
49 50
CultivationandDiagnosis
Gram stain of specimen presumptive identification
hemolytic; optochin sensitivity
Quellung test or capsular swelling reaction
51
Traditionally treated with penicillin G or V
Increased drug resistance
Two vaccines available for high risk individuals:
Capsular antigen vaccine for older adults and other high risk
individualseffective 5 years
Conjugate vaccine for children 2 to 23 months
52
FamilyNeisseriaceae
Gramnegative cocci
Residents of mucous membranes of warmblooded animals
Genera include Neisseria, Moraxella, Acinetobacter.
2 primary human pathogens:
Neisseria gonorrhoeae
Neisseria meningitidis
53
GenusNeisseria
Gramnegative, beanshaped, diplococci
None develop flagella or spores.
Capsules on pathogens
Pili
Strict parasites, do not survive long outside of the host
Aerobic or microaerophilic
Oxidative metabolism
Produce catalase and cytochrome oxidase
Pathogenic species require enriched complex media and CO
2. 54
130
55
Neisseriagonorrhoeae:
TheGonococcus
Causes gonorrhea, an STD
Virulence factors:
pili, other surface molecules for attachment; slows phagocytosis
IgA protease cleaves secretory IgG
56
Strictly a human infection
In top 5 STDs
Infectious dose 1001,000
Does not survive more than 12 hours on fomites
57 58
Gonorrhea
Infection is asymptomatic in 10% of males and 50% of females.
Males urethritis, yellowish discharge, scarring and infertility
Females vaginitis, urethritis, salpingitis (PID) mixed
anaerobic abdominal infection, common cause of sterility and
ectopic tubal pregnancies
Extragenital infections anal, pharygeal, conjunctivitis,
septicemia, arthritis
59 60
131
61
GonorrheainNewborns
Infected as they pass through birth canal
Eye inflammation, blindness
Prevented by prophylaxis immediately after birth
62
DiagnosisandControl
Gram stain Gramnegative intracellular (neutrophils)
diplococci from urethral, vaginal, cervical, or eye exudate
presumptive identification
2030% of new cases are penicillinaseproducing PPNG or
tetracycline resistant TRNG
Combined therapies indicated
Recurrent infections can occur.
Reportable infectious disease
63 64
Neisseriameningitidis:
TheMeningococcus
Virulence factors:
Capsule
Pili
IgA protease
Endotoxin
12 strains; serotypes A, B, C cause most cases
65
Prevalent cause of meningitis; sporadic or epidemic
Human reservoir nasopharynx; 330% of adult population; higher in
institutional settings
High risk individuals are those living in close quarters, children 6months3
years, children and young adults 1020 years.
Disease begins when bacteria enter bloodstream, pass into cranial
circulation, and multiply in meninges
Very rapid onset; neurological symptoms; endotoxin causes hemorrhage
and shock; can be fatal
66
132
67 68
ClinicalDiagnosis
Gram stain CSF, blood, or nasopharyngeal sample
Culture for differentiation
Rapid tests for capsular antigen
69
Treated with IV penicillin G, chloramphenicol
Prophylactic treatment of family members, medical
personnel, or children in close contact with patient
Vaccines exist for group A and C.
70
OtherGramnegativeCocciandCoccobacilli
Genus Branhamella
Branhamella catarrhalis found in nasopharynx: significant
opportunist in cancer, diabetes, alcoholism
Genus Moraxella
Bacilli; found on mucous membranes
Genus Acinetobacter
Gramnegative bacilli; nonliving reservoir; source of nosocomial
infections
71
133
MinistryofHealth
Graduates
TheGramNegativeBacilliofMedicalImportance
2
AerobicGramNegative
NonentericBacilli
Large, diverse group of nonsporeforming bacteria
Wide range of habitats large intestines (enteric), zoonotic,
respiratory, soil, water
Most are not medically important; some are true pathogens,
some are opportunists
All have a lipopolysaccharide outer membrane of cell wall
endotoxin
3
AerobicGramNegative
NonentericBacilli
Pseudomonas and Burkholderia an opportunistic pathogen
Brucella and Francisella zoonotic pathogens
Bordetella and Legionella mainly human pathogens
4
Pseudomonas:ThePseudomonads
Small gramnegative rods with a single polar flagellum
Free living
Primarily in soil, sea water, and fresh water; also colonize plants and
animals
Important decomposers and bioremediators
Frequent contaminants in homes and clinical settings
Use aerobic respiration; do not ferment carbohydrates
Produce oxidase and catalase
Many produce water soluble pigments
5
Pseudomonasaeruginosa
6
134
PseudomonasAeruginosa
Common inhabitant of soil and water
Intestinal resident in 10% normal people
Resistant to soaps, dyes, quaternary ammonium disinfectants, drugs,
drying
Frequent contaminant of ventilators, IV solutions, anesthesia equipment
Opportunistic pathogen
7
SkinrashfromPseudomonas
8
PseudomonasAeruginosa
Common cause of nosocomial infections in hosts with burns, neoplastic
disease, cystic fibrosis
Complications include pneumonia, UTI, abscesses, otitis, and corneal
disease
Endocarditis, meningitis, bronchopneumonia
Grapelike odor
Greenishblue pigment (pyocyanin)
Multidrug resistant
Cephalosporins, aminoglycosides, carbenicillin, polymixin, quinolones,
and monobactams
9
Pseudomonas(left)and
Staphylococcus(right)
10
RelatedGramNegativeAerobicRods
Genera Burkholderia, Acinetobacter, Stenotrophomonas
Similar to pseudomonads
Wide variety of habitats in soil, water, and related environments
Obligate aerobes; do not ferment sugars
Motile, oxidase positive
Opportunistic
11
Burkholderia
Burkholderia cepacia Burkholderia cepacia active in
biodegradation of a variety of substances; opportunistic
agent in respiratory tract, urinary tract, and occasionally skin
infections; drug resistant
B. pseudomallei generally acquired through penetrating
injury or inhalation from environmental reservoir; wound
infections, bronchitis and pneumonia, septicemia
12
135
AcinetobacterandStenotrophomonas
Acinetobacter baumanii nosocomial and community
acquired infections; wounds, lungs, urinary tract, burns,
blood; extremely resistant treatment with combination
antimicrobials
Stenotrophomonas maltophilia forms biofilms; contaminant
of disinfectants dialysis equipment, respiratory equipment,
water dispensers, and catheters; clinical isolate in respiratory
soft tissue, blood, CSF; high resistance to multidrugs
13
BrucellaandBrucellosis
Tiny gramnegative coccobacilli
2 species:
Brucella abortus (cattle)
Brucella suis (pigs)
Brucellosis, malta fever, undulant fever, and Bang disease a zoonosis
transmitted to humans from infected animals
Fluctuating pattern of fever weeks to a year
Combination of tetracycline and rifampin or streptomycin
Animal vaccine available
Potential bioweapon
14
Agglutinationtitertestforbrucellosis
15
FrancisellaTularensisandTularemia
Causes tularemia, a zoonotic disease of mammals endemic to the
northern hemisphere, particularly rabbits
Transmitted by contact with infected animals, water and dust or bites by
vectors
Headache, backache, fever, chills, malaise, and weakness
10% death rate in systemic and pulmonic forms
Intracellular persistence can lead to relapse
Gentamicin or tetracycline
Attenuated vaccine
Potential bioterrorism agent
16
BordetellaPertussis
Minute, encapsulated coccobacillus
Causes pertussis or whooping cough, a communicable childhood affliction
Acute respiratory syndrome
Often severe, lifethreatening complications in babies
Reservoir apparently healthy carriers
Transmission by direct contact or inhalation of aerosols
17
BordetellaPertussis
Virulence factors
Receptors that recognize and bind to ciliated respiratory epithelial
cells
Toxins that destroy and dislodge ciliated cells
Loss of ciliary mechanism leads to buildup of mucus and blockage of the
airways
Vaccine DTaP acellular vaccine contains toxoid and other Ags
18
Prevalenceofpertussis
intheUnitedStates
19
LegionellaPneumophilaand
Legionellosis
Widely distributed in water
Live in close association with amoebas
1976 epidemic of pneumonia afflicted 200 American Legion members
attending a convention in Philadelphia and killed 29
Legionnaires disease and Pontiac fever
Prevalent in males over 50
Nosocomial disease in elderly patients
Fever, cough, diarrhea, abdominal pain, pneumonia fatality rate of 330%
Azithromycin
20
21
Appearanceof
Legionella
pneumophila
EnterobacteriaceaeFamily
Enterics
Large family of small, nonsporeforming gramnegative rods
Many members inhabit soil, water, decaying matter, and are common
occupants of large bowel of animals including humans
Most frequent cause of diarrhea through enterotoxins
Enterics, along with Pseudomonas sp., account for almost 50% of
nosocomial infections
22
23
Bacteriathat
accountforthe
majorityofhospital
infections
Facultative anaerobes, grow best in air
All ferment glucose, reduce nitrates to nitrites, oxidase negative, and
catalase positive
Divided into coliforms (lactose fermenters) and noncoliforms (nonlactose
fermenters)
Enrichment, selective and differential media utilized for screening samples
for pathogens
24
137
Biochemicaltraitsforseparatingentericgenera
25
Isolationmediaforenterics
26
api,biochemicaltestingofenterics
27
AntigenicStructures
andVirulenceFactors
Complex surface antigens contribute to pathogenicity and
trigger immune response:
H flagellar Ag
K capsule and/or fimbrial Ag
O somatic or cell wall Ag all have
Endotoxin
Exotoxins
28
Antigenicstructuresingramnegative
entericrods
29
ColiformOrganismsandDiseases
30
138
EscherichiaColi:TheMostPrevalent
EntericBacillus
Most common aerobic and nonfastidious bacterium in gut
150 strains
Some have developed virulence through plasmid transfer,
others are opportunists
31
PathogenicStrainsofE.Coli
Enterotoxigenic E. coli causes severe diarrhea due to heatlabile toxin and
heatstable toxin stimulate secretion and fluid loss; also has fimbriae
Enteroinvasive E. coli causes inflammatory disease of the large intestine
Enteropathogenic E. coli linked to wasting form infantile diarrhea
Enterohemorrhagic E. coli, O157:H7 strain, causes hemorrhagic syndrome
and kidney damage
32
Escherichiacoli
Pathogenic strains frequent agents of infantile diarrhea
greatest cause of mortality among babies
Causes ~70% of travelers diarrhea
Causes 5080% UTI
Coliform count indicator of fecal contamination in water
33
RapididentificationofE.coliO157:H7
34
OtherColiforms
Clinically important mainly as opportunists
Klebsiella pneumoniae normal inhabitant of respiratory tract, has
large capsule, cause of nosocomial pneumonia, meningitis,
bacteremia, wound infections, and UTIs
Enterobacter sp. UTIs, surgical wounds
Citrobacter sp. opportunistic UTIs and bacteremia
Serratia marcescens produces a red pigment; causes pneumonia,
burn and wound infections, septicemia and meningitis
35
AcapsulestainofKlebsiellapneumoniae
36
Serratiamarcescens
37
NoncoliformLactoseNegativeEnterics
Proteus, Morganella, Providencia
Salmonella and Shigella
38
Opportunists:ProteusandItsRelatives
Proteus, Morganella, Providencia ordinarily harmless saprobes
in soil, manure, sewage, polluted water, commensals of
humans and animals
Proteus sp. swarm on surface of moist agar in a concentric pattern
Involved in UTI, wound infections, pneumonia, septicemia, and infant
diarrhea
Morganella morganii and Providencia sp. involved in similar infections
All demonstrate resistance to several antimicrobials
39
Wavelike,swarmingpatternofProteusvulgaris
40
SalmonellaandShigella
Welldeveloped virulence factors, primary pathogens, not
normal human flora
Salmonelloses and Shigelloses
Some gastrointestinal involvement and diarrhea but often affect
other systems
41
TyphoidFeverandOtherSalmonelloses
Salmonella typhi most serious pathogen of the genus;
cause of typhoid fever; human host
S. choleraesuis zoonosis of swine
S. enteritidis includes 1,700 different serotypes based on
variation on O, H, and V
i
Flagellated; survive outside the host
Resistant to chemicals bile and dyes
42
140
TyphoidFever
Bacillus enters with ingestion of fecally contaminated food or water;
occasionally spread by close personal contact; ID 1,00010,000 cells
Asymptomatic carriers; some chronic carriers shed bacilli from
gallbladder
Bacilli adhere to small intestine, cause invasive diarrhea that leads to
septicemia
Treat chronic infections with chloramphenicol or sulfatrimethoprim
2 vaccines for temporary protection
43
Prevalenceofsalmonelloses
44
45
ThePhasesOfTyphoidFever
AnimalSalmonelloses
Salmonelloses other than typhoid fever are called enteric
fevers, Salmonella food poisoning, and gastroenteritis
Usually less severe than typhoid fever but more prevalent
Caused by one of many serotypes of Salmonella enteritidis;
all zoonotic in origin but humans can become carriers
Cattle, poultry, rodents, reptiles, animal, and dairy products
Fomites contaminated with animal intestinal flora
46
ShigellaandBacillaryDysentery
Shigellosis incapacitating dysentery
S. dysenteriae, S. sonnei, S. flexneri, and S. boydii
Human parasites
Invades villus of large intestine, does not perforate intestine or invade
blood
Enters Peyers patches instigate inflammatory response; endotoxin and
exotoxins
Treatment fluid replacement and ciprofloxacin and sulfatrimethoprim
47
Theappearanceofthelargeintestinalmucosa
inShigella
48
141
TheEntericYersiniaPathogens
Yersinia enterocolitica domestic and wild animals, fish, fruits,
vegetables, and water
Bacteria enter small intestinal mucosa, some enter lymphatic and
survive in phagocytes; inflammation of ileum can mimic appendicitis
Y. pseudotuberculosis infection similar to Y. enterocolitica, more
lymph node inflammation
49
NonentericYersiniaPestisandPlague
Nonenteric
Tiny, gramnegative rod, unusual bipolar staining and
capsules
Virulence factors capsular and envelope proteins protect
against phagocytosis and foster intracellular growth
Coagulase, endotoxin, murine toxin
50
GramstainofYersiniapestis
51
YersiniaPestis
Humans develop plague through contact with wild animals
(sylvatic plague) or domestic or semidomestic animals (urban
plague) or infected humans
Found in 200 species of mammals rodents, without causing
disease
Flea vectors bacteria replicates in gut, coagulase causes
blood clotting that blocks the esophagus; flea becomes
ravenous
52
Infectioncycleof
Yersiniapestis
53
PathologyofPlague
ID 350 bacilli
Bubonic bacillus multiplies in flea bite, enters lymph, causes
necrosis and swelling called a bubo in groin or axilla
Septicemic progression to massive bacterial growth;
virulence factors cause intravascular coagulation
subcutaneous hemorrhage and purpura black plague
Pneumonic infection localized to lungs, highly contagious;
fatal without treatment
54
142
Thebubo,classicsignofbubonicplague
55
Diagnosis depends on history, symptoms, and lab findings
from aspiration of buboes
Treatment: streptomycin, tetracycline, or chloramphenicol
Killed or attenuated vaccine available
Prevention by quarantine and control of rodent population in
human habitats
56
OxidasePositiveNonentericPathogens
Pasteurella multocida
Haemophilus influenzae
H. aegyptius
H. ducreyi
H. parainfluenzae
H. aphrophilus
57
PasteurellaMultocida
Zoonotic genus; normal flora in animals
Opportunistic infections
Animal bites or scratches cause local abscess that can spread
to joints, bones, and lymph nodes
Immunocompromised are at risk for septicemia and
complications
Treatment: penicillin and tetracycline
58
Haemophilus
Tiny gramnegative pleomorphic rods
Fastidious, sensitive to drying, temperature extremes, and disinfectants
None can grow on blood agar without special techniques chocolate agar
Require hemin, NAD, or NADP
Some species are normal colonists of upper respiratory tract or vagina (H.
parainfluenzae, H. ducreyi)
Others are virulent species responsible for childhood meningitis, and
chancroid
59
Haemophilus
H. influenzae acute bacterial meningitis, epiglottitis, otitis media,
sinusitis, pneumonia, and bronchitis
Subunit vaccine Hib
H. aegyptius conjunctivitis, pink eye
H. ducreyi chancroid STD
H. parainfluenzae and H. aphrophilus normal oral and
nasopharyngeal flora; infective endocarditis
60
MeningitisintheUnitedStates
61
Acuteconjunctivitis
62
144
MinistryofHealth
Graduates
TheGramPositiveBacilliofMedicalImportance
MedicallyImportant
GramPositiveBacilli
Three general groups:
1. Endosporeformers
Bacillus, Clostridium
2. Nonendosporeformers
Listeria, Erysipelothrix
3. Irregular shaped and staining properties
Corynebacterium, Proprionibacterium, Mycobacterium,
Actinomyces, Nocardia
2
3
SporeformingBacilli
Genus Bacillus
Genus Clostridium
4
GeneralCharacteristicsofthe
GenusBacillus
Grampositive, endosporeforming, motile rods
Mostly saprobic
Aerobic and catalase positive
Versatile in degrading complex macromolecules
Source of antibiotics
Primary habitat is soil
2 species of medical importance:
Bacillus anthracis
Bacillus cereus
5
Bacillusanthracis
Large, blockshaped rods
Central spores that develop under all conditions except in the living body
Virulence factors polypeptide capsule and exotoxins
3 types of anthrax:
Cutaneous spores enter through skin, black sore eschar; least
dangerous
Pulmonary inhalation of spores
Gastrointestinal ingested spores
6
145
ControlandTreatment
Treated with penicillin, tetracycline, or ciprofloxacin
Vaccines
Live spores and toxoid to protect livestock
Purified toxoid; for high risk occupations and military personnel;
toxoid 6X over 1.5 years; annual boosters
7 8
Bacilluscereus
Common airborne and dustborne; usual methods of disinfection and
antisepsis are ineffective
Grows in foods, spores survive cooking and reheating
Ingestion of toxincontaining food causes nausea, vomiting, abdominal
cramps and diarrhea; 24 hour duration
No treatment
Increasingly reported in immunosuppressed
9
TheGenusClostridium
Grampositive, sporeforming rods
Anaerobic and catalase negative
120 species
Oval or spherical spores produced only under anaerobic conditions
Synthesize organic acids, alcohols, and exotoxins
Cause wound infections, tissue infections, and food intoxications
10
11
GasGangrene
Clostridium perfringens most frequent clostridia involved in
soft tissue and wound infections myonecrosis
Spores found in soil, human skin, intestine, and vagina
Predisposing factors surgical incisions, compound fractures,
diabetic ulcers, septic abortions, puncture wounds, gunshot
wounds
12
146
VirulenceFactors
Virulence factors
toxins
alpha toxin causes RBC rupture, edema and tissue
destruction
collagenase
hyaluronidase
DNase
13
Pathology
Not highly invasive; requires damaged and dead tissue and anaerobic
conditions
Conditions stimulate spore germination, vegetative growth and release of
exotoxins, and other virulence factors.
Fermentation of muscle carbohydrates results in the formation of gas and
further destruction of tissue.
14
15
Immediate cleansing of dirty wounds, deep wounds,
decubitus ulcers, compound fractures, and infected incisions
Debridement of disease tissue
Large doses of cephalosporin or penicillin
Hyperbaric oxygen therapy
No vaccines available
16
ClostridiumdifficileAssociated
Disease(CDAD)
Normal resident of colon, in low numbers
Causes antibioticassociated colitis
Relatively noninvasive; treatment with broadspectrum antibiotics
kills the other bacteria, allowing C. difficile to overgrow
Produces enterotoxins that damage intestines
Major cause of diarrhea in hospitals
Increasingly more common in community acquired diarrhea
17
Mild uncomplicated cases respond to fluid and electrolyte
replacement and withdrawal of antimicrobials.
Severe infections treated with oral vancomycin or
metronidazole and replacement cultures
Increased precautions to prevent spread
18
Tetanus
Clostridium tetani
Common resident of soil and GI tracts of animals
Causes tetanus or lockjaw, a neuromuscular disease
Most commonly among geriatric patients and IV drug
abusers; neonates in developing countries
19
Pathology
Spores usually enter through accidental puncture wounds,
burns, umbilical stumps, frostbite, and crushed body parts.
Anaerobic environment is ideal for vegetative cells to grow
and release toxin.
Tetanospasmin neurotoxin causes paralysis by binding to
motor nerve endings; blocking the release of
neurotransmitter for muscular contraction inhibition; muscles
contract uncontrollably
Death most often due to paralysis of respiratory muscles
20
21
Treatment aimed at deterring degree of toxemia and infection and
maintaining homeostasis
Antitoxin therapy with human tetanus immune globulin; inactivates
circulating toxin but does not counteract that which is already bound
Control infection with penicillin or tetracycline; and muscle relaxants
Vaccine available; booster needed every 10 years
22
ClostridialFoodPoisoning
Clostridium botulinum rare but severe intoxication usually
from home canned food
Clostridium perfringens mild intestinal illness; second most
common form of food poisoning worldwide
23
BotulinumFoodPoisoning
Botulism intoxication associated with inadequate food
preservation
Clostridium botulinum sporeforming anaerobe; commonly
inhabits soil and water
24
Pathogenesis
Spores are present on food when gathered and processed.
If reliable temperature and pressure are not achieved air will be
evacuated but spores will remain.
Anaerobic conditions favor spore germination and vegetative growth.
Potent toxin, botulin, is released.
Toxin is carried to neuromuscular junctions and blocks the release of
acetylcholine, necessary for muscle contraction to occur.
Double or blurred vision, difficulty swallowing, neuromuscular symptoms
25 26
InfantandWoundBotulism
Infant botulism caused by ingested spores that germinate
and release toxin; flaccid paralysis
Wound botulism spores enter wound and cause food
poisoning symptoms
27
Determine presence of toxin in food, intestinal contents or feces
Administer antitoxin; cardiac and respiratory support
Infectious botulism treated with penicillin
Practice proper methods of preserving and handling canned foods;
addition of preservatives.
28
ClostridialGastroenteritis
Clostrium perfringens
Spores contaminate food that has not been cooked
thoroughly enough to destroy spores.
Spores germinate and multiply (especially if
unrefrigerated).
When consumed, toxin is produced in the intestine;
acts on epithelial cells, acute abdominal pain,
diarrhea, and nausea
Rapid recovery
29
GramPositiveRegular
NonSporeFormingBacilli
Medically important:
Listeria monocytogenes
Erysipelothrix rhusiopathiae
30
Listeriamonocytogenes
Nonsporeforming Grampositive
Ranging from coccobacilli to long filaments
14 flagella
No capsules
Resistant to cold, heat, salt, pH extremes and bile
Virulence attributed to ability to replicate in the
cytoplasm of cells after inducing phagocytosis;
avoids humoral immune system
31 32
Primary reservoir is soil and water; animal intestines
Can contaminate foods and grow during
refrigeration
Listeriosis most cases associated with dairy
products, poultry, and meat
Often mild or subclinical in normal adults
Immunocompromised patients, fetuses and
neonates; affects brain and meninges
20% death rate
33
DiagnosisandControl
Culture requires lengthy cold enrichment process.
Rapid diagnostic tests using ELISA available
Ampicillin and trimethoprimsulfamethoxazole
Prevention pasteurization and cooking
34
Erysipelothrixrhusiopathiae
Grampositive rod widely distributed in animals and
the environment
Primary reservoir tonsils of healthy pigs
Enters through skin abrasion, multiples to produce
erysipeloid, dark red lesions
Penicillin or erythromycin
Vaccine for pigs
35
GramPositiveIrregular
NonSporeFormingBacilli
Medically important genera:
Corynebacterium
Proprionibacterium
Mycobacterium
Actinomyces
Nocardia
36
Pleomorphic; stain unevenly
20 genera; Corynebacterium, Mycobacterium, and
Nocardia greatest clinical significance
All produce catalase, possess mycolic acids, and a
unique peptidoglycan.
37
Corynbacteriumdiptheriae
Grampositive irregular bacilli
Virulence factors assist in attachment and growth.
diphtherotoxin exotoxin
2 part toxin part B binds and induces endocytosis;
part A arrests protein synthesis
38
39
Reservoir of healthy carriers; potential for diphtheria
is always present
Most cases occur in nonimmunized children living in
crowded, unsanitary conditions.
Acquired via respiratory droplets from carriers or
actively infected individuals
40
41
2 stages of disease:
1. Local infection upper respiratory tract
inflammation
sore throat, nausea, vomiting, swollen lymph nodes;
pseudomembrane formation can cause asphyxiation
2. Diptherotoxin production and toxemia
target organs primarily heart and nerves
42
DiagnosticMethods
Pseudomembrane and swelling indicative
Stains
Conditions, history
Serological assay
43
Antitoxin
Penicillin or erythromycin
Prevented by toxoid vaccine series and boosters
44
GenusProprionibacterium
Propionibacteriumacnes most common
Grampositive rods
Aerotolerant or anaerobic
Nontoxigenic
Common resident of sebaceous glands
Causes acne
45
Mycobacteria:AcidFastBacilli
M. leprae
M. avium complex
M. fortuitum
M. marinum
M. scrofulaceum
M. paratuberculosis
46
GenusMycobacterium
Grampositive irregular bacilli
Acidfast staining
Strict aerobes
Produce catalase
Possess mycolic acids and a unique type of
peptidoglycan
Do not form capsules, flagella or spores
Grow slowly
47 48
152
Tubercle bacillus
Produces no exotoxins or enzymes that contribute to
infectiousness
Virulence factors contain complex waxes and cord
factor that prevent destruction by lysosomes or
macrophages
49
EpidemiologyofTuberculosis
Predisposing factors include: inadequate nutrition,
debilitation of the immune system, poor access to
medical care, lung damage, and genetics.
Estimate 1/3
rd
of world population and 15 million in
U.S. carry tubercle bacillus; highest rate in U.S.
occurring in recent immigrants
Bacillus very resistant; transmitted by airborne
respiratory droplets
50
CourseofInfectionandDisease
Only 5% infected people develop clinical disease
Untreated, the disease progresses slowly; majority
of TB cases contained in lungs
Clinical tuberculosis divided into:
primary tuberculosis
secondary tuberculosis (reactivation or reinfection)
disseminated tuberculosis
51
PrimaryTB
Infectious dose 10 cells
Phagocytosed by alveolar macrophages and multiply
intracellularly
After 34 weeks immune system attacks, forming
tubercles, granulomas consisting of a central core
containing bacilli surrounded by WBCs tubercle
If center of tubercle breaks down into necrotic
caseous lesions, they gradually heal by calcification.
52
53
SecondaryTB
If patient doesnt recover from primary tuberculosis,
reactivation of bacilli can occur.
Tubercles expand and drain into the bronchial tubes
and upper respiratory tract.
Gradually the patient experiences more severe
symptoms.
violent coughing, greenish or bloody sputum, fever,
anorexia, weight loss, fatigue
Untreated, 60% mortality rate
54
153
ExtrapulmonaryTB
During secondary TB, bacilli disseminate to regional
lymph nodes, kidneys, long bones, genital tract,
brain, and meninges.
These complications are grave.
55
Diagnosis
1. In vivo or tuberculin testing
Mantoux test local intradermal injection of purified
protein derivative (PPD); look for red wheal to form in
4872 hours induration; established guidelines to
indicate interpretation of result based on size of
wheal and specific population factors
2. X rays
3. Direct identification of acidfast bacilli in specimen
4. Cultural isolation and biochemical testing
56
57
ManagementandPreventionofTB
624 months of at least 2 drugs from a list of 11
One pill regimen called Rifater (isoniazid, rifampin,
pyrazinamide)
Vaccine based on attenuated bacilli CalmetGuerin
strain of M. bovis used in other countries
58
Mycobacteriumleprae:
TheLeprosyBacillus
624 months of at least 2 drugs from a list of 11
One pill regimen called Rifater (isoniazid, rifampin,
pyrazinamide)
Vaccine based on attenuated bacilli CalmetGuerin
strain of M. bovis used in other countries
59
EpidemiologyandTransmissionof
Leprosy
Endemic regions throughout the world
Spread through direct inoculation from leprotics
Not highly virulent; appears that health and living
conditions influence susceptibility and the course of
the disease
May be associated with specific genetic marker
60
154
CourseofInfectionandDisease
Macrophages phagocytize the bacilli, but a weakened
macrophage or slow T cell response may not kill bacillus.
Incubation from 25 years; if untreated, bacilli grow
slowly in the skin macrophages and Schwann cells of
peripheral nerves
2 forms possible:
Tuberculoid superficial infection without skin disfigurement
which damages nerves and causes loss of pain perception
Lepromatous a deeply nodular infection that causes severe
disfigurement of the face and extremities
61
Diagnosing
Combination of symptomology, microscopic
examination of lesions, and patient history
Numbness in hands and feet, loss of heat and cold
sensitivity, muscle weakness, thickened earlobes,
chronic stuffy nose
Detection of acidfast bacilli in skin lesions, nasal
discharges, and tissue samples
62
Treatment by longterm combined therapy
Prevention requires constant surveillance of high risk
populations.
WHO sponsoring a trial vaccine
63
InfectionsbyNonTuberculosis
Mycobacteria(NTM)
M. avium complex third most common cause of
death in AIDS patients
M. kansaii pulmonary infections in adult white
males with emphysema or bronchitis
M. marinum water inhabitant; lesions develop
after scraping on swimming pool concrete
M. scrofulaceum infects cervical lymph nodes
M. paratuberculosis raw cows milk; recovered
from 65% of individuals diagnosed with Crohns
disease
64
Actinomycetes:FilamentousBacilli
Genera Actinomyces & Nocardia are nonmotile
filamentous bacteria related to mycobacteria.
May cause chronic infection of skin and soft tissues
Actinomyces israelii responsible for diseases of the
oral cavity, thoracic or intestines actinomycoses
Nocardia brasiliensis causes pulmonary disease
similar to TB.
65
155
MinistryofHealth
Graduates
CerebrospinalFluid(CSF)Culture
FormationandPhysiology
~First recognizedby Cotugno in 1764, CSF is the third major
fluid of the body.
Physiologic System
1.To supply nutrients to the nervous system
2.To remove metabolic wastes
3.To produce a mechanical barrier to cushion the brain and spinal
cord against trauma.
Meninges Layers
1.Dura mater - outer layer
2.Arachnoid mater - middle layer
3.Pia mater - inner layer
2
CSF flows through the subarachnoid space between
the arachnoid and pia mater
20 ml of fluid produced every hr in choroids plexus
and reabsorbed by arachnoid villi
3
Aimofthetest
Diagnosis of the bacteria or fungal meningitis by
microscopic examination and culture with
identification and susceptibility test of the
isolated organism
4
Infection of CSF
Common bacterial pathogen
Haemophilus influenzae Salmonella (rare)
Neisseria meningitidis Brucella (rare)
Streptococcus pneumoniae Treponema pallidum (rare)
GroupA & B streptococci Listeria monocytogenes
Gramnegativebacilli
Viruses
Enteroviruses(coxsackieviruses
A andB, echoviruses)
Herpessimplex virus
Mumpsvirus Arboviruses (togavirus,bugavirus and
other)
Parasite
Freelivingamoebae Toxoplasma (rare)
Toxoplasma gondii Strongyloides strecoralis
Entamoeba histolytica
Microbes that cause chronic meningitis
M. tuberculosis Blastomyces dermatitides
Cryptococcus neoformans Candida spp.
Coccidoides immitis Nocardia
Histoplasma capsulatum Actinomyces
Note:CSFisasterilebodyfluidanddoesnotcontainanycommensals;however,careshouldbetakennotto
contaminatethespecimenwithskinnormalfloraduringcollection
5
CSF
Examination
Microbiology
Testing
CellCount
Biochemical
Testing
glucose
proteinand
globulin
6
156
Specimencollection
Who will collect the specimen:
Only physicians.
Methods and precautions:
lumbar puncture
Ventricular puncture
CSF usually collected in three sterile tubes
Tube 1 used for chemical and serologic test
(tubes are frozen)
Tube 2 used for microbiology lab
( room temp.)
Tube 3 used for hematology (cell count)
( refrigerated)
Quantity of specimen:
Mini. 5-10 ml of CSF is recommended for culture.
7
Specimentransport
Time relapse before
processing the sample
- CSF is an emergency
specimen and should
be processed
immediately.
Storage
- Room Temperature,
Do not refrigerate.
8
Criteriaofspecimenrejection
Non sterile container and the general causes for rejection as
mislabeling, un-labeling, insufficient amount.
Specimenmore than 30 minutes.
9
CSFprocessing
VisualdetectionofEtiologicagent
10
Macroscopicinspection
Reporttheappearance:
clear,slightlyturbid,
cloudyordefinitely
purulent(lookinglike
pus),
containsblood
containsclots
Normalc.s.f.Appears
clearandcolourless.
11
InitialProcessing
centrifugation of all specimens
greater than 1 mL in volume for at
least 15 minutes at 1500x g.
The supernatant is removed to a
sterile tube, leaving approximately
0.5 mL of fluid in which to suspend
the sediment before visual
examination or culture.
The supernatant can be used for to
test for the presence of antigens or
for chemistry evaluations.
Note:if the specimen turbid it is not
necessary to make the initial processing as
mentioned above.
12
157
Microscopicexamination
13
StainingofCSF:
After thoroughly mixing the sediment heaped drop is placed on the surface on a
sterile slide. The sediment should never spread out on the slide surface, because
this increases the difficulty of finding small numbers of microorganisms, the drop of
sediment is allowed to air dry, and heated or methanol fixed and stained by gram
stain, and another slide stained by Methylene blue in parallel.
14
Gramstainof N.meningitidis inCSF
withassociatedPMNs.
N.meningitidis mayoccurintracellularlyorextracellularlyinPMNleukocytesandwill
appearasgramnegative,coffeebeanshapeddiplococci.
15
Gramstainof S.pneumoniae withWBCs
S.pneumoniae mayoccurintracellularlyorextracellularlyandwillappearasgram
positive,lanceolatediplococci,sometimesoccurringinshortchains.
16
Gramstainof H.influenzae
H.influenzae aresmall,pleomorphicgramnegativerodsorcoccobacilliwith
randomarrangements.
17
Indiainkstain:
India ink stain: the large polysaccharide capsule of cryptococcus neoformans
allows these organisms to be visualized After mixing the CSF and ink to make a
smooth suspension, a coverslip is applied to the drop and the preparation is
examined under high power magnification for characteristics encapsulated
yeast cells and by oil immersion.
18
Wetmountpreparation
Wetmount:amoebasarebestobservedbyexaminingthoroughlymixed
sedimentasawetpreparationunderphasecontrastmicroscopyorlight
microscopywiththecondenserclosedslightly.
Amoebasarerecognizedbytypicalslow,methodicalmovementinone
directionbyadvancingpseudopodia.
158
Culture
19
After vortexing the sediment and preparing smears Several drops of the
sediment shoud be inoculated to each medium. Plates should be incubated
at 37
o
C for at least 72 hours. The broth should be incubated in air for at
least 5 days.
These media will support the growth of almost all bacterial pathogens and
several fungi.
Preliminaryidentification
20
H.InfluenzaeOnChocolateagar
21
H.parainfluenzaeonlyVfactor H.influenzaebothVandXfactors
Sheep blood agar contains hemin but not NAD, Several bacterial species including
Staphylococcus aureus, produce NAD as a metabolic byproduct,therfore tiny colonies
of Haemophilus spp. May be seen growing on blood agar very close to colonies
that can produce V factor.
Satellitismphenomenon
22
Streptococcuspneumoniae
23
streptococcusagalactiae
24
159
Susceptibilitytesting
25
Postspecimenprocessing
26
Interfering factors
Patientonantibiotictherapy.
Impropersamplecollection.
Result reporting
Resultsofthemicroscopyandallpositivecultures
ofCSFarereportedimmediatelytothetreating
physician.
Turn around time
Gramstainresultisreportedwithin30minutesof
specimenreceipt
PositiveCultureresults=3 5days
NegativeCultureresults=23days
CSFprocessing
DirectdetectionofEtiologicagent
27
Latex agglutination testing
(antigen detection)
Bacteria
N. meningitidis groups A, B, C, Y and
W135
E. coli K1 antigen
H. influenzae type b
S. pneumoniae
S. agalactiae
Cryptococcus neoformans
Rapid diagnostic tests (RDTs)
RDT for meningococcal meningitis
RDT for pneumococcal meningitis
Molecular methods
28
160
MinistryofHealth
Graduates
BloodCulture
WhatisaBloodCulture?
Abloodcultureisa
laboratorytestinwhich
bloodisinjectedinto
bottleswithculture
mediatodetermine
whether
microorganismshave
invadedthepatients
bloodstream.
2
NeedforBloodCulture?
No microbiological test is more essential to the
clinician than the blood culture. The finding of
pathogenic microorganisms in a patients
bloodstream is of great importance in terms of
diagnosis, prognosis, and therapy.
L.BarthReller,Clin.Infect.Diseases,1996
3
ProofinBloodborneInfection
A clinically suspected infection is ultimately confirmed by
isolation or detection of the infectious agent. Subsequent
identification of the microorganism and antibiotic
susceptibility tests further guide effective antimicrobial
therapy.
Bloodstream infection is the most severe form of infection
and is frequently lifethreatening, and blood culture to detect
circulating microorganisms has been the diagnostic standard.
4
Diseases.
Blood is normally sterile
The presence of bacteria in the bloodstream may represent:
(1) a transient bacteremia;
(2) bacteremia secondary to infection at a primary site, such as the
lungs; or
(3) an infection of the bloodstream, known as septicemia or sepsis.
Septicemia is a serious disease characterized by
chills, fever, prostration, and the presence of
bacteria and/or their toxins in the bloodstream.
5
Depending on the clinical situation, bacteremia may be:
Transient
Comes and goes
Usually occurs after a procedural manipulation (ex. Dental
procedures)
Intermittent
Can occur from abscesses at some body site that is seeding the
blood
Continuous Bacteremia
Infective endocarditis
6
Primary Bacteremia:
blood stream bacterial invasion with no preceding or
simultaneous site of infection with the same
microorganism
Secondary Bacteremia:
isolation of a microorganism from blood as well as
other site(s)
7
Mostlikelypathogens.
8
Clinicalspecimens.
Venous blood
infants: 0.5 2 ml
children: 2 5 ml
adults: 5 10 ml
Requires aseptic technique
Multiple blood specimens should be drawn to detect
intermittent bacteremia or to confirm or rule out
contaminated cultures.
9
The general rule is to collect two or three blood specimens, each
inoculated with at least 10 to 20 mL of blood, per 24hour period.
Specimens should be collected at intervals no closer than 3 hours,
to demonstrate that the bacteremia is continuous.
Specimen collection should be done before initiation of
antimicrobial therapy. If antimicrobial therapy is to be initiated
immediately, two specimens should be collected from different
sites.
Blood collected through peripheral or indwelling central venous
catheters is often contaminated with members of the indigenous
microflora of the skin.
10
Specimenpreservation.
Transporttimeatroomtemperature
shouldbelessthan2hours.
11
Contaminants.
Duringcollection,bloodspecimensfrequently
becomecontaminatedwithmembersofthe
indigenousmicrofloraoftheskin.
Coagulasenegativestaphylococci,especially
S.epidermidis,areespeciallycommon
contaminants.
12
162
Gramstaininformation.
Gram staining of blood specimens at the time of
collection is rarely of value.
It can be helpful when bacteria are observed in
hematology smears, as in cases of
meningococcemia, Streptococcus pneumoniae
infection, or other infections in which the
concentration of bacteria in the bloodstream is very
high (10
4
bacteria/mL or higher).
13
Culturemedia.
Traditional blood culture systems
o Conventional broth method
o Biphasic methods
1st used for recovery of Brucella spp from blood.
o Lysis centrifugation methods
Modern blood culture systems
o most often are twobottle systems
o support the growth of most aerobes or anaerobes
that could be present in the blood.
14
characteristicsof
continuousmonitoringbloodculturesystems
15 16
Screeningtests.
Traditional blood culture systems:
bottles were examined by clinical microbiology laboratory
(CML) professionals at regular intervalsat least once daily
for evidence of bacterial growth, such as hemolysis, bubbles,
or turbidity.
The newer systems:
detect gas production or other metabolic activities of any
microorganisms present.
Detection techniques include infrared spectroscopy, color
change in an indicator, and pressure measurement.
17
Laboratorydiagnosis.
Positive blood cultures are:
1. Gram stained:
If bacteria are observed, a preliminary report (often telephonic)
should be sent to the clinician stating the cellular morphology,
cellular arrangement, and Gram reaction of the organism(s)
observed.
18
2. Inoculated onto appropriate media:
The types of media inoculated are most often based on the Gram
stain observations.
Anaerobic media should be inoculated if the cellular morphology
is suggestive of a particular species of anaerobe, or when only the
anaerobic bottle is positive.
When no organisms are observed on Gram stain, a routine media
is inoculated, such as blood agar, chocolate agar, and MacConkey
agar (MAC)
Some CMLs routinely inoculate media to be incubated
anaerobically.
19
3. Further workup:
depends on the type(s) of organisms that grow on these media.
It is important to keep in mind that more than one pathogen may
be present.
20
Factorsaffectingisolationofcausativeorganisms:
The possible types of bacteremia (presence of
bacteria in blood)
Specimen collection methods
Blood volumes
Number & timing of blood cultures
Previous antimicrobial therapy
Interpretation of results
Type of patient population being served by the
laboratory.
21
Safetyprecautions
Do not puncture the site twice as this may cause infection.
When injecting blood into culture bottles be careful not to
prick your fingers.
Needles should not be recapped, but discarded in a safety
container.
Follow all precautions necessary for prevention of blood
borne disease (standard precautions)
NOTE: working with blood cultures, keep the cultures within
a Biosafety Cabinet or
behind a shield, or
wear a face mask and always
wear gloves, lab coat
because blood cultures contain material from patients that
may harbor blood born pathogens. Follow all precautions
necessary for prevention of bloodborn disease (standard
precautions).
22
Intravenouscathetertips
23
Principle.
When colonization of an indwelling catheter is suspected
of being the focus of septicemia, the catheter may be
cultured to determine its status.
The number of CFU of bacteria on the catheter directly
relates to whether it is the source of infection or not. If
the isolate is identical to that of blood culture it is most
properly the source of infection.
A semi quantitative culture technique of Maki et al is
used to distinguish between low density colonization
(contamination) and significant colonization and real
infection.
164
Specimencollectionandtransport.
The skin around the catheter is
carefully disinfected with
alcohol and then iodine
preparation.
Catheter is removed.
A short section (approximately
5cm), including the area just
beneath the skin is cut off.
Catheter tip is sent to the
microbiology laboratory in
sterile container without liquid.
Tips should be cultured within
2 hrs of collection to avoid
desiccation of microorganisms.
25
Cultureprocedure.
Use sterile forceps to transfer
catheter tip from transport
container to a blood agar plate.
Using light pressure, roll
catheter tip back and forth
across agar surface at least 4
times. It is essential that the tip
has good contact with the
surface of the agar. If the tip is
bent and hard to roll, use
forceps to pick up tip, and rub
all surface onto agar.
Incubate plate overnight at
3537C in CO2 incubator
After incubation count
colonies.
26
Interpretationofculture.
A positive culture (equal to more than 15 CFU)
correlates well with the catheter tip serving as a
source of infection.
Identify and perform susceptibility testing on each
organism that produces 15 CFU
More than 2 organisms in quantities > 15 CFU
generally represent contamination of the tip.
27
Reportingresults
For < 15 CFU reported as:
no growth.
or < 15CFU without identification or susceptibility.
Report > 15 CFU, with the identification and
susceptibility pattern ,ward the report as follows:
(significant colonization > 15 CFU).
Three different species with > 15 CFU isolated, generally
represents contamination of the tip during catheter
withdrawal and reported as: 3 different species isolated
and this properly represents contamination).
165
MinistryofHealth
Graduates
Urine Culture
TheUrinarySystem
Upperurinarytract:
Pairedkidneys
Aureterforeachkidney
Lowerurinarytract:
Urinarybladder
Urethra
2
UrinaryTractInfection
(UTI)
aninfectionofoneormore
structuresintheurinary
system.
arecharacterizedasbeing
eitherUpper orLower based
onanatomiclocationofthe
infection.
3
Termsrelatingtoinfectiousdiseaseofurinarytract
4
Factorspredisposetourinarytractinfection:
Aremorecommoninwomen
Incomplete bladder emptying:
Bladder outflow obstruction
Neurological problems (e.g.multiple sclerosis, diabetic
neuropathy)
Gynaecological abnormalities (e.g.uterine prolapse)
Foreign bodies:
Urethral catheters
Ureteric stent
stone
Loss of host defences:
Atrophic urethritis and vaginitis in postmenopausal women
Diabetesmellitus
5
Symptoms of UTIs
Typical features of cystitisand urethritis include:
Abrupt onset of frequency of micturition (urination)
Scalding pain in the urethra during micturition (dysuria)
Lower back pain, abdominal pain and tenderness over bladder
Suprapubic pain during and after voiding
Intense desire to pass more urine after
micturition due to spasm of inflamed bladder (urgency)
Urine that may appear cloudy and have an unpleasant smell
Presence of blood in the urine (haematuria)
Cystitis has more acute onset and severe symptoms
Systemic symptomssuggestiveof pyelonephritis:
Fever above 38.3C
Loin pain
may be indication for hospitalization
6
166
Symptoms continued..
Prostatitis is suggestedby:
Pain in the lower back, perirectal area and testicles
High fever, chills and symptoms similar to bacterial cystitis
Inflammatory swelling of prostate, which can lead
to urethral obstruction
Urinary retention, which can cause abscess formation or s
eminal vesiculitis
7
Possiblepathogens
I BACTERIA
Grampositive
Staphylococcussaprophyticus
Haemolyticstreptococci
Gramnegative
Escherichiacoli
Proteusspecies
Klebsiella strains
*SalmonellaTyphi
*SalmonellaParatyphi
*Neisseria gonorrhoeae
*Thesespeciesarenotprimarilypathogensoftheurinarytract, butmaybefoundinurine.
AlsoMycobacteriumtuberculosis,Leptospira interrogans,Chlamydia,Mycoplasma
andCandidaspecies.
8
II PARASITES
Schistosoma haematobium,Trichomonas vaginalis,andoccasionally
Enterobius vermicularis,Wuchereria bancrofti andOnchocerca
volvulus.
Findingintestinalparasitesinurineindicatesfaecalcontamination.
9
E.coliisthecommonesturinarypathogencausing6090%of
infections.Somestrainsaremoreinvasive,e.g.capsulatedstrains
areabletoresistphagocytosis,otherstrainsaremoreadhesive.
Candida urinaryinfectionisusuallyfoundindiabeticpatientsand
thosewithimmunosuppression.
UTIscausedbyPseudomonas,Proteus,Klebsiella speciesandS.
aureus,areassociatedwithhospitalacquiredinfections,often
followingcatheterizationorgynaecologicalsurgery.
Proteus infectionsarealsoassociatedwithrenalstones.
S.saprophyticus infectionsareusuallyfoundinsexuallyactive
youngwomen.
10
ProbabilitiesofUTIincaseofnegativeurineculture
M.tuberculosisisusuallycarriedinthebloodtothe
kidneyfromanothersiteofinfection.Itisoften
suspectedinapatientwithchronicfeverwhenthere
ispyuriabuttheroutinecultureissterile.
Pyuriawithanegativeurineculturemayalsobe
foundwhenthereisinfectionwithChlamydia
trachomatis,Ureaplasma,orN.gonorrhoeae,or
whenapatienthastakenantimicrobials.
11
Commensals
Thebladderandurinarytractarenormallysterile.
Theurethrahowevermaycontainafewcommensals andalsothe
perineum(widevarietyofGrampositiveandGramnegative
organisms)whichcancontaminateurinewhenitisbeingcollected.
Withfemalepatients,theurinemaybecomecontaminatedwith
organismsfromthevagina.
Vaginalcontaminationisoftenindicatedbythepresenceof
epithelialcells(moderatetomany)andamixedbacterialflora.
Mosturinespecimenswillcontainfewerthan10*4contaminating
organismspermlprovidingtheurinehasbeencollectedwithcare
tominimizecontaminationandthespecimenisexaminedsoon
aftercollection(Why?)beforethecommensalshavehadtimeto
multiplysignificantly.
12
167
Microflorathatcolonizesurethra:
Coagulase negativestaphylococci
Viridians(exceptaerococcus uriae) &nonhemolyticstreptococci
Lactobacilli
Diphtheroids (exceptCorynebacterium urealyticum)
NonpathogenicNeisseria species
Gramnegativebacilli(Enterobacteriaceae)
Anaerobiccocci,propionibacterim spp
Anaerobicgramnegativecocci andbacilli
Commensal Mycobacteriumspp
Occasionalyeast
13
Clinicalspecimen
Thevalueoflaboratorytestingproceduresand
interpretationisdependentonthequalityofthe
specimencollected.
Preventionofcontaminationbynormalvaginal,perineal,
andanteriorurethralfloraisthemostimportant
considerationforcollectionofaclinicallyrelevanturine
specimen.
Goodpatienteducationisessential(collectionafter
instructiongiventopatientsbyhealthcareworker).
14
Clinicalspecimen
(Methodsofcollection)
Cleanvoidedmidstreamurine (MSU)
mostcommonmethodofurinecollection
moreconvenient
leasttraumatic
normalfloracancontaminatethespecimen
notsuitableinchildren
15
Adhesivebag:
Suitableforinfantorchildren
aurinarycollectionbag(plastic
bagwithanadhesivesealon
oneend)isattachedoverthe
labiaingirlsoraboy'spenisto
collectthespecimen
FollowtheBroomhalletal
method
Bytappingjustabovethe
pubiswithtwofingersplace
onsuprapubic regionafter1
houroffeed,tappingonatthe
rateof1tap/secondfora
periodof1minute,ifnot
successfultappingisrepeated
onceagain.
Thechildspontaneouslypass
theUrineandtobecollected
inasterilecontainer
16
Cathetercollection:
Inoutcatheter:shouldbe
restrictedtothosepatients
whoareunabletoproducea
midstreamsampleand
performedwithaseptic
technique.
Indwellingcatheters:
Specimensaspiratedthrough
thesoftrubberconnector
betweenthecatheter&the
collectingtubingnotfrom
catheterbag.
17
Suprapubicaspirateofurine(SPA)
Goldstandardtestforchildrenandforcasesdifficulttointerpret
18
168
Collecturineinsterilewidemouthcontainer.
Shouldbeplacedinbiohazardlabeledspecimen
transportbags.Thespecimeninonecompartment
andtherequestforminthesecond.
19
Collectioncontainers
Properlylabeledcontaineratleast
with:
Name
ID
Source
Doctor
Date/hour
20
1ml
Volumeslessthan1mlcallthewardandrequest
additionalsample.
Ifunabletoobtainadditionalsample,informthe
wardthatthespecimenwillnotbecultured.
Documentonproblemlogbookandstorespecimen
inrefrigerator(problematicspecimenstobeheldfor
1week).
21
AcceptableVolumes
Transporturinetothelaboratoryassoonaspossible
aftercollection.
Culturewithin2hoursaftercollection
Ifanydelayrefrigerateat4C&culturewithin24
hours.
22
SpecimenTransport
Dipslidetechnique:
Commerciallyavailableslide
Coveredoneachsidewithculturemedium
Thenimmersedinfreshlyvoidedurine
Senttolaboratoryforincubation35C
Semiquantitative
Suitableforgeneralpracticesthataresituateddistancefroma
laboratory
23
SpecimenTransport
Boricacid withfinalconcentration1.8%intheurine.
Itwillpreserveurinecount
Alsowhitebloodcells
Fewbacterialstrainsareinhibitedbyboricacid.
24
SpecimenTransport
169
Specimenhandlinginthelaboratory
Specimensshouldberefrigeratedimmediatelyupon
receiptinthelaboratoryunlesstheyareprocessed
atonce.
Specimenswhicharereceivedmorethan2hafter
collectionwithoutevidenceofrefrigerationshould
berefrigerated,andarequestforarepeatspecimen
shouldbetelephoned.
Wheninformationoncollectiontimeandmethodis
notprovided,sameprocedureshouldbefollowedor
commentonthefinalreportshouldbeincluded.
25
Routinecultureofurineinanybrothmedium
Cultureofcentrifugedsediment
Routinecultureforanaerobes(doneonlyon
Suprapubicaspirates).
Inoculateurineinmultipledifferentmedia
Cultureofspecimensdelayed>2hrswithout
refrigeration
Cultureoffoleyscathetertips
Direct,unstandardaizedantimicrobialsusceptibility
exceptinemergency
26
UnsuitableProcedures
LaboratoryExaminationofUrine
1. Describetheappearanceofthespecimen:
Report:
Colourofspecimen
Whetheritisclearorcloudy(turbid)
Note:
Normalfreshlypassedurineisclearandpaleyellowtoyellowdependingon
concentration.
Whenlefttostand,acloudinessmaydevelopduetotheprecipitationofurates inan
acidurineorphosphatesandcarbonatesinanalkalineurine.
Urates maygivetheurineapinkorangecolour.
27
Note:Otherchangesinthecolourofurinecanbecausedbytheingestionofcertain
foods,herbs,anddrugsespeciallyvitamins.
28
2. Examinethespecimensmicroscopically:
Urineisexaminedmicroscopicallyasawetpreparation
todetect:
Significantpyuria,i.e.WBCsinexcessof10cells/lofurine
Redcells
Casts
Yeastcells
Trichomonasvaginalismotiletrophozoites
Schistosomahaematobiumeggs
Bacteria(providingtheurineisfreshlycollected)
29
Reportthefollowing
1. Bacteria(reportonlywhenthe
urineisfreshlypassed):
Usuallyseenasrods,butsometimes
cocci orstreptococci.
Bacteriuria isusuallyaccompanied
bypyuria (puscellsinurine).
Note:Inaurinaryinfection,protein
andnitriteareoftenfoundinthe
urine.
WithE.coliinfections,theurineis
markedlyacid.
Analkalineurineisfoundwith
Proteusinfections.
Largecellularcast,puscells,redcells,and
bacteria(bacilliinbackground)inurine
sedimentasseenwiththe40objective.
Large cellular
cast
30
170
2. White blood cells (pus
cells):
Theseareround,1015min
diameter,cellsthatcontain
granules
Inurinaryinfectionstheyareoften
foundinclumps.Inurine
sediments,whitebloodcells(WBC)
areusuallyreportedas:
Few:Upto10WBCs/HPF(high
powerfield,i.e.using40objective)
Moderatenumber:1140/HPF
Many:Morethan40WBC/HPF
Urinesedimentshowingpuscells(larger
granulatedcells)andredcellsasseenwith
the40Xobjective.
31
Note:Afewpuscellsarenormallyexcretedin
urine.Pyuriaisusuallyregardedassignificant
whenmoderateormanypuscellsarepresent,
i.e.morethan10WBC/l.
Bacteriuriawithoutpyuriamayoccurin
diabetes,entericfever,bacterialendocarditis,
orwhentheurinecontainsmany
contaminatingorganisms.
Pyuriawithasterileroutineculturemaybe
foundwithrenaltuberculosis,gonococcal
urethritis,C.trachomatisinfection,and
leptospirosis,orwhenapatientwithurinary
infectionhasbeentreatedwithantimicrobials.
Pus cells
32
3. Red blood cells:
Thesearesmallerandmorerefractile thanwhitecells.
Theyhaveadefiniteoutlineandcontainnogranules.
Theyareusuallyreportedasfew,moderateormanyin
numberperhighpowerfield.
Whentheurineisisotonic,theyhavearinged
appearance.
Whentheurineishypertonic,i.e.moreconcentrated
thanthefluidinsidetheredcells,fluidwillbedrawn
outofthecellsandtheywillappearsmallerthan
normalandoftencrenated (spiky).
Whenhaematuria (redcellsinurine)isdueto
glomerulonephritis (inflammationoftheglomeruli of
thekidneys),theredcellsoftenvaryinsizeandshape
(dysmorphic).
Insicklecelldisease,sickled redcellscansometimesbe
seenintheurine.
Red cells
33
Haematuria maybefoundinurinary
schistosomiasis (usuallywithproteinuria),
bacterialinfections,acuteglomerulonephritis,
sicklecelldisease,leptospirosis,infective
endocarditis,calculi(stones)intheurinary
tract,malignancyoftheurinarytract,and
haemorrhagicconditions.
Note:Thefindingofredcellsintheurineof
womenmaybeduetomenstruation.
34
4. Casts:
Thesecanusuallybeseenwith
the10objectiveprovidedthe
condenseririsisclosed
sufficientlytogivegoodcontrast.
Theyconsistofsolidifiedprotein
andarecylindricalinshape
(Why?)becausetheyareformed
inthekidneytubules.
Hyaline cast in urine as seen
with the 40 objective.
Thefollowingcastscanbefoundinurine:
1. Hyalinecasts,whicharecolourlessandempty.
Theyareassociatedwithdamagetothe
glomerular filtermembrane.Afewmaybeseen
followingstrenuous(hard)exerciseorduring
fever.
2. Waxycasts,whicharehyalinecaststhathave
remainedinthekidneytubulesalongtime.They
arethickeranddenserthanhyalinecasts,often
appearindentedortwisted,andmaybeyellowin
colour.Theyusuallyindicatetubulardamageand
cansometimesbeseeninrenalfailure.
3. Cellularcasts,whichcontainwhitecellsorred
cells
Redcellcastsappearorangered.Theyindicate
haemorrhageintotherenaltubulesor
glomerular bleeding.
Whitecellcastsarefoundwhenthereis
inflammationofthekidneypelvisortubules.
Yellowbrownpigmentedcastsmaybeseenin
theurineofjaundicedpatients.
4. Granularcasts,whichcontainirregularsized
granulesoriginatingfromdegeneratecellsand
protein.Theyarealsoassociatedwithrenal
damage.
Different casts which
may be found in urine.
36
171
5. Epithelial cells:
Theseareeasilyseenwiththe10
objective.Theyarenucleatedand
varyinsizeandshape.
Theyareusuallyreportedasfew,
moderate,ormanyinnumberper
lowpower(10objective)field.
Itisnormaltofindafewepithelial
cellsinurine.Whenseeninlarge
numbers,however,theyusually
indicateinflammationofthe
urinarytractorvaginal
contaminationofthespecimen.
Epithelialcells,redcellsand
occasionalpuscellinurinesediment
asseenwiththe10objective.
37
6. Yeast cells:
Thesecanbedifferentiatedfromred
cellsbytheirovalshapeandsomeof
theyeastsusuallyshowsingle
budding.
Ifindoubt,runadropofdilute
aceticacidunderthecoverglass.
Redcellswillbehaemolyzed bythe
acid,butnotyeastcells.
Yeastcellsareusuallyreportedas
few,moderate,ormanyperHPF.
Theycanbeseenintheurineof
womenwithvaginalcandidiasis,and
occasionallyinspecimensfrom
diabeticsandthosewith
immunosuppression.
YeastcellsandpseudohyphaeofCandida
albicansinurinesedimentasseenwiththe
40objective
38
7. Trichomonasvaginalis:
T.vaginalis isfoundintheurineof
womenwithacutevaginitis
(occasionallyseenintheurineofmen).
Thetrichomonads arealittlelarger
thanwhitecellsandareusuallyeasily
detectedinfreshurine(Why?)because
theyaremotile.
Theymovebyflagellaandan
undulating(wavy)membrane.
YeastcellsandTrichomonasvaginalisin
urinesedimentasseenwiththe40
objective
39
8. Eggs of Schistosoma
haematobium:
Recognizedbytheirlargesize(about
145X55m)andspineatoneend.
Theurinewillcontainredcellsand
protein.
EggofSchistosomahaematobium
andredcellsinurinesedimentas
seenwiththe40objective
40
9. Others:
41
Culturetheurinespecimen
Semiquantitativemethod:
Calibratedplatinumloopthatdelivers0.001mlof
urine
Disposable,sterile,plasticcalibratedloop1l
42
172
CLEDagar(cystine lactoseelectrolytedeficientmedia)
*supportthegrowthofmanyuropathogenand
*inhibittheswarmingofproteus spp.
*differentiatebetween:
lactose(yellow)&nonlactose(blue,grey,green)organisms,
containBromothymol blueindicator.
*supportthegrowthofcertainstaphylococci,streptococci&
Candida.
Bloodagar(BAP).
MullerHintonagar(forsensitivity)
Calibrateddisposableloop0.001l
43
Materials
Wellmixedurine
Useacalibratedloopvertically
(0.001l).
InoculatethesurfaceofCLED
agarandBAP
makingastraightlinedownthe
centeroftheplate,thenaseries
ofcloseperpendicularstreaks
throughtheoriginalline.
Incubate aerobicatmosphere
at35C37Cfor24hour
44
Procedure
Culture
45 46
ExamineandreporttheculturesCLEDagarculture
Lookespeciallyforcoloniesthatcouldbe:
Escherichiacoli(performindole andbetaglucaronidase testsfor
rapididentification.
Proteusspecies.
Pseudomonasaeruginosa.
Klebsiella strains.
Staphylococcusaureus.
Staphylococcussaprophyticus.
Enterococcusfaecalis
47
Appearanceofsomeurinarypathogenson
CLEDagar
E.coli:Yellow(lactosefermenting)opaquecoloniesoftenwith
slightlydeepercolouredcentre.
Klebsiella species:Largemucoid yelloworyellowwhitecolonies.
Proteusspecies:Transluscent bluegreycolonies.
P.aeruginosa:Greencolonieswithroughperiphery(characteristic
colour).
E.faecalis:Smallyellowcolonies.
S.aureus:Deepyellowcoloniesofuniformcolour.
S.saprophyticus andothercoagulase negativestaphylococci:
Yellowtowhitecolonies.
Note:Contaminatingorganismsusuallyproduceafewcoloniesof
mixedgrowth.Mosturinaryinfectionsshowgrowthofasingle
typeoforganismalthoughmixedinfectionscanoccurespeciallyin
chronicinfectionsorfollowingcatheterization orgynaecological
surgery.
48
173
Counttheapproximatenumberofcolonies.
Estimatethenumberofbacteria,i.e.colonyformingunits
(CFU)permlofurine.Reportthebacterialcountas:
1. Lessthan 10000 organisms/ml(104/ml),notsignificant.
2. 10000100000/ml (104105/ml),doubtfulsignificance(suggest
repeatspecimen).
3. Morethan 100000/ml (105/ml),significantbacteriuria.
Example
If25E.colicoloniesarecountedandamlloopwasused,the
approximatenumberofCFUper1/500mlofurine:500x25=12500
Suchacountwouldbereportedas:10000100000E.
coli/ml
49
Reportingbacterialnumbers
Abacterialcountof10
5
organisms/mlormorefromafresh
cleancatchurinespecimen,indicatesaurinaryinfection.
Acountof10
4
10
5
/ml,couldmeaninfectionor
contamination.Arepeatspecimenisindicated.
Acountoflessthan104/mlisnearlyalwaysdueto
contamination unlesstheurinewasculturedafter
antimicrobialtreatment hadbeenstarted.
Itisimportant,however,tointerpretculturecountsinrelation
tothepatientsclinicalcondition.
UTIswithlowerculturecountsareoftenobtainedfrom
catheterizedpatients orthosewithurinaryobstruction.
50
Interpretationofbacterialcounts
Performsusceptibilitytestingonurineswithsignificant
bacteriuria,particularlyfrompatientswithahistoryof
recurringUTI.
CulturesfrompatientswithaprimaryuncomplicatedUTImay
notrequireasusceptibilitytest.
51
Antimicrobialsusceptibilitytesting
174
MinistryofHealth
Graduates
Respiratory Tract Infections
Respiratorysystemdividedintoupperandlower
tracts
RespiratoryTractInfections
2
(LRT)involve:
trachea
bronchialtree(bronchi&bronchioles)
lungtissue,orboth.
Purpose:forventilation,gasexchange
Presentingsymptomsusually:
Cough,chestpain,dyspneaandvaryingdegreesof
sputumproduction
Lowerrespiratorytractinfections:
3
Infectionsofthe(LRT)mayinvolve:
trachea(tracheitis)
bronchialtree(bronchitis,andbronchiolitis)
lungtissue(alveolitis andpneumonia),orboth.
Lowerrespiratorytractinfections
4
Presentingsymptomsusually:
Fever
Cough
Chestpain
Dyspnea
Varyingdegreesofsputumproduction
Changesonchestxrays
Respiratorydistress,mayrequiremechanicalventilation
Ptsonrisk(agingpopulation+immunocompromising
conditions).
5
Floraofrespiratorytract
NonspecificmechanismscanprotectLRTforminfection:
*Nasalhairs
*Convolutedpassages
*Mucusliningnasalturbinate's
*SecretoryIgA&nonspecificantibacterialsubstances(lysozyme).WBCs,
macrophages
*Reflexes:suchascoughing,sneezing,&swallowing.
Anyparticlesescapedfromairflowturbulence&mucociliarysweeping
activityenteralveoli,macrophagesingestthem&carrythemtothe
lymphatic's.
*inadditiontonormalfloraofnasopharynx&oropharynxhelptoprevent
colonizationoftheupperrespiratorytractbypathogenicmicroorganisms
(undercertainsituationforunknownreasonsorduetopreviousdamage
byviralinfection,lossofsomehostimmunity,orphysicaldamageto
respiratoryendothelium).
6
175
Sputum(earlymorning
sputumsamplesshouldbe
obtained)
Thisisthemostcommonly
obtainedspecimen
Noninvasivemethodsto
investigatethelower
respiratorydisease.
Sputumshouldbecollected
undersupervisionbefore
startingantibiotics.
Bloodcultureshouldalwaysbe
collected.
Sterilewidemouthcontainertightly
fittedscrewcaplid
7
Typesofspecimensreceived: EndotrachealandTracheostomysecretions
Aspiratesobtainedthrough(ETT)/tracheostomy
reflectsthecolonizingfloraofthetrachea.
8
Bronchoscope
Uptoonethirdofpatientswithbacterial
pneumoniamaybeunabletoproduceasputum
specimen,evenunderoptimalconditions
usingfiberopticBronchoscopyforobtaining
transbronchialbiopsyand
Bronchialbrush
(particularlyinpatientswithlungabscesses
orothersuspecteddeeppulmonaryinfections).
9
Bronchoalveolarlavagefluid(BAL):
Involvesinjectionof
30to50mlof
physiologicsaline
throughafiberoptic
bronchoscope,the
salineaspiratedand
submittedforsmear
preparationand
culture.(This
techniqueisusefulin
diagnosispneumonia
inintubatedpatients
undergoingventilation
10
11
SputumCollection
Approachesusedtoimprovethequalityofthespecimen
obtainedinclude:
Patientshouldbestanding,orsittinguprightinbed
Takedeepbreathtofullthelungs,andemptyinonebreath,
coughinghardanddeeplyaspossible.
Obtainingthespecimenpriortoantibiotictreatment
Rinsingthemouthpriortoexpectoration
Nofoodforonetotwohourspriortoexpectoration
12
176
Sputum>2mlshouldnotbeprocessedunlessobviouslypurulent
Only1sampleper24hrsubmitted
Scoringsystemshouldbeusedtorejectspecimenthatreoral
contamination
Inoculationoftheculturemediaimmediatelyafterthespecimenis
obtainedorimmediatelyafterprompttransporttothe
microbiologylab
13
SputumCollection Sputum
Timing
*Theoptimaltimingfor
sputumspecimencollection
isthefirstmorningsample
(accumulatedsecretions
duringsleep)
containers
*Specimensshouldbe
collectedinasterilewide
mouthcontainer or
respiratorytrapapparatus,
withoutanypreservative,
withatightlyfittedscrew
caplid.
14
Transport
Allspecimensshouldbetransportedtothe
laboratorypromptly&processedassoonaspossible
aftercollectionwithin12 hrsorrefrigeratedtillitis
processedlateronthesameday.
Prolongedstorageat4Cmaydiminishthequantity
ofmorefragileorganismsasHaemophilus.
15
Handleallsamplesusinguniversalprecautions.
Allmanipulationoflowerrespiratorytract
specimensshouldbeperformedinabiohazard
safetycabinetbecauseofthepossibilityofoccult
Tuberculosis.
16
Transport
*Excessivedelayintransportspecimensreceived>2hrs
aftercollectionwillbeprocessedwiththecomment:
(Specimendelayedintransport,interpretsresultswith
caution).
*specimensreceivedimproperlyorunlabelled.Callward
toseeifanothersamplecanbecollected,ifnot,the
nursemustcometothelaboratoryandpositively
identifythespecimenandpersonallyrelabelthe
specimen.Includeinyourfilecommentthatthe
specimenwasrelabeledbynursenameinthe
laboratory.
*leakingsputumsamplesarerejected&wardnotifiedto
recollect,ifnotpossible,processspecimeninthehood
usinggloves.
17
Suboptimalspecimens
NB:
*therequestformshouldclearlymentiontheunusual
organismstobelookedforlike:
Corynebacteriumdiphtheriae
Bordetellapertussis
Legionellaspecies,Nocardiaspecies
18
177
Inducedsputum
Patientswhoareunabletoproducesputummaybe
assistedbyrespiratorytherapytechnician.
Byallowingthepatienttobreathaerosolized
dropletsofasolutionofsodiumchloridefor10min.
suchspmayavoidtheneedforamoreinvasive
proceduressuchasbronchoscopyorneedle
aspirationinmanycases.
19
Diagnosisoftheseinfectionsfrequentlyiscomplicated
bythecontaminationofspecimenswithupper
respiratorytract(URT)secretionsduringcollection.
BecausetheorganismswhichcommonlycauseLRTare
thesameasthosewhichnormallycolonizetheURT
(throughwhichsputummustpassduringexpectoration).
Techniquesfortheexaminationofsputummustbeable
todistinguishbetweenthetwosourcesofthe
organisms;thelaboratoryshouldensurethatan
appropriatespecimenisprocessed.
Thespecimenmustbemicroscopicallyexaminedbothto
assessitsqualityandtolookfororganismsassociated
withaninflammatorycellresponse.
20
Sampleswereprocessedimmediately
Microscopicexamination:
Gramstained todeterminethepresenceof:
1. Squamousepithelialcellsindicateoropharyngeal
contamination.
2. WBCsindicatelowerrespiratorytractinfection.
3. PredominantorganismsassociatedwithPMNsto
identifythemostlikelypathogen.
Sputumplantedtoblood,chocolateand
MacConkeyagar.
21
*Important
Gramstained
Allexpectoratedsputumspecimensarescreenedfor
oropharyngealcontamination.
Selectapurulentportionofthespecimengentlyrollthe
materialevenlyonaslidethendogramstain
Examinethefilmforthepresenceof:
Squamousepithelialcells (SEC)and
Polymorphnuclearleucocytes usinglowpower(10)inatleast
2040representativesfieldsandthentaketheaverage.
Examinetheslideunderoilimmersionandreportthe
predominantanddifferentmorphologicaltypesoforganisms
seeninareasofinflammation.
22
23 24
178
Goodqualityspecimens
Quantifyno&typesofinflammatorycells
Notethepresenceofbronchialepithelialcells
ConcentrateonareaswithWBCswhenlookingfor
organisms
Determineifthereisapredominantorganism(>10per
oilimmersionfield)
*Semiquantitateandreportorganismwith
descriptive
*Ifnopredominantorganismispresent,report
mixedgrampositiveandgramnegativeflora
25
Sputumgramstained Interpretationofgramstain
26
Reportingmicrobialobservations
Type/numberoforganisms/HPF
Grampositivecocci
Gramnegativecocci
Gramnegativerod
Grampositiverod
*PF:(lowpowerfield)x10(examine1020fields)
*HPF:(highpowerfield)oilimmersion
27
None Few Moderate Many
BAC/HPF 0 15 630 >30
Rejectioncriteriaforsputum
1. Sputum:
10SECs/LPF
Note:
IfthenumberofWBCsis10timesthenumberofSECs
&thereis3to4+ofasinglemorphotypeofbacteria,
acceptthespecimenforculture.
28
Rejectioncriteriafortrachealaspirates
2. Trachealaspiratesfromadults:
10SECs/LPF
or noorganismsseen.
3. Trachealaspiratesfrompediatricpatients:
noorganismsseen.
Note:ifnoorganismsareseeninaspecimenwithnumerous4+WBCs
andcellulardebris
(PseudomonasandHaemophilluscanbemissedinsuchsmears,because
theycannotbedistinguishedamongthecellulardebris).
29
Reporting
1. Smearcontains10Squamousepithelialcellper
lowpowerfield,suggestiveofpoorquality;culture
notperformed.Pleaserecollectifclinically
indicated.
2. Smearisnegativeforbacteriaafterexaminationof
40fields;culturenotperformed.Contact
laboratoryiffurtherstudiesareclinicallyindicated.
30
179
Followup
Notifythecaregiverthatthespecimenwillnotbe
cultured.
31
Note
1. Neutropenicpatientareculturedregardlessthe
presenceofWBCs.
2. Dontrejectsputumandendotrachealaspirates
forLegionellaorAFB,orspecimensfromcystic
fibrosispatientsaspurulentcellresponsesarenot
typicallyelicited.
32
CultureMedia
Purulentportionofthesputumiscultureddirectlyonto
theplatesandthenmakeathinsmearonamicroscopic
slide
SheepBloodagar
Chocolateagar
MacConkeyagar
Sabauroud dextroseagarplate(ifanyyeastcellsor
pseudohyphae areseeninthegramstain)
*Fortranstracheal aspiratesaddanaerobicculturealso.
33
Incubation
Incubateplatesin5%CO
2
at35Cfor1824hrs, All
culturesarekeptforaminimumof2daysbefore
discardingplatesandfinalizingtheculture.
34
InterpretationofCultureMedia
Allplatesareexaminedafter24hrs.incubation,
ifthereisnogrowth,reincubateforanother24hrs
35
Lookforpredominantgrowthofa
potentiallowerrespiratorytract
pathogensinclude:
Streptococcuspneumonia
Haemophilusinfluenzae
Branhamellacatarrhalis
Betahemolyticstreptococcus
Pseudomonas,Klebsiella,andother
gramnegativebacilli
Staphaureus
Aspergillus,Yeast,fungus
Normalrespiratoryflorathatcan
beisolatedfromexpectorated
sputumwhichismixedwith
oropharyngealsecretions,consists
of:
Alphaandgamma
streptococci
Diphtheroids
Neisseriaspeciesnonpathogenic
Coagulasenegativestaph
36
180
Examineforandalwaysreport
Streptococcuspyogens
GroupBstrespstsococci inpediatricpopulation
Bordetella spp
Yerisnia pestis
Nocardia spp
Bacillusanthracis
Cryptococcusneo
Molds,notconsideredsaprophyticcontaminants
Neisssersia gonorrhoeae
Francisella tularensis
37
Correlatetheamountandtypeoforganismsgrown
onculturewiththepresenceofWBCsand
correspondingmorphotypesseenongramstain
Potentialpathogenspresentinamountslessthan
thenormalflora,shouldbeconsideredpartofthe
normalflora
*streptococcuspneumoniae
*haemophilusinfluenza
38
Reportgrowthsofpotentialpathogensinthethird
andfourthquadrants
(however,aflexibleapproachisessential,andon
occasionslownumbersoforganismsmaybe
reported).
39
ReportingResults
AIfnoneoftheaboveorganismsareisolatedreportas
(nopathogensisolated)
BIfanypathogenisolated,itisreportedina
semiquantitativemannerasfollow:
Scant growthin1
st
quadrantonly
Few growthin1
st
and2
nd
quadrant
Moderate growthin1
st
,2ndand3
rd
quadrant
Heavygrowthinall4quadrants
40
41
Usethefollowingasaguidelineforcultureswithmultiplepotential
pathogensfromsputum&endotrachealsuction:
1or2potentialpathogens,fullidentificationwithsensitivityseen
aspredominantongramstain.
3ormorepotentialpathogens+withor withoutnormalflora
reportpreliminaryidentificationwithoutsensitivitiese.g
[manynormalfloraincludingmanystreptococcuspneumonia,
haemophilus spp.,pseudomonas.etc]
3ormorecoliforms +withnormalfloradonotperform
identificationorsensitivityandreportingas
[manynormalfloraincludingmixedcoliforms].
3ormorecoliforms withoutnormalfloradonotperform
identificationandsensitivity,reportas
[Mixedcoliforms,nonormalflorapresent].
42
181
AlphastrepruleoutS.pneumonia
YeastruleoutCryptococcusneoformansonly
Saureus,gramnegativebacilli
*<normalflora,quantityandlimitID,no
susceptibility
*Addcommentthatorganismnotpredominanton
stain
IDmould,MycobacteriaorNocardiaspp
43
Transtracheal aspirates,bronchiallavage andbrush
cultures(Deeprespiratoryspecimens)
shouldbesterile,withnonormalrespiratoryflora
Howeverthespecimenmaybecontaminatedwith
oropharyngealfloraduringcollection.Ifnormalflorais
present,listorganismswithanormalflora&listthe
potentialpathogensseparately.
e.g. manynormalfloraconsistingof:
Alphahemolyticstreptococci
Coagulase negativestaphylococci
Manypseudomonasaeruginosa
Isolate,identify,andperformantibioticsensitivityonthe
predominantgrowth
44
Bronchialalveolarlavage(BAL)orbronchial
washing
Themethodologyofchoiceforthebronchoscopic
diagnosisofpneumoniaespeciallyventilator
associatedpneumonia.
45 46
Storage
Specimenshouldbeprocessedwithin2hrsof
collectionotherwiserefrigerationshouldbeutilized.
Prolongedstorageat4Cmaydiminishthe
quantitiesofthemorefastidiousandfragile
bacteria.
47
ProcessingProcedure
Centrifugethespecimenat3000rpmfor10mins.
Vortexthesedimentfor10to20seconds.
Culturethesediment on
Bloodagarplate incubateinCO
2
at3537C
Bloodagarplateincubateanaerobicallyfor48hoursat37C
ChocolateagarplateincubateinCO
2
MacConkeyagarplateincubateaerobicallyat37C
Reportaseither(nopathogenisolated)or/scantyor/moderateor/
heavygrowthof
48
182
Quantitativeexaminationof(BAL)
Principle:
Colonycountsinexcessof10*4cfu/mldiscriminate
betweencolonizationandinfection
Materials:
Bloodagarplate incubateinCO
2
at3537C
ChocolateagarplateincubateinCO
2
MacConkeyagarplateincubateaerobicallyat37C
49
ProcessingProcedure
Centrifugethespecimenat3000rpmfor10mins.
Vortexthesedimentfor10to20seconds.
Usingasterileloopselectloopful ofthesuspensionforgram
staining
Dilutetheoriginalsuspensionwith9.0mlsterilesalinetomake
1:10dilution
Usingcalibratedloop0.01ofthedilutedsampleandsteakthe
platesinthefashionofaurinecolonycount
Incubatetheplatesat3537Cforafull24hrs andread
Greaterthan10coloniesrepresents104cfu/ml
Identifyanycolonymorphptypes presentinnumbers>10*4cfu/ml
andsubmitthemforappropriatesusceptibilitytesting
50
Reporting
Reportthegramstainresultsintermsofthepresenceof
puscells,
epithelialcellsand
bacterial.
Asemiquantitativegradingsystemissufficient.
Reportwithidentificationandsensitivities,anyisolatesoccurringin
numbers>10*4cfu/ml.
Reportnegativeresultsas<10*4cfu/mlofrespiratoryorganisms
isolatedorasnopathogensisolated
*cultureofanaerobesinBALspecimensisnotroutinely
recommended.
51 52
BloodagarCO
2
ChocolateagarCO
2
MacConkeyagaraerobic
Bloodagaranaerobicsetuponlyifanaerobic
infectionissuspected,e.g.
Transtrachealaspiration,
Bronchialbrushing,
Lungabscess,and
Fibrosisoflung
53
Media
Causesofpneumoniaacquiredoutsideor
insidehospital
54
Bacterialcauses:
S.pneumoniae,
H.influenza,
S.pyogenes,
B.catarrhalis,
S.aureus,
Anaerobes,
Occasionallyaerobicgramnegativerods(kleb.).
Atypicalpneumonia
Mycoplasmapneumonia,chlamydia,legionellapneumophila.
Tuberculosis
183
Ministry of Health
Parasitology
184

Parasitology

Medical Parasitology Lab
- Slide 8 Collection of Fecal Specimen
* Urine will destroy the amoebic trophozoites and dirt will interfere with examination
- Slide 12 Preservation methods for fecal specimens
Bayers solution - This technique was found to be very useful, particularly for cyst.
- Slide 16 SAF
Need to expand out CB, IHK into actual words for the students. What is CB and IHK?
- Slide 92 Zinc Sulphate technique
* A sedimentation technique is also recommended to detect these infections.
- Slide 132 Reagents and method
* A 10 vol. (volume) H2O2 solution means that 1 volume will give 10 volumes of oxygen at NTP
on complete degradation.
- Slide 135 False Reactions
1.1- Aspirin and NSAIDs other than acetaminophen should not be taken for 7 days prior to
specimen collection to prevent possible gastrointestinal Irritation.
1.2.3- To avoid this it is best if the patient does not eat these food for at least 1 day before the
test specimen is obtained.
2- Vitamin C and iron supplements containing vitamin C should be avoided for 3 days prior to
collections, because ascorbic acid is a strong reducing agent that interferes with the peroxidase
reaction.
- Slide 136 Interpretation
Need to restrict consumption of red meat etc before doing this test.
- Slide 141 Diarrhea
The total fecal osmolality is close to the serum osmolality (290 mOsm/kg)
Stool PH: Human feces is normally 7-8
- Slide 151 Procedures
Neutral Fat Stain Procedure : 60 large orange-red droplets indicates malabsorption.
Split Fat Stain Procedure: 100 orange-red droplets measuring 675 m indicates malabsorption

185
MinistryofHealth
Graduates
MedicalParasitologyLab
1. Introduction
Sample Collection and Preservation Methods
Wet mount Preparation
Normal saline 0.85%
Iodine
BMB
2. Artifacts
3. Concentration Techniques
Modified Formal Ether Sedimentation technique
Acid Ether Sedimentation technique
4. Flotation Techniques
By using Sheathers solution
By using Sodium Chloride solution
By using Zinc Sulphate
Topics
2
5. Staining of parasites
6. Detecting of Blood Parasites
Thick and thin Blood smear
7. Counting of Helminthes Eggs in Feces
8. Chemical Tests
Fecal PH test
Testing feces for Occult Blood
Fecal fat test
Stool reducing sugar test
Topics(cont.)
3
1. To familiarize the student with the most widely
used techniques for detection of parasites.
2. To be able to identify the parasite stages.
3. To teach the student, how to deal with risk
samples.
GeneralLabObjectives
4
1. Waste residue of indigestible material (cellulose
during the previous 4 days)
2. Bile pigments and electrolyte.
3. Intestinal secretions, including mucus.
4. Leukocytes that migrate from the bloodstream
5. Epithelial cells that have been shed.
6. Bacteria and Inorganic material (1020%) chiefly
Calcium and Phosphates. Undigested and
unabsorbed food
Whatisthestoolorfeces?
5
Fecal specimen are examined for protozoa, helminthes larvae
or eggs.
The stages of protozoa found in stool samples are
trophozoites and cysts or oocysts.
The stages of helminthes usually found in the stool samples
are eggs and larvae, though whole adult worms or segment
of worms may also be seen.
Adult worms and segment of tape worms are usually visible
to naked eye, but eggs, larvae, cyst, oocyst and trophozoites
can be seen only with the microscope.
In order to see these structure, the fecal material must be
properly collected and examined.
FecalSpecimen
6
186
No technique is 100% successful in detecting parasites by
single stool examination
At least three serial stools must be examined before a patient
can be considered free from infections
Because of the intermittent passage of certain parasites, the
possibility of finding organisms is increased by examining
multiple specimens.
It is suggested that 3 different stool motions should be
examined both pretreatment and post treatment (to ensure
eradication of documented pathogenic protozoa).
NumberofSpecimensandCollectionTime
7
Because of fragile nature of many intestinal parasites, and the
need to maintain their morphology for accurate identification,
reliable microscopic diagnosis can not made unless the stool
is collected properly.
The stool specimen must be enough for satisfactory
examination of fresh feces uncontaminated by urine, dirt,
water or other body secretion such as menstrual blood.
If the sample is too small (less than the size of a walnut or
similar) or contaminated with urine, it should not be
accepted. Ask the patient to pass another specimen.
CollectionofFecalSpecimen
8
Collect the specimen in a clean dry screwcapped container
Collect the stool with a clean wooden tongue depressor or
similar object.
The container should be free from antiseptics and
disinfectant.
Random specimen: suitable for qualitative testing for blood
and microscopic examination.
Timed specimen: for quantitative fecal testing such as fecal
fat testing, because of the variability of bowel habit and the
transit time required for food to pass through the digestive
tract, so the most representative sample is a complete 3 day
collection.
CollectionofFecalSpecimen(cont.)
9
The container with the specimen should be clearly
labeled with the following:
o Patients name and patient identifier number.
o Date and time of collection.
All samples should be accompanied by a requisition
form from the physician giving relevant clinical details
and recent travel history.
Samples and forms from patient with a confirmed or
suspected diagnosis of certain infectious diseases such
as AIDS or hepatitis should be clearly labeled with
Biohazard
10
Most viable parasites are susceptible to desiccation or
temperature variation.
If time lapse between collection and observation is
considerable, i.e. more than 24 hours, it may be
necessary to add some form of preservative to feces
specimen to retain morphology.
Formed samples can be kept in a refrigerator at 4 C
for a short time, but not in incubator.
Any whole worms or segments passed should be
placed in a separate container
11
Preservation allows fecal samples to be examined after a
delay in delivery or testing.
Many methods for the preservation of stool samples and
permanent staining procedures.
The most common fixatives are:
Polyvinyl Alcohol, PVA
Merthiolate Iodine Formalin, MIF
Sodium acetate Acetic acid Formalin, SAF
Formalin.
Bayers solution* (Not a very well known technique not mentioned
anywhere else so suggest deleting this)
The preservatives used have different effect on the various
stages of the parasites.
Preservationmethodsforfecalspecimens
12
187
Formalin 4% has been used for many years as an all purpose
fixative that is appropriate for helminthes eggs and larvae and
for protozoan cyst.
The fixative has a long shelf life.
Concentration methods, like formalin ethyl acetate
concentration can be performed from the preserved stool
samples without loss of concentration abilities.
The major disadvantage of formalin is that permanent
staining procedures can't be performed from formalin
preserved stool samples.
Formalin
13
This fixative is recommended for the preservation of the
trophozoite and cyst stages of the intestinal protozoa, and
also suitable for helminthes eggs and larvae.
The preservation of the two stages of protozoa is excellent.
The PVA is a plastic resin that serves as adhesive for the stool
material.
Has a long shelf life. ( months to years ).
Concentration methods cant performed from the specimen
preserved by PVA.
PVA
14
The greatest advantage of this fixative is that a
permanent stain can be prepared from stool
specimen preserved by PVA, giving excellent result
with trichrome staining.
Specimen preserved by PVA cant be used with
immunoassay kits.
Toxic, because contain mercury compound.
PVA(cont.)
15
SAF
Good routine fixative for protozoan cyst and trophozoites, helminthes
eggs, and larvae.
Has long shelf life. ( months to years).
The preserved stool samples permits concentration techniques, most
monoclonal detection kits, and permanent staining.
Unlike the PVA, the SAF fixative has poor adhesive properties when SAF
preserved samples are used to prepare permanent stained smears. (
Mayers albumin has been recommended as an adhesive.
The combination of SAF preserved material and CB, IHK, and mod. Ziel
Neelsen provides excellent staining of protozoan where staining of SAF
preserved material with Trichrome gives poor results.
16
SAF(cont.)
Specific advantages of the use of SAF are:
SAF preserved material can be used for
concentration techniques and permanent stained
smears (CB, IHK).
SAF preserved material can be used for some
immunoassay methods.
SAF is easy to prepare and has a long shelf life.
Unlike the PVA, the SAF fixative contain no mercury
compounds. It is therefore much less toxic than PVA
17
This fixative was originally developed as
a screening procedure for intestinal parasites.
MIF combines preservation and staining for most kinds
and stages found in feces.
Its contains Merthiolate, Iodine, and Formalin.
The preserved material permits concentration
techniques.
The major disadvantages are the short shelf life ( due
to iodine) and permanent stained smears cant be
prepared from MIF preserved material.
MIF
18
188
Fixativeusedforthepreservationofstoolsamples:an
overviewoftheadvantagesanddisadvantages:
Formalin4% PVA SAF MIF
Toxicity +/ +++
(duotoHg)
+/ +/
Shelflife Long
(months)
Long
(months/years)
Long
(months/years)
Limited
Preparation Easy Difficult Easy Easy
Qualityof
fixation
Egg:++ Egg:++ Egg:++ Egg:++
Cyst:++ Cyst:+++ Cyst:++ Cyst:++
Trophs:+/ Trophs:+++ Trophs:+++ Trophs:+/
Formalin ether Possible Not possible Possible Possible
19
1. Cestodes
Wash in water to remove the mucus. Large tapeworms such as Taenia can
be washed for several hours to relax the musculature, and can then be
fixed in 10% formol saline b/w two glass slides to give flatter specimens.
2. Trematodes
These should be treated in a similar manner to cestodes, and mounted
with the ventral sucker uppermost
3. Nematodes
Adult are washed in saline to remove mucus. Worms up to about 7 cm in
length are fixed in hot(6070C) 70% alcohol, which straightens out living
worms, except those which have natural curvatures at the head or the
tail. Alternatively, they can be fixed in hot 5% formalin.
Large worms such Ascaris lumbricoides can be fixed and preserved in cold
5% formalin
Preservationofworms
20
A stool analysis is a series of tests done on a stool
(feces) sample to help diagnose certain conditions
affecting the digestive tract .
These conditions can include infection (such as from
parasites, viruses, or bacteria), poor nutrient
absorption, or cancer.
StoolAnalysis
21
Laboratory analysis includes macroscopic, microscopic
examination, chemical tests, and microbiologic tests.
The stool may be checked for color, consistency, weight
(volume), shape, odor, and the presence of mucus and
parasites stages.
The stool may be examined for hidden (occult) blood, fat,
meat fibers, bile, white blood cells, and sugars called
reducing substances.
The pH of the stool also may be measured.
A stool culture is done to find out if certain bacteria may be
causing an infection.
StoolAnalysis(cont.)
22
FecalSampleExamination
1. Macroscopic Examination
o Color
o Consistency
o Abnormal features
o Adult worm or segment
2. Microscopic Examination
o WBC/HPF
o RBC/HPF
o Mucus
o Yeast
o Cyst, trophozoite, or both
o Larvae, egg, or both
3. Chemical Examination
o Fecal pH test
o Fecal fatty acid testing
o Testing feces for Occult Blood
o Stool reducing substances testing
23
1. Color:
Brown is normal color, results from the intestinal
oxidation of stercobilinogen to urobilin.
Bright red to dark red to black stools occur when iron or
bismuth is taken or when there is intestinal hemorrhage.
Pale yellow stools indicate the biliary obstruction,
steatorrea, and also associated with diagnostic
procedures that use barium sulfate.
White stools occur when there is obstructive jaundice.
Green stool may observed in patient taking oral antibiotic,
because of oxidation of bilirubin to biliverdin.
MacroscopicExamination
24
189
2. Consistency:degreeof
moisture,willbeaguideasto
whetherthetrophozoite
stageorthecyststageof
protozoaislikelytopresent.
Formed,writeF
Soft,writeS
Loose,writeL
Watery,writeW
MacroscopicExamination(cont.)
25
3. Abnormal features:
If mucus is present writ M, and B if blood is present.
The presence of mucus coated stool is indicative for intestinal
inflammation or irritation.
4. Adult worm or segments
The feces may have adult helminthes or segments present such as
Ascaris lumbricoides, Entrobius vermicularis, or Taenia spp. gravid
segment, these can be seen by naked eye.
And frequently motile for several days and may migrate to the top of
the container.
MacroscopicExamination(cont.)
26
o Ifseveralspecimensarereceivedatthesametime;
thosecontainingbloodandmucusshouldbe
examinedfirst,followedbyliquidspecimens.These
specimensarethemostlikelytocontainamoebic
trophozoites(whichdiesoonafterbeingpassed),
andmustbeexaminedwithin1hourafterpassage.
o Formedspecimensmaybeexaminedatanytime
duringthefirstday,butshouldnotbeleft
overnight(cystmaydisintegrate).
o Excessivebulkystoolsmayindicateconditionssuch
asgiardiasis.
Notice
27
Wet mount is the simplest and easiest technique
for the examination of feces, and this method
should be performed in all laboratories at
peripheral level.
A wet mount can be prepared directly from fecal
material or from concentrated specimens.
The basic types of wet mount that should be used
for each fecal examination are normal saline
(0.85% NaCl), iodine, and buffered methylene
blue.
MicroscopicExaminationofwetmount
28
The Saline Wet Mount
Is used for the initial microscopic examination of
stool specimens.
It is employed primarily to demonstrate worms
eggs, larvae. Protozoan trophozoites, and cysts.
This type of mount can also reveal the presence
of red blood cells and white blood cells.
If the presence of amoebic trophozoites is
suspected, warm saline (37C) should be used.
MicroscopicExaminationofwetmount(cont.)
29
The Iodine Wet Mount
Is used mainly to stain glycogen and the nuclei
of cysts, if present.
Cysts can usually be specifically identified in this
mount.
Trophozoite can not be revealed by this type of
wet mount, because iodine kill trophozoite.
30
190
The Buffered Methylene Blue Wet Mount
Should be prepared each time amoebic trophozoites
are seen in a saline wet mount, or when their presence
is suspected.
BMB stains amoebic trophozoites, but not stain
amoebic cysts, flagellate trophozoites or flagellate cysts.
BMB stain is appropriate only for fresh unpreserved
specimens.
BMB stains live organism only, it isnt used on preserved
samples in which the organism have been killed
Wait for five minutes to allow the stain to penetrate the
trophozoites. It will overstrain the trophozoites in 30
minutes.
31
Formedstool:taketheportionofstoolfroman
areatoincludeinsideandoutsidepartsofthe
specimen.
Stoolwithmucus:ifmucusispresent,labela
secondslidewiththepatientsnameornumber.
Putadropofsalineontheslide,pickupasmall
portionofmucusandmixwiththesaline.
Trophozoites,ifpresent,aresometimesmore
readilyfoundinmucusthaninthesolidpartsof
thestool.
Loosewaterystool:ifmucusisnotpresent,pick
upasmallportionofthestool(anypart)andmix
withthesaline.
Notice
32
Materials and reagents:
Microscope slides.
Cover slips.
Applicator sticks.
Marker or pen for labeling.
Reagents:
Saline solution (isotonic)
Lugols iodine (1% solution)
Buffered Methylene Blue (BMB)
MakingDirectsmearMicroscopy
33
Examinetheslideon
microscope:
o 10X
o 40X
Wetmountprocedures
34
If no parasites are found:
No ova, cysts or parasites seen, and specify
whether this result was obtained by direct
examination or by a concentration method
(name method used).
Never state categorically: No parasites
If any parasites are seen, write the scientific name of the
parasite with stages
Example: Giardia lambilia cyst
ResultofExamination
35
Arifacts
36
191
Artifacts: other things, living or artificial, present in
the stool that are not parasites and could mislead
the laboratory worker.
Note: Artifacts not to be mistaken for cysts.
Definition
37
Roundoroval,
sometimeswithangular
irregularedges,contain
onelargevacuoletaking
upalmostthewhole
cell,thecompressed
cytoplasmformsa
granularringroundit.
Blastocystis
38
Blastocystis
39
Oval,oftenwithbuds,
oftencontaineccentric
clusterof36small
granules.
Somerelatedformsof
yeastarerectangular,with
averyclearovalcytoplasm
inside:arthrospores.
Yeast
40
Yeast in an iodine-stained concentrated wet mount of stool. Yeast in wet mount may be
confused for Giardia lamblia cyst.
Yeast Giardialambliacyst
Yeast
41
Roundorslightlyelongated,
withanirregularoutline.
Containrefractilecytoplasm,
clearandgranularwithtiny
vacuoles.
Nucleusindistinct,
sometimeswithastar
shapedfalsekaryosome.
Leukocytes
42
192
Puscanbeseenbythe
nakedeyeasopaque,
greyishstreaks(not
transparentlikemucus).
Underthemicroscopeit
appearsasamassofmore
orlessdegenerate
leukocytes
Pus
43
Bacteria
44
Theseareprotozoathatmaybeparasiteofmenwithout
causinganysignificantpathogeniceffects,ormaybefoundin
transitinstoolfollowingtheconsumptionofinfectedfoods.
Theyappearinstoolinaformresemblingcystcalledoocysts
orsporocysts.
Anelongatedoval,sometimestaperedatonepole.
Therethreetypes:
a. 4sporozoites(smallbananashapedrods),each
containingasmallroundnucleus,sometimesafewlarge
granulesmassedatonepole.
b. Onelargeroundgranularcell.
c. Refractilegranulescompletelyfilltheinterior.
Coccidia
45
Coccidia(cont.)
46
Coccidia(cont.)
47
Airbubbles
48
193
Oildroplet
49
Fungal sporein awet mount of stool. Such spores may beconfused for thecysts of
Entamoeba spp.
Fungalspore Entamoebahistolytica/disparcyst
Fungalspores
50
Plantfiber
51
Strongyloidesstercoralislarvae
52
Misdiagnosis can lead to improper treatment
53
Hairfiber
54
194
Maybeconfusedwithhelmintheseggs
Plantcell
55
Plantmaterialinaniodinestainedconcentratedwetmountofstool.
Thismaterialcanbeconfusedforahookwormegg
Plantcell
56
CharcotLeydencrystals
57
Epithelialcells
58
Macrophages
59
RBCs
60
195
Pollengrains
61
Pollengrains
62
Starchgranules
63
Crystals
64
Non parasitic objects may be misidentified as parasites. The
differentiation of the most common pseudoparasites is as follow:
1. Protozoan cyst: may be confused with air bubbles, fat globules or yeasts.
protozoan trophozoites and macrophages.
Trophozoites of Entamoeba histolytica/dispar must be motile and
hematophagus.
Macrophages found in cases of intestinal amoebiasis are distinguishable
from amoebic trophozoites by possessing a larger nucleus and, although
they can haematophagus, they are only motile for a very short time. Their
pseudopodia are small, blunt and granular.
Non parasiticstructurefoundinstool
65
3. Ova, their general shape, except for Entrobius, is perfectly
symmetrical, distinguishing them from various objects found in
stools.
4. Trichuris and Taenia ova may be confused with pollen grains.
5. Ascaris ova may be confused with vegetable cells, the latter
having smooth, thick walls but irregular shape.
6. Strongyloides or hookworm larvae can be confused with hair or
vegetable fibers. The latter are usually tapered at one end and
the other being blunt and with no internal structure.
Free living nematode larvae may be found in concentrates if
contaminated water is used
Cont.
66
196
7. Fasciola ova resemble vegetable cells.
8. Insect egg may be found in stools as spurious infection. Mite
eggs may be confused with hookworm eggs.
9. Dipylidium caninum eggs sacs can look similar to vegetable
cells.
10. Other structure found in stool are crystals, Charcot Leyden
are the breakdown products of eosinophil cells and may be
present in stools or sputum. Can also indicate parasitic
infection present.
11. Starch granules are sometimes seen in stool. When
undigested, they appear as concentric rings and stain blue
with iodine, when partially digested, they stain red.
Cont.
67
Concentrationtechniques
68
The microscopic examination of feces is required for
the recognition and identification of intestinal
parasites:
1. Direct Microscopy:
Advantages
Useful for the observation of motile protozoan
trophozoites.
Disadvantages
May not detect ova, cysts and larvae which are
present in scant numbers.
69
2. Concentration techniques :
Advantages
Maximizes the numbers of organisms detected which may be
too scanty to be seen by direct microscopy alone.
Worm eggs, larvae, and protozoan cysts may be recovered.
Disadvantages
Destroys trophozoite stages. Most concentration methods
destroy trophozoites stages.
Will not pick up Enterobius vermicularis as these are lost
during the process due to eggs deposited on outside of feces
not throughout the feces.
The purpose of concentrating feces is to increase possibility to
finding ova, cyst, or larvae in samples that not be able to seen by
direct microscopy.
70
1. Sedimentation method
Modified FormalinEthyl Acetate sedimentation
technique
Acid Ethyl Acetate sedimentation technique
2. Flotation method
Saturated Salt Solution technique
Sheathers Sugar Centrifugal Flotation technique
Zinc Sulphate Centrifugal Flotation technique
ConcentrationMethods
71
ModifiedFormalinEthylAcetatesedimentation
SedimentationMethods
72
Formalin Ethyl acetate method is the recommended concentration
procedures.
Most types of worm eggs (round worms, tapeworms, schistosomes, and
other fluke eggs), larvae, and protozoan cysts may be recovered by this
method.
Advantages:
1. Speed: one sample can be processed in 5 minutes.
2. Broad spectrum: it will recover most ova, cyst and larvae.
3. The morphology of most parasites is retained for easy identification.
Disadvantages:
1. Requires several pieces of apparatus which does not make it an easy.
2. The preparation contains some debris.
3. Formalin is an irritant.
4. Hymenolepis nana and Fasciola spp. do not concentrate well.
ModifiedFormalinEthylAcetateSedimentation
73
Libra
Applicator stick
Glass centrifugal tubes
Beaker
Wire sieve
Vortex or whirlimixer
Centrifuge.
Reagent:
Reagent I: 10% formalin solution in distilled water.
Reagent II: ethyl acetate.
MaterialsandMethod
74
1. Emulsify 1 gm. of feces in 7 ml of 10% formalin in a
centrifuge tube.
2. Strain the suspension through a brass wire sieve,
and collect in beaker.
3. Pour the filtrate into a 15 ml boiling tube and add 3
ml of ethyl acetate, then mix well 15 sec on vortex
or whirlimixer or 1 min by hand.
4. Transfer the ethyl acetate formalin suspension
back into the washed centrifuge tube, and
centrifuge at 3,000 rpm for 1 min.
Procedures
75
5. Loosen the fatty layer and debris at the top of the
tube with an applicator stick and invert the tube
quickly to discard the supernatant.
6. On righting the tube, a few drops only should
remain with the sediment, mix the sediment well
and transfer one drops onto a glass slide and cover
it with coverslip.
7. Scan the whole coverslip using 10x objective,
turning into 40x for confirmation of identification
of parasites.
Procedures(cont.)
76
Acid ethylacetatesedimentationtechnique
SedimentationMethods
77
Libra
Applicator stick
Glass centrifugal tubes
Beaker
Wire sieve
Vortex or whirlimixer
Centrifuge.
Reagent:
Reagent I: 15% Hydrochloric acid.
MaterialsandMethod
78
198
1. Mix thoroughly 1 gm. feces with 3 ml of 15% of
hydrochloric acid and then mix well.
2. Add and additional 56 ml of 15% HCl and mix.
3. Strain the suspension through a wire sieve into
beaker.
4. Place suspension in a glass centrifuge tube and
make up to the 10 ml with distilled water.
5. Add 4 ml of ethyl acetate, stopper the tube and
shake vigorously 20 30 sec using vortex.
Procedures
79
6. Centrifuge 23 min at 1500 rpm, the suspension
now will be layered.
7. Loosen plug of debris with applicator stick and
immediately pour off liquid.
8. Transfer one drops onto a glass slide and cover it
with coverslip.
9. Scan the whole coverslip using 10x objective,
turning into 40x for confirmation of identification
of parasites.
Procedures(cont.)
80
FlotationMethod
ConcentrationTechniques
81
1. Flotation method
Saturated Salt Solution technique
Sheathers Sugar Centrifugal Flotation technique
Zinc Sulphate Centrifugal Flotation technique
ConcentrationMethods
82
These method use the high specific gravity of a
solution to float the lighter ova and cyst. They can be
improved by centrifugation.
Advantage:
Easy to perform .
Disadvantage:
Delay in examination can result in distortion.
Larvae and some fluke eggs do not concentrate.
Frequent checking of specific gravity.
Flotationtechnique
83
SaturatedSaltSolution
FlotationTechniques
84
199
1. Boil granular sodium chloride in excess in water to produce a saturated
solution which when cooled has a specific gravity of 1.18 1.2.
2. Half fill a wide mounted flat bottomed container with the saturated
salt solution.
3. Emulsify 1gm of feces in the solution and strain it to remove the debris
from the surface.
4. Pour the filtrate into meniscus and fill it to the top with saturated salt
solution.
5. Lay a glass slide or large coverslip over the top, avoiding any bubbles
being trapped.
6. Leave for 20 min before quickly inverting the slide.
7. Scan for ova using the 10x objectives.
MaterialsandMethod
85
Advantages:
It is cheap preparation using simple apparatus.
It concentrates nematode ova well.
Disadvantages:
It doesnt concentrate cysts.
SaturatedSaltsolutiontechnique
86
Sheathers SugarCentrifugalFlotationTechnique
87
Sheathers sugar solution:
Table sugar 500gm
Distilled water 320ml
Phenol crystal ( melt in hot water bath) 6.5gm
MaterialsandMethod
88
1. Soften 1gm of feces with water to a soft.
2. Strain the aqueous suspension through a wire sieve.
3. Mix 1 part aqueous suspension with 2 part of
Sheather's sugar solution.
4. Pour into a centrifuge tube, centrifugation 1500 rpm
for 10 minutes.
5. Pour the supernatant into a meniscus and add a
sufficient solution to bring the meniscus to the top.
6. Place a coverslip and wait for 10 minutes.
7. Examine under microscope.
Procedures
89
Advantages:
Reveals most nematode eggs and protozoan cyst.
Disadvantages:
Flukes eggs and tape worm eggs are not demonstrate
well.
Also most nematode larvae are not demonstrate well.
SheathersSugarsolutiontechnique
90
200
ZincSulphatemethod
91
Advantages:
Zinc sulphate centrifugal flotation technique is useful
for the recovery of protozoan cysts and helminthes
eggs.
Disadvantages:
Large trematode eggs, some tape worm eggs, and
infertile Ascaris lumbricoides eggs are not concentrated
by this method.
ZincSulphatetechnique
92
Zinc sulphate solution with specific gravity 1.18.
Mix 330gm dry zinc sulphate in 670ml distilled water.
Use the hydrometer or densitometer to adjust specific
gravity around 1.18
MaterialsandMethod
93
StainingofParasites
94
A. Saline wet mount:
In saline wet mount, trophozoites and cyst of amoeba,
flagellates and ciliate may be seen.
Cyst will appear as round or oval, refractile structure.
Trophozoite of amoeba may be round or irregular, but
trophozoite of flagellate are usually pyriform (elongated,
pear shaped).
In freshly passed feces (the stool must not be more than
1hour old), motile trophozoites may be seen.
Motility can be very helpful in identifying species,
especially in case of flagellates.
Protozoainwetmount
95
Organism may be detected with the low power (10x)
objective, but a high power (40x) dry objective will be
necessary to identify reliable the structure as a cyst or
trophozoite.
With the high power dry objective, you can see
motility, inclusions like erythrocytes and yeast in
amoebic trophozoites, chromatoid bodies in amoebic
cyst.
Also, we can see the shape and structural detail
(sucking discs, spiral grooves, or filaments) of flagellate
trophozoites and cysts.
Continued
201
However, its necessary to regulate carefully the
microscope illumination so that the objects appear
clearly.
Too much or too little light will interfere with your
observations.
Its also necessary to focus up and down to see all the
layer of the specimen.
Remember to examine the whole coverslip area in a
systematic manner to reduce the chances of
overlooking organisms.
Continued
97
B. Iodine wet mount:
Iodine wet mount are examined for amoebic and flagellate cysts but
not trophozoites because its killed by iodine.
In the iodine wet mount, cytoplasm of the cyst will stain yellow or
light brown, and nuclei will stain dark brown.
Cyst stained with iodine can be detected with 10x objective, but they
are not refractile as in saline mount. 40x used to see the
characteristics of the cysts.
In iodine stained cysts of Entamoeba, the arrangement of peripheral
chromatin and the position of karyosome can be seen. If the
peripheral chromatin isnt present, the cyst is not an Entamoeba spp.
These peripheral chromatin bodies stain light yellow.
Sometimes, young cysts contain glycogen, this stains dark brown with
iodine.
Continued
98
Buffered Methylene Blue wet mount:
If you see amoebic trophozoites, or structures that
resemble trophozoites, you should prepare and examine
BMB mount.
After 510 minutes of staining, the trophozoite sometimes
remain motile, but often curl up in BMB preparation. For
that do not confuse curled trophozoites with cysts do not
stain with BMB solution.
In the trophozoites, the nucleus and inclusion (RBC, yeast)
will stain dark blue while the cytoplasm will stain light
blue.
Continued
99
Occasionally, some trophozoite will not stain, so you
should look for well stained organisms.
Look for peripheral nuclear granules (granules in
membrane around the nucleus), if these are present,
the trophozoite is an Entamoeba ssp. and you must
identify the species.
Continued
100
Eggsmaybeeasilydetectedandidentifiedinsaline
mounts.
Theyshouldnotbestained(stainsmayinterferewith
identification).
Mosteggsarelargeenoughtoberecognizedwiththe
lowpower(10x)objective,butafewsmalleggswill
requireahighpowerdrylens(40x)objective.
Insalinemounts,larvaemaybepresent,anditseasily
recognizedwith10xobjective.
Helminthesinwetmount
101
In saline mounts, larvae of Strongyloides stercoralis
may be seen, but Hookworm larvae are not usually
present if the sample is fresh.
If the sample is old and contain Hookworm infection,
then larvae may be present.
SO, it may be necessary to distinguish between these
two species.
Activity
Findthewaystodifferentiatebetweenspeciesinold
samples.
Continued
102
202
CountingofHelminthesEggs StollsMethod
103
The intensity of an intestinal helminthes infection may
sometimes be indicated by the concentration of its
eggs in feces.
Eggs counts can be value in epidemiological surveys.
The approximate number of eggs per gram of feces
can be calculated by using formal ether technique.
When a more accurate count is required the Stolls
method can be used.
Countinghelmintheseggsinfeces
104
1. Weigh 3 grams of feces in a screw cap container.
2. Add 42 ml of water to give (1/15) dilution of the feces.
If the feces are formed specimen, use sodium hydroxide 0.1mol/l
solution instead of water.
3. Using a rod, break up the feces and mix it with the water.
4. Cap the container and shake hard to complete in mixing.
5. Using a graduated plastic bulled pipette, or a Pasteur pipette marked to
measure the required volume, pick 150 l of the suspension and
transfer this slide.
6. Cover the slide with long coverslip if available, or two squares
coverslips.
7. Examine systematically the entire preparation, using 10x objective.
Include the count any eggs laying outside the edges of the coverslip
because these are contained in 150 l sample.
Stollstechniqueprocedures
105
8. Multiply the number of eggs counted by 100 to give the number of eggs
per gram of feces.
If the specimen isnt formed, the following additional calculation is
necessary to give the number of eggs per gram.
Fluid specimen *5
Unformed watery specimen *4
Unformed soft specimen *3
Semiformed specimen *2
9. Calculate the number of eggs per day, by multiplying the number of
eggs per gram by the total weight of 24hrs fecal specimen.
10. Calculate the number of burden worm by dividing the number of eggs
per gram on number of eggs the parasite laying per day.
Continued
106
The Center For Disease Control, Atlanta
Protozoa Helminthes
Rare 25/coverslip 25/coverslip
Few 1/510HPF 12/LPF
Moderate 12/HPFor1/ 23
HPF
1 /510HPF
Many Several/HPF Several/LPF
Interpretationofresults
107
DetectionofBloodParasites
Thick&Thin
BloodSmear
108
203
The most commonly used technique for blood examination
is stained blood films.
Geimsa stain is usually used to stain the films.
Delafilds haematoxylin stain is used for microfilariae.
Either thick or thin films may be used depending on the
circumstances.
The thick film is more sensitive in detecting parasite and
also saves time in examination.
Good film thickness can be gauged by being able to just still read
printing through the film when slide placed on a printed page.
The thin film technique cause very little distortion of the
parasite, and permits species identification when it may
not be possible in thick films, but many fields must be
examined to detect parasite when they are few in number.
BloodExamination
109
Therefore, both thick and thin films must always be
prepared when searching for plasmodia and
trypanosomes.
If a precise identification can not be made from thick film,
the thin film will be available.
Thick films should be used when searching for
microfilariae.
The most economical use of slides is achieved by making a
combination thick and thin slide. (not recommended due
to differing drying times)
However, combination films must dry thoroughly 810 hrs.
to overnight before they can be satisfactorily stained.
Slides for malaria should be stained in the same day.
Continued
110
The thin films will dry quickly and can be stained as soon as they are dry,
and examine for parasites.
If parasites are not seen in the thin film, stain the thick film using Fields
stain, and examine for parasites.
Direct wet mounts of fresh whole blood (or centrifuged blood) are usually
used for detection of microfilariae and trypanosomes, this only gives
evidence of infection and stained films are necessary for confirmation of
species present.
In areas where malaria, trypanosomes, and/or microfilariae may all
present, both wet and stained films should be prepared and examined.
If neither trypanosomes nor microfilariae occur in region, only stained
smears need to be made for detection of plasmodia.
Continued
111
For optimum staining, the thick and thin films should be
made on separate slides and different concentrations used
for staining.
When its done, good quality staining of thick film is of
primary importance. Best results are obtained if the blood
smear have dried overnight.
Fixation of thin blood film done by adding 3 drops of
methanol, or dipping it in a container of methanol for few
seconds, with prolonged fixation it may be difficult to
demonstrate Schuffners dots and Maurers dots.
To permit dehemoglobinization, thick film should not be
fixed; therefore avoid exposure to methanol or methanol
vapor
ExaminationofThick&Thinbloodsmear
112
Focus on film with 10x objective and search for microfilariae. They are
easily detected with 10x objective.
If microfilariae are present, switch to oil immersion objective and
identify the species.
Also, look for malaria parasites with oil immersion objective, at least 100
fields should be examined.
Microscopy of thick film should reveal the following features:
1. The background should be clean, free from debris, with a pale
mottled gray color derived from the lysed erythrocytes.
2. Leukocyte nuclei are stained a deep, rich purple.
3. Malaria parasite are well defined with deep red chromatin and
pale purplish blue cytoplasm.
ReadingofThickFilm
113
Focus with the 10x objective on the thin terminal end of the film where
the RBCs are in one layer, put oil drop on the slide and switch to the oil
immersion objective.
When examining fro malaria parasites and trypanosomes, at least 200
fields should be examined.
Microscopy of thin film should reveal the following features:
1. The background should be clean and free from debris; erythrocytes
are stained a pale greyish pink.
2. Neutrophil leukocytes have deep purple nuclei and well defined
granules.
3. Malaria parasite are well defined with deep red chromatin and pale
purplish blue cytoplasm.
4. Like plasmodia, the cytoplasm of trypanosomes stain blue, the
nucleus and kinetoplast stain red or purple.
ReadingofThinFilm
114
204
In thin films, look at the appearance of the parasite and the appearance
of the RBCs containing the parasites.
1. The appearance of the parasites
2. The appearance of the RBC containing the parasites:
Size & Shape: Is the parasitized cell the same size as the blood
cell without parasite or Is it enlarged?
Stippling: Is the RBC filled with pink or red staining dots?
Schuffners stippling in the ghost of the erythrocyte can some
times be seen at the edges of the film and indicate infection with
Plasmodium vivax or P. ovale,.
Maurers dots show as stippling in erythrocytes containing the
larger ring forms of Plasmodium falciparum.
Identificationofmalarialparasites
115
Thicksmear Thinsmear
LysedRBCs,manylayer FixedRBCs,singlelayer
(largevolume)
0.25l blood/100fields
(smallvolume)
0.005l blood/100fields
Goodscreeningtest
(positiveornegative)
Goodspeciesdifferentiation
Savetimeinexamination Requiresmoretimetoread
Lowdensityinfectioncanbedetected
asbloodelementsmoreconcentrate
(more sensitive)
Lowdensityinfectionscanbemissed
(lesssensitive)
Comparison
116
BloodParasite
Microfilariae
Trypanosoma
Leishmania
Plasmodium
Plasmodium
falciparum
Plasmodiumvivax
Plasmodiumovale
Plasmodiummalariae
BloodProtozoa
117
TrypanosomaTrypomastigotes
118
Leishmaniapromastigotes
119 120
205
121 122
123
P. vivax
P. ovale
P.
malariae
P.
falciparum
Ringform
124
P. vivax
P. ovale
P.
malariae
P.
falciparum
Trophozoiteform
125
P. vivax
P. ovale
P.
malariae
P.
falciparum
Schizontsform
126
206
P. vivax
P. ovale
P.
malariae
P.
falciparum
Gametocyteform
127
SpeciesDifferentiationOnThinFilms
Feature P.Falciparum P.vivax P.ovale P.malariae
EnlargedinfectedRBC + +
InfectedRBCshape round round,distorted oval,fimbriated round
StipplinginfectedRBC Maurersclefts
Schuffner's
spots
Schuffner'sdots none
Trophozoiteshape
smallring,
applique
largering,
amoeboid
largering,
compact
smallring,
compact
Chromatindot oftendouble single large single
Matureschizont
rare, 1230
merozoites
1224
merozoites
412merozoites
(scattered)
612merozoites
( rosette)
128
FecalChemicalTests
TestingforFecalOccultBlood
129
Hematemesis: bleeding into the gastrointestinal tract
may be rapid with the vomiting of blood.
Melaena: the passage of blood through the rectum.
When the bleeding is chronic with only small amounts
of blood being passed in the feces.
If the blood or its products is not recognized in the
feces, it is referred to Occult blood (hidden Blood).
FecalOccultBloodTestFOBT
130
Chemical tests to detect occult blood are based on the
principle that hemoglobin and its derivatives react in
a similar way to peroxidase enzymes (pseudo
peroxidase activity).
Chromogen such as guaiac, O toluidine, 4
aminophenazone or benzidine will be used as
indicator for oxidation reaction.
Hemoglobin and its derivatives catalyze the transfer of
oxygen from hydrogen peroxide to Guaiac, oxidation
of the colorless chromogen produces a blue color.
Hemoglobin + H
2
O
2
+ Guaiac Oxidized guaiac + H
2
O
O
2
Pseudo-peroxidase
Blue color
PrincipleoftheTest
131
Reagents:
Acetic acid 10% v/v
Alcohol 95% v/v
4aminophenazone (4aminoantipyrine)
Hydrogen Peroxide (H
2
O
2
) 10 vol. solution*
Working 4aminophenazone reagent:
The amounts given are sufficient for 1 test with positive and negative
controls.
Prepare fresh as follows:
Alcohol 95% v/v 15 ml
Acetic acid 10% v/v 1ml
4aminophenazone 0.4g
Dissolve the 4aminophenazone in alcohol solution and immediately
before use add the acetic acid. Mix well
Reagentsandmethod
132
207
1. Dispense about 7ml of distilled water into a test tube
2. Add a sample of feces about 1gm., use a glass or plastic rod to emulsify
the feces.
3. Allow the fecal particles to settle or centrifuge the emulsified specimen.
4. Take 3 completely clean tubes and label them as :
T: Patient's test.
Neg.: Negative control.
Pos.: Positive control.
5. Add into each tube as follow:
T 5ml supernatant fluid from emulsified feces.
Neg. 5ml distilled water.
Pos.: 5ml distilled water in which 5ul of whole blood has been
mixed.
Procedures
133
6. Add 5ml of working 4aminophenazone reagent on top of the fluid in
each tube .Do Not Mix
7. Add 10 drops of the 10 vols. Hydrogen peroxide solution. Do Not Mix,
allow to stand for 1 minute.
8. Look for the appearance of a blue color where 4aminophenazone
reagents meets the sample or control solutions.
Neg. control: this should show no color change.
Pos. control: this should show a positive reaction.
Color Result
Nocolorchange Negative
Paleblue Positive+
Dark blue Positive+ +
Blue black Positive+++
Continued
134
False positive:
1
1. Aspirin and antiinflammatory medications.
2. Red meat (contain Myoglobin) and fish.
3. Green vegetables (Melons), and Horseradish.
4. Menstrual and hemorrhoid contamination.
5. Some intestinal bacteria that produce peroxidase enzymes.
False negative:
2
1. Vitamin C greater than 250 mg/dl
2. Iron supplements containing vitamin C
FalseReactions
135
The commonest cause of positive occult blood tests in
tropical and other developing countries are Hookworm
infections, peptic ulcer, and bleeding from esophagus
or liver cirrhosis.
Other causes include carcinoma in gastrointestinal
tract, erosive gastritis duo to alcohol or drugs, or
swallowed blood from nosebleeds.
If the test is negative but there is high clinical
suspicion, a further two specimen should be tested
to detect bleeding which be intermittent.
Interpretation
136
Therefore, to prevent falsepositive reactions, the
sensitivity of the test must be increased.
Many commercial testing kits are available for occult
blood testing with guaiac reagent.
The kits contain guaiac impregnated filter paper, to
which the fecal specimen and hydrogen peroxide are
added.
Two or three filter paper areas are provided for
application of material taken from different areas of the
stool, and positive and negative controls are also
included.
CommercialMethods
137 138
208
iFOBT: The immunochemical fecal occult blood test,
Hemoccult ICT, is specific for the globin portion of
human hemoglobin and uses anti-human hemoglobin
antibodies.
BecauseHemoccult ICT isspecific for humanbloodin
feces, it doesnot requiredietaryor drugrestrictions.
It is moresensitiveto lower GI bleeding that couldbe
an indicator of colon cancer or other gastrointestinal
disease.
Can be used for patients who are taking aspirin and
other anti-inflammatorymedications.
Continue...
139
FecalChemicalTests
FecalFatTesting
&
ReducingSugars
140
Diarrhea is defined as an increase in daily stool weight above 200 with
increased liquidity and frequency of more than three times per day.
Diarrhea classification can be based on four factors: duration of the
illness, mechanism, severity, and stool characteristics.
Diarrhea lasting less than 4 weeks is defined as acute, and diarrhea
persisting for more than 4 weeks is termed chronic diarrhea.
The major mechanisms of diarrhea are secretory, osmotic, and altered
motility.
The laboratory tests used to differentiate these mechanisms are fecal
electrolytes (fecal sodium, fecal potassium), fecal osmolality, and stool
pH.
Diarrhea
141
Secretory Diarrhea: Bacterial, viral, and protozoan
infections produce increased secretion of water and
electrolytes, which override the reabsorptive ability of
the large intestine
Osmotic Diarrhea: Incomplete breakdown or
reabsorption of food presents increased fecal material to
the large intestine, resulting in the retention of water
and electrolytes in the large intestine which in turn
results in excessive watery stool.
Altered Motility: Altered motility describes conditions of
enhanced motility (hypermotility) or slow motility
(constipation).
Continued
142
Malabsorption is defined as the impaired absorption of
nutrients by the intestine.
Maldigestion is defined as the impaired digestion of
food.
Maldigestion and malabsorption contribute to osmatic
diarrhea.
Specific nutrient absorptive disorders:
Carbohydrate Malabsorption
Fat malabsorption/Maldigestion
Protein Malabsorption
Mineral and Vitamin Malabsorption
Malabsorption&Maldigestion
143
Investigations of Carbohydrate Malabsorption:
D xylose Test
Lactose Intolerance
Stool Reducing Sugar
Investigations of Fat Malabsorption:
Fecal Fat Test
Investigations of Protein Malabsorption:
Albumin Level

1
antitrypsin
Investigations of Mineral and Vitamins Malabsorption:
Stool Electrolyte (Na
+
, K
+
)
InvestigationsofMalabsorption
144
209
Stool Reducing Sugar
This test measures unabsorbed sugars in stool. It is used
to evaluate the body's ability to digest carbohydrates, or
to absorb nutrients from food and drinks.
Testing for fecal reducing substances detects congenital
disaccharidase deficiencies as well as enzyme
deficiencies due to nonspecific mucosal injury.
Fecal carbohydrate testing is most valuable in assessing
cases of infant diarrhea and may be accompanied by a
pH determination.
The copper reduction test is performed using a Clinitest
tablet and one part stool emulsified in two parts water .
InvestigationofCarbohydrateMalabsorption
145
A result of 0.5 g/dL is considered indicative of
carbohydrate intolerance.
The Clinitest on stools can distinguish between diarrhea
caused by abnormal excretion of reducing sugars and
those caused by various viruses and parasites.
Sucrose is not detected by the Clinitest method because it
is not a reducing sugar.
A positive result would be followed by more specific serum
carbohydrate tolerance tests, the most common being the
Dxylose test for malabsorption and the lactose tolerance
test for maldigestion.
Continued
146
The Fecal Reducing Substances test is performed in a
laboratory, on a sample of stool as small as 5 grams.
Unfortunately this sample needs to be delivered to the
laboratory ASAP, preferably within 1 hour.
This is because lactose (or other sugars) in the stool will
normally be broken down by chemical processes within
24 hours after the specimen is produced
Adults and Children :
Normal: = 0.25 g/dl
Suspicious: 0.250.5 g/dl
Abnormal: > 0.5 g/dl
Continued
147
Steatorrhea: is the presence of excess fat in feces, Stools
may also float due to excess lipid, have an oily appearance
and be especially foulsmelling.
There is increased fat excretion, which can be measured by
determining the fecal fat level.
Possible biological causes can be lack of bile acids, defects
in pancreatic enzymes maldigestion, and defective
mucosal cells malabsorption.
The absence of bile acids will cause the feces to turn gray
or pale.
Quantitative fecal fat analysis is used as a confirmatory test
for steatorrhea
InvestigationofFatMalabsorption
148
Fecal fat testing can be done by either qualitative
microscopy method or quantitative methods.
1. Qualitative Microscopy Method:
Is the simplest form of the fecal fat test, a random
fecal specimen is submitted to the hospital laboratory
and examined under a microscope after staining with
a Sudan III dye. Visible amounts of fat indicate some
degree of fat malabsorption.
The staining procedure consists of two parts, the
neutral fat stain and the split fat stain.
InvestigationofFatMalabsorption
149
Neutral fats stain are readily stained by Sudan III and appear as
large orangered droplets, often located near the edge of the
coverslip.
Observation of more than 60 large orangered droplets/HPF can be
indicative of steatorrhea.
The split fat stain representing total fat content can provide a
better indication.
The breakdown of neutral fats by bacterial lipase and the
spontaneous hydrolysis of neutral fats may lower the neutral fat
count.
Observation of more than 100 large orangered droplets/HPF can
be indicative of steatorrhea
This also precludes the comparison of the two slide tests to
determine whether maldigestion or malabsorption is causing
steatorrhea
QualitativeMicroscopyFatTesting
150
210
1. Homogenize one part stool
with two parts water.
2. Mix emulsified stool with
one drop 95% ethyl alcohol
on slide.
3. Add two drops saturated
Sudan III in 95% ethanol.
4. Mix and coverslip.
5. Examine under high power
6. Count orange droplets per
highpower field
Neutral Fat Stain Procedure Split Fat Stain Procedure
1. Mixemulsifiedstoolwith
onedropof36%aceticacid.
2. Addtwodropssaturated
SudanIII.
3. Mixandcoverslip.
4. Heatgentlyalmosttoboiling.
5. Examineunderhighpower.
6. Countandmeasurethe
orangedropletsperhigh
powerfield
Procedures
151
Quantitative fecal fat analysis is used as a confirmatory test for
steatorrhea.
Quantitative fecal analysis requires the collection of at least a 3day
specimen.
The patient must also maintain a regulated intake of fat (100 g/dl) prior to
and during the collection period.
Refrigerating the specimen prevents any bacterial degradation.
The method routinely used for fecal fat measurement is the Van de
Kamer titration, although gravimetric methods are available.
Fecal lipids are converted to fatty acids and titrated to a neutral endpoint
with sodium hydroxide.
QuantitativeFatTesting
152
The fat content is reported as grams of fat or the
coefficient of fat retention per 24 hours.
The coefficient of fat =
Normal values based on a 100 g/dl intake are 1 to 6 g/dl
or a coefficient of fat retention of at least 95%.
Although the Van de Kamer titration is the gold standard
for fecal fat, the acid Steatocrit is a rapid test to estimate
the amount of fat excretion.
It is similar to the microhematocrit test.
( Dietary Fat Fecal Fat )
Dietary Fat
X 100
Continued
153
The acid steatocrit is a reliable tool to monitor a patients
response to therapy and screen for steatorrhea in pediatric
populations.
Acid steatocrit % =
An acid steatocrit value less than 31% was considered normal
while a value greater than 31% indicated steatorrhea in
adults.
Acid steatocrit is higher in infants and droppped with age . An
acid steatocrit of less than 10% is indicative of steatorrhea in
children.
Fatty layer length in cm
(Fatty layer length in cm + solid layer
length)
X100
Continued
154
Calculate the fecal fat in grams per 24 hours.
In adult:
Fecal fat in g/24 hrs.= [0.45 X acid steatocrit %] 0.43
In children up to 15 years:
Fecal fat in g/24 hrs.= [0.1939 X acid steatocrit %] 0.2174
Continued
155
211
MinistryofHealth
Graduates
TheParasitesofMedicalImportance
Protozoa
The study of eucaryotic parasites, protozoa and
helminths
Cause 20% of all infectious diseases
Less prevalent in industrialized countries;
increasingly common in AIDS patients
Parasitology
2
Singlecelled, animallike microbes, most having
some form of motility
Estimated 100,000 species, approximately 25 are
important pathogens
Life cycles vary
Most propagate by simple asexual cell division of the
active feeding cell (trophozoite).
Many undergo formation of a cyst.
Others have a complex life cycle that includes asexual
and sexual phases.
TypicalProtozoanPathogens
3 4
InfectiveAmebas
5
Alternates between a large trophozoite, motile by
means of pseudopods and a smaller nonmotile cyst
Trophozoite has a large nucleus and lacks most other
organelles.
Humans are the primary hosts.
Ingested
Carried by 10% of world population
EntamoebahistolyticaandAmebiasis
6
212
7
Cysts are swallowed and arrive at the small intestine;
alkaline pH and digestive juices stimulate cysts to
release 4 trophozoites.
Trophozoites attach, multiply, actively move about
and feed.
Asymptomatic in 90% of patients
Ameba may secrete enzymes that dissolve tissues
and penetrate deeper layers of the mucosa.
Causing dysentery, abdominal pain, fever, diarrhea
and weight loss
Entamoebahistolytica
8
Lifethreatening manifestations are: hemorrhage,
perforation, appendicitis, and tumorlike growths,
amebomas.
May invade liver and lung
Severe forms of disease result in 10% fatality rate.
Effective drugs are iodoquinol, metronidazole, and
chloroquine
Entamoebahistolytica
9
Caused by Naegleria fowleri and Acanthamoeba
Ordinarily inhabit standing water
Primary acute meningoencephalitis is acquired
though nasal contact with water or traumatic eye
damage.
Infiltration of brain is usually fatal.
AmebicInfectionsoftheBrain
10
An occupant of the intestines of domestic animals
such as pigs and cattle
Acquired by ingesting cystcontaining food or water
Trophozoite erodes intestine and elicits intestinal
symptoms.
Healthy humans are resistant.
Rarely penetrates intestine or enters blood
Treatment tetracycline, iodoquinol, nitrimidazine
or metronidazole
AnIntestinalCiliate:Balantidiumcoli
11 12
TheFlagellates
13
Small, pearshaped
4 anterior flagella and an undulating membrane
Exist only in trophozoite form
3 infect humans:
T. vaginalis
T. tenax
T. hominis
Trichomonads:Trichomonas species
14
15
Causes an STD called trichomoniasis
Reservoir is human urogenital tract
50% of infected are asymptomatic.
Strict parasite, cannot survive long outside of host
3 million cases yearly, a top STD
Female symptoms foulsmelling, greentoyellow
discharge; vulvitis; cervicitis; urinary frequency and
pain
Male symptoms urethritis, thin, milky discharge,
occasionally prostate infection
Metronidazole
Trichomonasvaginalis
16
Pathogenic flagellate
Unique symmetrical heart shape with concave
ventral surface that acts like a suction cup
Cysts are small, compact, and multinucleate.
Reservoirs include beavers, cattle, coyotes, cats, and
humans.
Cysts can survive for 2 months in environment.
Usually ingested with water and food
ID 10 to 100 cysts
GiardialambliaandGiardiasis
17 18
Cysts enter duodenum, germinate, travel to jejunum
to feed and multiply
Causes giardiasis diarrhea, abdominal pain
Diagnosis is difficult because organism is shed in
feces intermittently.
Treatment: quinacrine or metronidazole
Agent is killed by boiling, ozone, and iodine
19
Obligate parasites that live in blood and tissues of
human host
Cause lifethreatening and debilitating zoonoses
Spread in specific tropical regions by bloodsucking
insects that serve as intermediate hosts
Have complicated life cycles and undergo
morphological changes
Categorized according to cellular and infective stages
Hemoflagellates:VectorBorneBloodParasites
20
21
Distinguished by their infective stage;
trypomastigote is an elongate, spindleshaped cell
with tapered ends, eellike motility
2 types of trypanosomiasis:
T. brucei African sleeping sickness
T. cruzi Chagas disease endemic to
Central and South America
TrypanosomaspeciesandTropanosomiasis
22
Spread by tsetse flies
Harbored by reservoir mammals
Two variants of disease caused by 2 subspecies:
T.b.gambiense Gambian strain; West Africa
T.b. rhodesiense Rhodesian strain; East Africa
Biting of fly inoculates skin with trypomastigotes,
which multiplies in blood and damages spleen,
lymph nodes and brain.
Trypanosoma brucei andAfricanSleeping
Sickness
23
Chronic disease symptoms are sleep disturbances,
tremors, paralysis and coma.
Trypanosomes are readily demonstrated in blood,
spinal fluid or lymph nodes.
Treatment before neurological involvement
melarsoprol, eflornithine
Control involves eliminating tsetse fly.
24
25
Causes Chagas disease
Reduviid bug (kissing bug) is the vector.
Infection occurs when bug feces is inoculated into a
cutaneous portal.
Local lesion, fever, and swelling of lymph nodes,
spleen, and liver
Heart muscle and large intestine harbor masses of
amastigotes.
Chronic inflammation occurs in the organs
(especially heart and brain).
Treatment nifurtimox and benzonidazole
Trypanosomacruzi
26
27
Leishmaniasis zoonosis transmitted among
mammalian hosts by female sand flies that require a
blood meal to produce eggs
Endemic to equatorial regions
Promastigotes are injected with sand fly bite,
convert to amastigote and multiply; if macrophage is
fixed the infection is localized; systemic if
macrophage migrates.
LeishmaniaspeciesandLeishmaniasis
28
29
Cutaneousoriental sore, Baghdad boil localized
ulcerated sore
Espunda skin and mucous membrane infection of
the head; chronic infection
Systemicvisceral high intermittent fever; weight
loss, enlarged spleen, liver, and lymph nodes
Kala azar is the most severe and fatal form if untreated.
30
216
Sporozoans
Lack locomotor organelles in the trophozoite state
Alternate between sexual and asexual phases and
between different animal hosts
Most form specialized infective bodies that are
transmitted by arthropod vectors, food, water, or
other means.
Plasmodium
Toxoplasma
Cryptosporidium
Apicomplexanparasites
31
Dominant protozoan disease
Obligate intracellular sporozoan
4 species: P. malariae, P. vivax,
P. falciparum and P. ovale
Female Anopheles mosquito is the primary vector;
blood transfusions, mother to fetus
300500 million new cases each year
2 million deaths each year
Plasmodium:TheAgentofMalaria
32
2 distinct phases of malarial parasite development:
asexual phase human host
Infected female mosquito injects asexual sporozoite
which localizes in liver; it then undergoes schizogony
generating 2,00040,000 merozoites which enter
circulation in 516 days depending on species.
Merozoites attach to and enter red blood cells, convert
to trophozoites and multiply; red cell bursts releasing
merozoites that differentiate into gametes.
33
Sexual phase mosquito host
Mosquito draws infected RBCs; gametes fertilize
forming diploid cell which forms sporozoites in
stomach.
Sporozoites lodge in salivary glands; available to infect
human host
34
35
Symptoms include episodes of chillsfeversweating,
anemia, and organ enlargement.
Symptoms occur at 4872 hour intervals as RBCs
rupture; interval depends on species.
P. falciparum most malignant type; highest death
rate in children
Diagnosis by presence of trophozoite in RBCs,
symptoms
Increasing drug resistance
Therapy is chloroquine, quinine, or primaquine.
Plasmodium
36
217
Zoonotic in domestic animals and birds
CoccidianParasites
37
Intracelllular apicomplexan parasite with extensive
distribution
Lives naturally in cats that harbor oocysts in the GI
tract
Acquired by ingesting raw meats or substances
contaminated by cat feces
Most cases of toxoplasmosis go unnoticed except in
fetus and AIDS patients who can suffer brain and
heart damage.
Treatment: pyrimethamine and sulfadiazine
ToxoplasmagondiiandToxoplasmosis
38
39
Sarcocystis parasites of cattle, swine, and sheep
Domestic animals are intermediate hosts; they pick
up infective cysts while grazing on grass
contaminated with human feces.
Humans are infected when the meat is consumed.
Symptoms include diarrhea, nausea, and abdominal
pain.
No specific treatment
Sarcocystis andSarcocystosis
40
An intestinal pathogen
Infects a variety of mammals, birds, and reptiles
Exists in tissue and oocyst phases
1990s 370,000 cases in Milwaukee, WI due to
contaminated water; filtration required for removal
Ingestion of oocysts which give rise to sporozoites
that penetrate intestinal cells
Causes gastroenteritis, headache, sweating,
vomiting, abdominal cramps, diarrhea
AIDS patients may suffer chronic persistent diarrhea.
No effective drugs
Cryptosporidium:ANewlyRecognized
IntestinalPathogen
41
Intracellular intestinal parasite with oocyst stage
Transmitted in fecally contaminated food or drink
Infection usually asymptomatic or selflimited
Symptoms include malaise, nausea and vomiting,
diarrhea, fatty stools, abdominal cramping, and
weight loss.
Treat with sulfadiazine and pyrimethamine, when
required
IsosporabelliandCoccidiosis
42
Emerging protozoan pathogen; causes cyclosporiasis
Oralfecal transmission; fresh produce and water
Oocysts enter small intestine and invade the
mucosa.
Symptoms of watery diarrhea, stomach cramps,
bloating, fever, muscles aches
Diagnosis can be complicated.
Treatment: trimethoprim and sulfamethoxazole
CyclosporacayetanensisandCyclosporiasis
43
First protozoan found to cause a disease redwater
fever of cattle
First protozoan found to be associated with a vector
tick
Human babesiosis relatively rare zoonosis
Associated with infected rodents
Infection resembles malaria.
BabesiaspeciesandBabesiosis
44
219
MinistryofHealth
Graduates
TheParasitesofMedicalImportance
Helminth
The study of eucaryotic parasites, protozoa and
helminths
Cause 20% of all infectious diseases
Less prevalent in industrialized countries;
increasingly common in AIDS patients
Parasitology
2
Adults are large, multicellular animals with
specialized tissues and organs.
Adult worms mate and produce fertilized eggs that
hatch; larvae then mature in several stages to adults.
The sexes may separate or are hermaphroditic.
Adults live in the definitive host.
Eggs and larvae may develop in the same host, the
external environment of the intermediate host.
A transport host experiences no parasitic
development.
Four basic patterns of life and transmission
ASurveyofHelminthParasites
3 4
Pathology arises from worms feeding on and
migrating through tissues, accumulation of worms
and worm products.
Diagnosis based on blood cell count (eosinophilia),
serological tests; eggs, larvae, or adult worms in
feces; sputum, urine, blood, or tissue biopsies.
Antihelminthic drugs suppress a helminthic
metabolic process that differs from the human
process, inhibit the worms movement, prevent it
from holding position, and act locally in the
intestine.
Helminths
5 6
220
Most abundant animal groups; 50 species that affect
humans
Enlongated, cylindrical worms with protective
cuticles, circular muscles, a complete digestive tract,
and separate sexes
Ascaris lumbricoides, Trichuris trichiura, Enterobius
vermicularis, hookworms, Strongyloides stercoralis,
Trichinella spiralis, filarial worms
Nematode(Roundworm)Infestations
7
A large intestinal roundworm
Most cases in the U.S. occur in the southeastern states
Indigenous to humans
Ascaris spends its larval and adult stages in humans;
release embryonic eggs in feces, and are spread to other
humans; food, drink, or contaminated objects
Ingested eggs hatch into larvae and burrow through the
intestine into circulation and travel to the lungs and
pharynx and are swallowed.
Adult worms complete cycle in intestines and reproduce
200,000 eggs/day.
Ascarislumbricoides
8
Worms retain motility, do not attach.
Severe inflammatory reactions mark the migratory
route.
Allergic reactions can occur.
Heavy worm loads can retard physical and mental
development.
Ascarislumbricoides
9
Whipworm
Humans sole host
Trichuriasis has its highest incidence in the tropics.
Eggs hatch in intestines, larvae attach, penetrate the
outer wall and develop into adults.
Females lay 3,0005,000 eggs daily.
Worms can pierce capillaries, cause localized
hemorrhage, and allow bacteria to leave intestine.
Heavy infestations can cause dysentery, rectal
prolapse can be fatal in children.
TrichuristrichiuraandWhipwormInfection
10
Pinworm or seatworm
Enterobiasis most common worm disease of children
in temperate zones
Eggs are picked up from surroundings and
swallowed.
After hatching in the small intestine, they develop
into adults.
Anal itching occurs when mature females emerge
from intestine to release eggs.
Selfinoculation is common.
Tape test used to pick up eggs in anal area
Enterobius vermicularis andPinwormInfection
11
Characteristic curved ends and hooked mouths
Necator americanus and Ancylostoma duodenale
Humans shed eggs in feces, which hatch into filariform
larvae and burrow into the skin of the lower legs.
Larvae travel from blood to lungs, proceed up bronchi
and throat and are swallowed.
Worms mature and reproduce in small intestine and
complete, the cycle.
May cause pneumonia, nausea, vomiting, cramps and
bloody diarrhea
Blood loss is significant anemia.
Hookworms
12
13
Threadworm
Tiny roundworms completes life cycle in humans or
moist soil.
Larvae penetrate skin and migrate to lungs, are
swallowed and complete development in the
intestine.
Can reinfect the same host without leaving the body
Heavy worm loads can cause pneumonitis and
eosinophilia, bloody diarrhea, liver enlargement,
bowel obstruction and malabsorption.
Strongyloides stercoralis andStrongyloidiasis
14
Life cycle entirely within mammalian host
Acquired from eating undercooked pork or bear
meat
Larvae migrate from intestine to blood vessels,
muscle, heart, and brain, where it forms cysts
First symptoms flulike, diarrhea
Second symptoms muscle and joint pain, shortness
of breath, pronounced eosinophilia
No cure after larva have encysted
TrichinellaspiralisandTrichinosis
15 16
Complete their life cycle in human blood,
lymphatics, or skin
Filarial worms; elongate, filamentous bodies, spread
by biting arthropods
Cause chronic, deforming disease
Wuchereria bancrofti elephantiasis
Onchocerca volvulus river blindness
Loa loa eye worm
TissueNematodes
17
Tropical infection spread by mosquitoes
Vector deposits larvae which move into lymphatics
and develop into adults.
Chronic infection causes blockage of lymphatic
circulation and elephantiasis, massive swelling in the
extremities.
Wucherereia bancrofti andBancroftian
Filariasis
18
222
Transmitted by biting black flies
Larvae develop into adults in subcutaneous tissues.
Adult females migrate via the blood to the eyes,
provoking inflammatory reactions. Affects optic
nerve.
Coinfection with Wolbachia bacteria causes river
blindness.
Treatment: tetracycline and ivermectin
OnchocercavolvulusandRiverBlindness
19
Spread by bite of small flies
Temperaturesensitive worm migrates around/under
the skin and may enter the eye.
Treatment pull worm from a small hole in
conjunctiva or diethylcarbamazine
Loaloa:TheAfricanEyeWorm
20
Flatworms with ovoid leaflike bodies
Have digestive, excretory, neuromuscular, and
reproductive systems
Lack circulatory and respiratory systems
Animals such as snails or fish are usually the
intermediate hosts and humans are the definitive
hosts.
TrematodesorFlukes
21
Schistosomiasis prominent parasitic disease
Schistosoma mansoni, S. japonicum,
S. haematobium
Adult flukes live in humans who release eggs into
water; early larva (miracidium) develops in
freshwater snail into a 2nd larva (cercaria).
This larva penetrates human skin and moves into the
liver to mature; adults migrate to intestine or
bladder and shed eggs, giving rise to chronic organ
enlargement.
BloodFlukes:Schistosomes
22
Zoonotic
Liver flukes:
Opisthorchis (Chlonorchis) sinesis cycles between
mammals and snails and fish; humans are infected
by eating inadequately cooked fish containing
cercaria, larvae crawl into bile duct, mature and
shed eggs into feces; snail are infected.
Fasciola hepaticacycles between herbivores, snails,
and aquatic plants; humans are infected by eating
raw aquatic plants; fluke lodges in liver.
23
Lung fluke:
Paragonimus westermani cycles between
carnivorous animals, snails, and crustaceans;
humans infected by eating undercooked
crustaceans; intestinal worms migrate to lungs.
24
223
Flatworms
Long, very thin, ribbonlike bodies composed of sacs
(proglottids) and a scolex that grips the intestine
Each proglottid is an independent unit adapted to
absorbing food and making and releasing eggs.
Taenia saginata
Taenia solium
Cestode(Tapeworm)Infestations
25
Beef tapeworm
Very large, up to 2,000 proglottids
Humans are the definitive host.
Animals are infected by grazing on land
contaminated with human feces.
Infection occurs from eating raw beef in which the
larval form has encysted.
In humans, larva attaches to the small intestine and
becomes an adult.
Causes few symptoms; vague abdominal pain and
nausea; proglottids in stool
Taeniasaginata
26
27
Pork tapeworm
Infects humans through ingesting cysts or eggs
Eggs hatch in intestine, releasing tapeworm larva
that migrate to all tissues and encyst.
Most damaging if they lodge in heart muscle, eye, or
brain
May cause seizures, psychiatric disturbances
Taeniasolium
28
Arthropods exoskeleton and jointed legs; includes
arachnids and crustaceans; many must feed on
blood and tissue fluid of host during life cycle;
ectoparasites
Those of medical importance transmit infectious
microbes in the process of feeding biological
vectors
TheArthropodVectorsofInfectiousDisease
29
Insects
Mosquitoes require an aquatic habitat; females
take blood meal transmitting disease: malaria,
filariasis, zoonoses
Fleas highly motile, flattened bodies; feed on
warmblooded animals; carry zoonotic diseases:
plague, murine typhus
Lice small, soft; attach to head and body hair
feeding inconspicuously on blood and tissue fluid;
release feces that contaminate wound; epidemic
typhus, relapsing fever
Flies tsetse fly, sand fly
30
224
Arachnids
Ticks cling on vegetation and attach to host on
contact; larvae, nymph and adults get blood meal
by piercing skin of host
hard ticks Dermacentor, Ixodes small compact, rigid
bodies; transmit rickettsial, borrelial, and viral diseases
soft or argasid ticks Ornithodoros flexible outer
bodies; transmit relapsing fever
31 32
225
Ministry of Health
Clinical Chemistry
226
MinistryofHealth
Graduates
TechnologyinClinicalChemistry(1)
Introduction
The function of the Clinical Biochemistry Lab is to
perform quantitative and qualitative tests on
variable body fluids as well as on feces, tissues and
calculi to diagnose certain diseases, monitor their
progress or their response to treatment and to
screen for a disease in seemingly healthy individuals.
This requires certain technologies for applications of
suitable procedures of sound analytical methods.
2
Photometry
Many determinations in the clinical chemistry are based on
measurement of radiant energy emitted, transmitted,
absorbed or reflected under controlled conditions. The
measurement of the luminous intensity of light or the
amount of luminous light is called photometry.
The instruments depending on this photometric principle
include:
Spectrophotometers, flame emission spectrophotometers and
atomic absorption spectrophotometers.
Fluorescence, phosphorescence & chemiluminescence
techniques.
Fluorometry, turbidimetry and nephelometry depending on the
principles of fluorescence and light scattering measurements.
3
Spectrophotometry
Spectrophotometers are instruments used to measure
the intensity of light from the UV range through the
visible light.
Most spectrophotometric measurements in clinical
chemistry are made in the visible region (400 700 nm)
where the normally visible colors occur.
A substance (to be measured by colorimetric method)
must either have a characteristic color or participate in a
reaction which produces such a color. The amount of
color produced should be proportional to the amount of
substance being measured.
4
BasicComponentsofSpectrophotometer
5
TypesofSpectrophotometers
1. Singlebeam spectrophotometers:
o In this instrument, the light from the lamp travels along only one
pathway to the cell compartment (sample holder) and all
samples (blank, standard & tests) are read in the same position.
2. Doublebeam spectrophotometers:
o These instruments operate like singlebeam spectrophotometers
except they are designed to compensate for possible variations
in intensity of the light source.
o In this type, the light from the monochromator is split into two
beams; one beam is used as reference (directed through the
reference cuvet), the other for reading (transmitted directly
through the sample cuvet). So, any change in light intensity
affects both cuvettes simultaneously and thus is canceled out.
6
227
DoublebeamSpectrophotometers
1. Doublebeaminspace spectrophotometer:
7
DoublebeamSpectrophotometers
2. Doublebeamintime spectrophotometer:
8
AtomicEmissionSpectrophotometry(Flame
Photometry)
In the ground state of an atom, the electrons occupy the
lowest energy level.
In order to move from its energy level in the ground state to
an exited state, the energy of an electron must be raised by
the absorption of an amount of energy exactly equivalent to
that involved in the transition.
The electrons in the higher energy orbits are unstable and
tend to return to lower energy orbits (ground state).
In doing so, the energy previously absorbed is released as
quanta of light, the wave lengths of which are characteristic
of the substance, thus giving rise to the emission spectrum,
e.g. sodium is identified by yellow color, potassium produces
a violet color while lithium imparts a red color to a flame.
9
FlamePhotometer
10
AtomicAbsorptionSpectrophotometry
Atomic Absorption Spectrophotometry (AAS) is the
measurement of the absorption of light by free metallic
atoms.
It uses the heat of a flame to dissociate molecules to
free atoms, primarily at their lowest energy level
(ground state).
AAS measures the absorption of light of a unique wave
length by atoms in the ground state. The unique wave
length absorbed corresponds to the particular line
spectrum for that element.
With the proper light source, a particular cation can be
analyzed in a mixture of many cations.
11
AtomicAbsorptionSpectrophotometer
12
228
Fluorometry
Fluorometry is the technique for the measurement of fluorescence
emitted from certain substances.
It is used in the clinical chemistry laboratory for certain classes of
compounds, particularly when great sensitivity is required e.g.
drugs, hormones, vitamins, amino acids and porphyrins.
In fluorescence, the exciting radiation is electromagnetic usually
within UV or visible range.
The absorbed energy causes molecules of the substance involved
to pass into an excited state.
After a part of the energy has been lost, the molecule returns to its
ground state by reemission of a quantum of energy smaller than
that absorbed, thus the emitted radiation is of a lower frequency
and hence larger wave length.
In fluorescence, the excited state persists for less than 610
seconds.
13
Fluorometer
14
Chemiluminescence
Luminescence is the phenomenon of emission of light by an
electronically excited molecule on decay to ground state.
In chemiluminescence, the energy responsible for excitation
arises as a result of a chemical reaction.
In chemiluminescence, the molecule emitting the light is very
unstable chemically and thus it only exists transiently during
the reaction.
The emitted photons of an excited molecule can be
quantified accurately by using luminometer.
Chemiluminescence provides a sensitive and rapid method
for measuring a wide range of substances of biological and
clinical interest e.g. certain steroids, hormones, drugs and
immunoglobulins.
15
SimpleLuminometer
16
Electrophoresis
Electrophoresis is the migration of charged particles in a
liquid medium under the influence of an electric field.
A charged particle placed in an electrical field migrates
towards the anode (+) or cathode (), depending on the
net charge carried by the particle. The rate of migration
in a porous medium varies with its net charge and the
strength of the electrical field.
The electrophoresis apparatus is designed so that the
circuit between the two poles is bridged by the support
medium holding the sample, and the current flow is
partially carried by the components of the sample.
17
DiagramofElectrophoresisChamber
18
229
FactorsAffectingElectrophoresis
1. Sample:
Charge Size Shape
2. Electric field:
Current Voltage Resistance Heat
3. Buffer:
Composition Concentration pH
4. Support media:
Adsorption Electroosmosis Molecular sieving
19
TypesofElectrophoresis
1. Moving boundary electrophoresis.
2. Zone electrophoresis.
3. Isoelectric focusing.
4. High voltage electrophoresis.
5. Twodimensional electrophoresis.
20
BlottingTechniques
Southern Blotting:
This technique is widely used in molecular biology for
identifying a particular DNA sequence; determining the
presence, position, and number of copies of a gene in a
genome; and typing DNA.
Northern Blotting:
This technique is used to separate and detect RNAs and
RNA fragments instead of DNAs or DNA fragments.
Western Blotting:
It is a method used to separate, detect, and identify one
or more proteins in a complex mixture.
21
230
MinistryofHealth
Graduates
TechnologyinClinicalChemistry(2)
Electrochemistry
Several types of analytical methods are based on
electrochemical phenomenon.
Applications of electrochemistry:
1. Determination of pH of solutions by pH meter.
2. Determination of electrolytes by ion selective
electrode (ISE).
3. Determination of arterial blood gases by blood gases
analyzer.
2
pHMeter
pH meter is the
electrometric method
which is now
commonly used for
the determination of
the pH of solutions.
This method depends
on using a glass
electrode to measure
the pH of unknown
solution against the
calomel reference
electrode.
3
IonSelectiveElectrodes(ISE)
A range of ionselective electrodes have
been developed and use more specific
media instead of glass. In all cases the
basic design of the electrode is the same.
All ISE are similar in their principles of
operation. The difference arises in the
mechanism by which a particular
electrode achieves its selectivity for the
desired ion.
According to the physical state of the
membrane, ISE can be classified into:
1. Solid state electrode (solid membrane):
e.g. Sodium electrode
2. Liquid membrane electrode:
e.g. potassium electrode
4
GasSensors
Except for the oxygen electrode (which is an amperometric), the
other gassensing electrodes used in clinical chemistry are
potentiometric sensors separated from the fluid being analyzed
by a thin, gas permeable membrane.
1. pCO
2
Electrode:
This is essentially a pH electrode system containing a calomel
and glass electrode.
2. pO
2
Electrode:
The oxygen electrode is not strictly an ISE. The electrode system is
based on the electrochemical reduction of oxygen (amperometry).
The pO
2
electrode is used to measure the oxygen pressure of
blood and other body fluids.
5
EnzymeElectrodes
These electrodes are biosensors which consist of immobilized
material in contact with a transducer which will turn the
biochemical signal into an electrical signal.
The substance (substrate for the enzyme) to be measured diffuses
to the enzyme where a product is formed and then the electrode is
affected.
Enzyme electrodes normally have to be kept refrigerated to keep
the life time as long as possible; even so, few have a life time of
over a month.
Glucose oxidase electrode:
It is used for detecting glucose.
Urease electrode:
It is a glass electrode that is sensitive to NH4+ and is covered with
urease polymerized in a polyacrylamide matrix.
It has been used for determination of urea concentration.
6
231
Chromatography
Chromatography is an analytical procedure by which a
multicomponent mixture is separated into its
constituents with a differential migration phenomenon.
It is a versatile and powerful tool for the separation and
quantification of many clinically relevant analytes.
The primary goal of the chromatographic process is to
separate a mixture into its individual components, which
are called solutes or analytes.
A chromatographic separation requires a sample to be
introduced into a flowing stream of gas or liquid (mobile
phase) that passes through a bed, layer, or column
containing the stationary phase.
7
Chromatography
The stationary phase may be particles of a solid or gel, or a liquid.
If it is liquid, it may be distributed on solid particles. The liquid may
be chemically bonded to the particles (bonded phase), or
immobilized onto them (immobilized phase).
As the mobile phase carries the sample past the stationary phase,
the solutes with lesser affinity for the stationary phase remain in
the mobile phase and travel faster and separate from those that
have a greater affinity for it.
Chromatographic separations can be used for the quantitative
screening, preparation and identification, and quantitative analysis
of a wide variety of chemical compounds. These include proteins,
peptides, amino acids, carbohydrates, lipids, hormones and nucleic
acids. Its greatest application is in the measurements of
therapeutic drugs and drugs of abuse.
8
ChromatographicTerms
Column:
A cylindrical tube for holding either the adsorbent or ion exchange resin.
Stationary phase:
Usually a solid or liquid adsorbent, e.g. paper or water.
Mobile phase:
A solvent or a gas used to separate the components.
Tanks:
Airtight containers in which development takes place.
Origin:
The point of application of substance to chromatogram.
9
Loading:
The amount of substance applied to the paper or support.
Development:
The process of allowing the solvent to move along the
column or paper.
Multiple development:
When the development is rerun, a number of times in a
solvent system to improve resolution.
Polarity:
A polar compound is one that is held by the stationary phase
whereas a nonpolar compound tends to move forwards in
the mobile phase.
10
Solvent front:
The level at which the elution fluid (eluent) has reached.
Relative fraction (RF):
A ratio of the distance the solute has traveled from the point of
origin to the distance traveled by the solvent front.
Elution:
The use of solvent to separate components.
Resolution:
The degree of separation of the components after development.
Location:
The detection of the components after development, either by
using a special or general reagent, or ultraviolet light.
11
SeparationMechanismsinChromatography
1. Surface adsorption:
o This is competition between the mobile phase (liquid or gas) and the
solutes for adsorption sites on the support (the stationary phase which is a
solid).
2. Partition:
o The separation is achieved as a consequence of the relative solubility of a
substance in the two phases (stationary and mobile).
3. Ion exchange:
o The principle feature underlying this form of chromatography is the
attraction between oppositely charged particles.
4. Molecular phase and size:
o The solutes are separated on the basis of the differences in their molecular
size.
5. Affinity:
o This affinity may be due to antigenantibody interaction or enzyme
substrate interaction.
12
232
TypesofChromatography
1. Column chromatography:
It is one of adsorption chromatography in which the separation of mixtures
occurs on a column of suitable adsorbent packed in a glass tube and
supported either by a plug of glass or cotton wool.
2. Thin layer chromatography (T.L.C.):
It is primarily adsorption chromatography in which a uniform thin layer of
adsorbent on a supporting glass plate is used.
Highperformance thinlayer chromatography (HPTLC) has been applied to a
system of TLC.
TLC is applied in the investigations of sugars, amino acids, lipids and drugs.
3. Paper chromatography:
It is a liquidliquid partition chromatography where the liquid stationary
phase is supported on the cellulose paper sheet.
Some compounds are better resolved by using a stationary organic phase
(nonpolar), and a mobile aqueous phase (polar); this is called the reversed
phase chromatography.
13
TypesofChromatography
4. Affinity chromatography:
Affinity chromatography is a separation mechanism
involving an interaction between biochemical species, e.g.
enzymesubstrate, hormonereceptor and antigen
antibody.
5. Ionexchange chromatography:
In ionexchange chromatography, solutes in a mixture are
separated by virtue of their difference in sign and
magnitude of ionic charge.
6. Gas chromatography:
Gas chromatography is a process by which a mixture of
compounds in volatilized form is separated into its
constituent parts by moving a mobile phase, which is an
inert, over a stationary phase.
14
GasLiquidChromatography
1. The stationary phase is a liquid.
2. The separation is mainly by partition between the carrier
gas and the liquid phase supported on the inert material
(stationary phase).
15
HighPerformanceLiquidChromatography
(HPLC)
HPLC is the most recently introduced form of chromatographic
separations.
HPLC has great potential in the analysis of nonvolatile compounds of
biological interest such as steroid, nucleotides and nucleosides, drugs and
their metabolites, amino acids and peptides, aromatic amines, lipids, etc.
16
ImmunochemicalTechniques
Radial Immunodiffusion
It is a passive diffusion method, in which a concentration
gradient is established for a single reactant, usually the
antigen. The antibody is uniformly dispersed in the gel
matrix.
Electroimmunoassay
In this method, a single concentration gradient is established
for the antigen when an applied voltage is used to drive the
antigen from the application well into a homogenous
suspension of antibody in the gel.
This produces a unidirectional migration of antigen and
results in a lower limit of detection.
17
Nephelometry
It is the detection of light energy scattered or reflected toward a
detector that is not in the direct path of the transmitted light.
Common nephelometers measures scattered light at right angle to
the incident light.
Light scattering measurements are best applied to immunoassays
of immunoglobulins, specific proteins and haptens such as
therapeutic drugs.
Two types of nephelometry are encountered:
1. Static nephelometry:
The nephelometer detects light scatter by utilizing an electronic
system designed to reduce interference from light scatter produced
by large contaminating particles such as dust.
2. Rate nephelometry:
This is a kinetic method in which the nephelometer has been coupled
with a microprocessing unit for the rapid determination of specific
proteins by means of immunoprecipitin reactions.
18
233
Turbidimetry
It is the Measurement of the decrease in the intensity of the incident
beam of light as it passes through a solution of particles due to
scattering, reflectance and absorption.
Turbidimetry is carried out at 180from the incident beam.
Labeled Immunochemical Assays
These are sensitive and specific methods for accurate quantitation of
biologically important compounds (such as peptides, hormones,
vitamins and drugs) which may occur in biological fluids or tissues in
low concentrations (in g/ml or pg/ml).
The two main types are encountered:
1. Competitive immunoassay (limited reagent assays).
2. Noncompetitive immunoassay (excess reagent, twosite, sandwich
assays).
19
Type
Labels
(Reportergroups)
B/Fseparation Signaldetection Sensitivity
Precipitation
immunoassay
Notrequired Notrequired
Nakedeye
Turbidity
Nephelometry
~10g/ml
Particle
immunoassay
Bloodcells,artificial
particles(gelatin,
latex)
Notrequired
Nakedeye,
Spectrophoto
metry,Particle
counting
~5ng/ml
Radioimmunoassay
Radioisotopes
(
125
I,
3
H)
Required Photoncounting ~5pg/ml
Enzyme
immunoassay
Enzymes Required
Spectrophoto
metry,Fluoro
metry,Photon
counting
~0.1pg/ml
Fluorescent
immunoassay
Fluorophores Required Photoncounting ~5pg/ml
Chemiluminascent
immunoassay
Chemiluminascent
compoundssuchas
luminalderivatives,
acridiniumesters
Required Photoncounting ~5pg/ml
LabeledImmunochemicalAssays
20
EnzymeImmunoassay(EIA)
Enzyme immunoassay uses enzymes as labels and is widely
used nowadays.
Enzymes can amplify signals depending on the turnover of
enzyme catalytic activity.
Enzyme Linked ImmunoSorbent Assay (ELISA) is one of EIA
techniques.
The principle in ELISA involves heterogeneous methodology
in which bound and free materials must be separated using a
series of washing steps.
Three major methods of ELISA are commonly used.
1. Competitive method.
2. Indirect method.
3. Doubleantibody sandwich method.
21
ELISA CompetitiveMethod
Its principle involves competition of patient antigen and labeled antigen
for antibody binding sites, where an antibody to the analyte is covalently
bound to the tube or well.
There is an inverse relation between the concentration of patient antigen
and the enzyme activity.
22
ELISA IndirectMethod
Antigen to the analyte immunoglobulin is covalently bound to the inside
of the tube or well.
The enzyme activity is directly proportional to the concentration of the
analyte.
23
ELISA DoubleantibodySandwichMethod
Antibody to the analyte antigen is covalently bound to the wall of
reaction vessel (well).
Activity is directly proportional to the analyte concentration.
24
234
Automation
Automation is the replacement of human manipulative
effort and facilities in the performance of a given process
by mechanical and instrumental devices that are
regulated by feedback of information so that an
apparatus is selfmonitoring or selfadjusting.
This term has been applied in the field of clinical
chemistry to describe the process by which an analytical
instrument performs many tests with only minimal
involvement of an analyst.
One of the benefits of automation is a reduction in the
variability of results and errors of analysis by eliminating
tasks that are repetitive and monotonous for a human
that can lead to boredom or inattention.
25
TermsinAutomation
Analyzer configuration: The format in which analytical instruments
are configured. Automated instruments are configured by the
manufacturer either as open or closed systems.
Batch analysis: A type of analysis in which many specimens are
processed in the same analytical run.
Centralized testing: A mode of testing where specimens are
transported to a central or core facility for analysis.
Continuousflow analysis: A type of analysis in which each
specimen in a patch passes through the same continuous stream
and is subjected to the same analytical reactions as every other
specimen and at the same rate.
26
TermsinAutomation
Discrete analysis: A type of analysis in which each
specimen in a patch has its own physical and chemical
space separate from every other specimen.
Discretionary multiplechannel analysis: A type of
analysis in which specimens in sequence can be analyzed
by any one or more of the available processes.
Multiplechannel analysis: A type of analysis in which
each specimen is subjected to multiple analytical
processes so that a set of test results is obtained on a
single specimen.
27
TermsinAutomation
Parallel analysis: A type of analysis in which all
specimens are subjected to a series of analytical
processes at the same time in a parallel fashion.
Point of care testing: A mode of testing where the
analysis is performed at the site where health care is
provided (e.g. bedside testing).
Randomaccess analysis: A type of analysis in which any
specimen can be analyzed by any available process in or
out of sequence with other specimens and without
regard to their initial order.
28
TermsinAutomation
Sequential analysis: A type of analysis, in which any
specimen in a batch enters the analytical process one
after another, and each result or set of results emerges
in the same order as the specimens are entered.
Singlechannel analysis: A type of analysis in which each
specimen is subjected to a single process so that only
results for a single analyte are produced.
Specimen throughput rate: The rate at which an
analytical system processes specimens.
29
235
MinistryofHealth
Graduates
UnitsandCalculationsinClinicalChemistry
UnitsofMeasurement
A meaningful measurement is expressed with both a number and a unit.
All quantitative measurements must be expressed in clearly defined units that are
accepted and understood by all scientists.
The system International Units (SI Units) have replaced the old system of
reporting and measurements that can be safely understood anywhere nowadays.
This system has functioned as the international authority for measurements.
The International Unit (U) is used to express enzyme activity in U/L.
An International Unit of enzyme activity is the amount of enzyme which under
defined assay conditions will catalyze the conversion of 1 mol of substrate per
minute. .
The following formula is used for the interconversion between the conventional
unit (mg/dl) and the SI unit (mmol/l):
When the molecular weight of a substance cannot be accurately determined (e.g.
albumin), the results are expressed in gm/l.
2
Chemical substance SI Unit Interconversion Factor
Conventional
Unit
Glucose mmol/l X 18 0.0555 X mg/dl
Urea mmol/l X 6 0.16666 X mg/dl
Creatinine mol/l X 0.0113 88.49 X mg/dl
Uric acid mol/l X 0.0169 59.172 X mg/dl
Sodium mmol/l X 1 1 X meq/dl
Potassium mmol/l X 1 1 X meq/dl
Calcium mmol/l X 4 0.250 X mg/dl
Inorganic phosphorus mmol/l X 3.1 0.3229 X mg/dl
Magnesium mmol/l X 2.432 0.4114 X mg/dl
Interconversion betweenSIandConventional
Units
3
Chemical
substance
SI Unit Interconversion Factor
Conventional
Unit
Ammonia mol/l X 1.7 0.5872 X g/dl
Iron mol/l X 5.6 0.1791 X g/dl
Bilirubin mol/l X 0.0588 17.1 X mg/dl
Total protein gm/l X 0.1 10 X g/dl
Albumin gm/l X 0.1 10 X g/dl
Immunoglobulins gm/l X 100 0.01 X mg/dl
Total lipids gm/l X 100 0.01 X mg/dl
Cholesterol mmol/l X 38.65 0.02586 X mg/dl
Triglycerides mmol/l X 88.5 0.01130 X mg/dl
Interconversion betweenSIandConventional
Units
4
Molarity(M)=Thenumberofmoleculesperliterofsolution.
CalculationsinClinicalChemistry
Concentrations based on Volume
Concentrations based on the amount of dissolved solute
per unit volume are the most widely used in
biochemistry laboratories.
Molar solutions are the solutions which contain one
gram molecule of the substance per liter.
One gram molecule (g mole) is the molecular weight of
the substance expressed in grams e.g.:
Molar solution of NaOH = 23 + 16 + 1 = 40
Molar solution of H
2
SO
4
= 1X2 + 32 + 16X4 = 98
Molar solution of H
3
PO
4
= 1X3 + 31 + 16X4 = 98
5
w t
g
M W
= m o l e s
To calculate M, we need to know the weight of dissolved
solute in gm (wtg) and its molecular weight (MW).
Mole = 10
3
millimole = 10
6
micromole = 10
9
nanomole.
A one M solution contains Avogadro's number of molecules
per liter.
Avogadro's number
= number of molecules per gmole.
= number of molecules per gmole.
= number of atoms per gatom.
= number of ions per gion.
= 6.023 X 10
23
6
236
Normal solutions are solutions which contain one gram
equivalent of the substance per liter.
To calculate N, we need to know the weight of dissolved
solute in gm (wtg) and its equivalent weight (EW).
One gram equivalent is the equivalent weight of the
substance expressed in grams.
7
Normality(N)=thenumberofequivalentsofsoluteperliterofsolution
wt
g
EW
= equivalents
Normal solution of NaOH = =

40
1
40
Normal solution of H
2
SO
4
= =
98
2
49
Normal solution of H
3
PO
4
= =
98
3
32.7
N =nM
W eight /volume percent(%w/v)= the weightingofas ol ute
pe r100ml ofsolution
The molarity (M) and normality (N) are related by:
where n = number of active radicals (number of replaceable H+
or OH per molecule, or the number of lost or gained electrons
per molecule). e.g. a 0.01 M solution of H2SO4 is 0.02N.
Weight/volume percent is often used for routine laboratory
solutions.
8
Milligram percent is often used in clinical laboratories.
A one M solution of a nondissociable solute is one Osmolar,
while a one M solution of a dissociable salt is n Osmolar where
n is the number of ions produced per molecule.
Thus, a 0.03 M solution of KCl is 0.06 Osmolar.
9
Mil ligranpercent(mg%)= theweightinmg ofasolute
per100mlofsolution
The concentration of many commercial acids are given in terms of %
w/w.
In order to calculate the volume of stock solution required for a given
preparation, we must know its density (p) or specific gravity (SG).
where:
wt
g
= weight of pure substance required in g.
vol
ml
= volume o stock solution needed in ml.
p
g/ml
= density of the substance.
% = fraction of total weight that is pure substance.
10
Weight/weightpercent(%w/w)=theweightingofa soluteper100gofsolution
wt
g
=vol
ml
Xp
g/ml
X%(asdecimal)
Molal solutions are the solutions which contain one
gram molecule of the substance per 1000 grams of
solvent.
Molality is used in certain physicochemical
calculations e.g. calculation of boilingpoint
elevation and freezingpoint depression.
11
Molality(m)=thenumberofmolesofsoluteper1000gofsolvent
237
MinistryofHealth
Graduates
BloodGlucoseTests
BloodSugar
What is blood sugar?
Glucose is the main blood sugar
2
WhatareSourcesofBloodGlucose
1. Dietary carbohydrates.
2. Liver glycogen (glycogenolysis).
3. Noncarbohydrate source (gluconeogenesis).
3
WhataretheNormalValuesofBloodGlucose
Concentration?
The normal fasting (8 12 hours after meal) plasma
glucose level is 70 100 mg/dl.
The postprandial (2 hours after meal) plasma
glucose level must return to the normal fasting level
or ideally just below the fasting level.
Random plasma glucose level is up to 140 mg/dl.
4
AlterationsinBloodGlucoseLevels
Hypoglycaemia is the decrease of fasting plasma
glucose level below 40 mg/dl.
Hyperglycaemia is the rise of fasting plasma glucose
level above 126 mg/dl.
5
Fasting values between 100 & 126mg/dl or random
values between 140 & 200 mg/dl are called impaired
glucose tolerance
Only a small proportion of subjects with such mild
impaired glucose tolerance develop diabetes
mellitus later.
It is not possible to predict the outcome of this
condition at the time of subject presentation.
6
238
Subjects must not be diagnosed as having diabetes
mellitus merely on the basis of impaired glucose
tolerance, because of the serious psychological,
social and economic implications.
However, because of the increased risk of vascular
complications, secondary causes of impaired glucose
tolerance should be excluded and the subjects
should be given dietary advice and, if overweight,
should be advised to lose weight.
7
RegulationofBloodGlucose
Normally, blood glucose level is maintained within
relatively constant range despite the various
disturbing factors.
This homeostasis is achieved by tissue and hormonal
regulation:
8
Tissue Regulation
1. Gastrointestinal tract (GIT)
2. Liver
3. Muscle and adipose tissue
4. Kidney
9
HormonalRegulation
AntiInsulin
Hormones
Insulin
10
AntiInsulinHormones
1. Adrenaline and glucagon.
2. Glucocorticoids and growth hormone.
3. Thyroxine.
DiabetesMellitus
Diabetes Mellitus is a metabolic syndrome
characterized by hyperglycaemia, glucosuria,
polyuria, polydipsia and polyphagia.
It is confirmed if one of the following is present:
1. A fasting plasma glucose level of more than 126 mg/dl
on two occasions.
2. A random plasma glucose level of more than 200
mg/dl on two occasions.
3. Both a fasting plasma glucose level of more than 126
mg/dl and a random plasma glucose level of more
than 200 mg/dl.
12
239
DiabetesMellitus
Diabetes Mellitus is usually excluded if:
A fasting plasma glucose level is less than 100 mg/dl on
two occasions.
Random plasma glucose levels are less reliable for
excluding than for confirming the diagnosis.
Causes:
1. Insulin deficiency.
2. Resistance to insulin action.
3. Decreased the ratio between insulin and antiinsulin
hormones.
13
DiabetesMellitus
Type-I Diabetes Mellitus Type-II Diabetes Mellitus
1. Insulin-dependent diabetes mellitus
(IDDM).
2. J uvenilediabetes.
3. Less common (10 20%).
4. Usually beforetheageof 25 years.
5. Patients areusually thin.
6. Severetype.
7. Ketoacidosis is morecommon.
8. No detectiblecirculating insulin.
9. Insulin deficiency.
10. Treated by insulin only.
1. Non-insulin-dependent diabetes
mellitus (NIDDM).
2. Maturity (Adult) onset diabetes.
3. Morecommon (80 90%).
4. Usually after theageof 25 years.
5. Patients areusually obese.
6. Milder type.
7. Ketoacidosis is less common.
8. High plasmainsulin level.
9. Insulin resistance.
10. Treated by oral hypoglycaemic
drugs and may beby insulin.
14
InvestigationsofSuspectedDiabetesMellitus
Specimen collection and storage:
Plasma or whole blood
Venous blood or capillary blood
Serum or plasma
Double void technique
Storage of 24h urine
15
Parameters for blood glucose investigations:
1. Urine glucose
2. Random blood glucose
3. Fasting blood glucose
4. Two hours postprandial blood glucose
5. Glucose tolerance test (GTT )
6. Glycosylated haemoglobin (HbA1C)
7. Plasma fructosamine
8. Plasma Insulin
9. Serum Cpeptide.
16
Indications
Procedure
Results
1. Normal blood sugar curve
2. Renal glucosuria curve
3. Diabetic blood sugar curve
(Mild diabetes Moderate diabetes Severe diabetes)
4. Lag curve
17
OralGlucoseToleranceTest(GTT)
18
240
IntravenousGlucoseToleranceTest
This test may be performed to eliminate factors related
to the rate of glucose absorption as in:
1. Poor absorption of orally administered glucose.
2. Inability to tolerate a large oral glucose load.
3. Altered gastric physiology (e.g. after gastrectomy).
Preparation of patients is the same as for the OGTT.
The dose of glucose is 0.5 g/kg of body weight with
a maximum of 35 g given as a 25 g/dl solution.
The dose is administered intravenously over 3 min 15
s, and blood is collected every 10 min after the mid
injection time for one hour.
19
241
MinistryofHealth
Institute Graduates
KidneyBiochemistry
RenalFunctions
A. Reabsorptive and Excretory Functions:
Retain the important required substances.
Rid the body of the metabolic waste end products.
B. Regulatory Function:
Maintenance of the optimal chemical composition
of body fluids.
Acidbase homeostasis.
2
RenalFunctions
C. Endocrine Function:
Hormones released by the kidney:
1. Rennin and prostaglandins.
2. Erythropoietin.
3. Calcitriol (1,25dihydroxycholecalciferol).
Hormones acting on the kidney:
1. Aldosterone.
2. Vasopressin (antidiuretic hormone = ADH).
3. Parathyroid hormone (PTH).
Hormones altered and inactivated by the kidney:
1. Insulin.
2. Glucagon.
3. Aldosterone.
D. Metabolic Function:
Gluconeogenesis in prolonged starvation.
3
CollectionandStorageofUrineSamples
Single specimens of urine (random or morning samples) are
used for ward examinations and qualitative tests.
Doublevoided specimen is the urine excreted during a time
period after complete emptying of the bladder by 15 30
min.
24 hurine collections are preferred for quantitative tests due
to the diurnal variation in the excretion of some substances.
The 24 hcollection is as follows:
At a suitable time (e.g. 8 Oclock morning), the patient empties his bladder
and the urine is discarded.
All urine passed during the following 24 hours is saved in specific container.
At the same time of the next morning, the patient empties his bladder and
the urine is added to the collected one.
4
CollectionandStorageofUrineSamples
For storage of urine sample, the following are
possible:
It is satisfactory in most cases to use specimens collected
in cool, clean containers. Urine sample can be stored for
about one week in the refrigerator at 2 8C.
Urine samples can be stored for many months at 20C
without any addition.
Concentrated hydrochloric acid (HCl), thymol or
chloroform can be used for urine storage.
Acid should not be used for proteins, creatinine and
steroids determination.
5
Nephrolithiasis
Condition characterized by the presence of renal calculi (stones).
Due to nutritional, environmental or genetic factors.
Kidney stones may be:
Single stones which may be composed of any of: calcium oxalate, uric acid, calcium
carbonate, calcium phosphate or magnesiumammoniumphosphate (triple phosphate).
Mixed stones which may be composed of two or more of the mentioned constituents.
Cystine or xanthine stones which are rare and found in the inherited metabolic
abnormalities: cystinuria or xanthinuria respectively.
Both qualitative and quantitative analyses of the chemical constituents of
kidney stones may be useful in establishing the etiology and in planning
adequate therapy.
Radiological examinations are required to explore the degree of intrarenal
calcification and papillary damage.
6
242
KidneyFunctionTests
I Glomerular Function Tests
1.Complete Urine Analysis:
A. Physical Examination:
Volume: 750 2000 ml / day.
Colour: pale yellow amber yellow deep yellow.
Odour: Urineferous aromatic ammoniacal.
Aspect: Clear.
Deposit: Nil.
Reaction: Acidic.
Specific gravity: 1015 1025.
7
KidneyFunctionTests
1. Complete Urine Analysis:
B. Chemical Examination:
Albumin: Nil.
Blood: Nil.
Glucose: Nil.
Acetone: Nil.
Urobilinogen: Trace.
Bilirubin: Nil.
8
KidneyFunctionTests
1.Complete Urine Analysis:
C. Microscopic Examination:
Red blood cells: 0 5 / HPF.
Pus cells: 0 5 / HPF.
Crystals: Nil.
Casts: Nil.
Ova and parasites: Nil.
Epithelial cells: Nil in males. (amount depends on
specimen collection)
Few squamous epithelial cells in
females. (amount depends on specimen collection) 9
KidneyFunctionTests
2.Blood Urea (20 40 mg/dl)
Urea is the end product of protein metabolism.
Urea is synthesized in the liver from ammonia and then passes to
the kidney to be excreted.
Blood urea is increased in renal failure and urinary obstruction, but
it is decreased in liver cell failure.
Sometimes blood urea is represented by blood urea nitrogen (BUN)
which normally ranges from 8.0 to 16.0 mg / dl. (needs to be SI
units)
Blood urea is neither sensitive nor specific for kidney function
because it is affected by dietary proteins, chronic constipation and
gastrointestinal bleeding.
10
KidneyFunctionTests
3.Plasma creatinine: (0.6 1.2 mg/dl in males)? SI units
(0.5 0.9 mg/dl in females)? SI units
Creatinine (creatine anhydride) is the end product of creatine metabolism.
It is formed in the muscles from creatine phosphate and then passes to the
kidney to be excreted.
Plasma creatinine is increased in renal failure and urinary obstruction, but it is
decreased in chronic muscle dystrophy diseases
Plasma creatinine is preferred to urea estimation as an index of renal function
because creatinine is produced endogenously and is not affected by
exogenous factors.
11
KidneyFunctionTests
4. Creatinine clearance : (90 130 ml/min in males)
(80 120 ml/min in females)
The most convenient method of obtaining an acceptable accurate
estimation of glomerular filtration rate (GFR).
Lower values of creatinine clearance are indicative of diminished
glomerular filtration rate.
Has particular value in the general assessment of renal function especially
when plasma analysis is invalid e.g. after renal dialysis.
12
243
KidneyFunctionTests
II Tubular Function Tests
1. Plasma electrolytes and minerals:
Estimation of plasma electrolytes (sodium, potassium, chloride and bicarbonate)
and minerals (calcium, inorganic phosphate and magnesium) are important in
assessing the tubular function of the kidney.
2. Measurement of urine specific gravity: (1015 1025)
This measurement is a useful guide to the adequacy of the renal concentrating
mechanism.
3. Measurement of urine osmolality: (300 900 mOsmol/kgH2O)
This measurement is considered more valid than specific gravity measurement in
assessing concentrating ability of the kidney.
Ratio of urine osmolality to serum osmolality (280 300 mOsmol/kgH2O) should
be calculated.
13
KidneyFunctionTests
II Tubular Function Tests:
4. Urine concentration test (Water deprivation test):
This is a test for renal concentration ability.
5. Vasopressin test (Pitressin test):
More pleasant for the patient than water deprivation
test, and depends only on renal tubular function.
6. Urine dilution test (Water load test):
Very simple but less sensitive than water deprivation
test.
14
KidneyFunctionTests
III Special Function Tests:
1. Urinary Microalbumin:
Normally, albumin is not present in urine because it is filtered from
the bloodstream by the kidneys.
Microalbuminuria occurs when there is an abnormally high
permeability for albumin in the renal glomerulus.
Microalbuminuria cannot be detected by urine dipstick methods but
there is specific Microalbumin urine test to determine the presence
of the albumin in urine.
Microalbuminuria is diagnosed from elevated concentrations (30 to
300 mg/L) on at least two occasions. An albumin level above these
values is called "macroalbuminuria", or just albuminuria.
15
KidneyFunctionTests
III. Special Function Tests:
1. Urinary Microalbumin:
To compensate for variations in urine concentration in spotcheck
samples, the albumin/creatinine ratio (ACR) is calculated.
Microalbuminuria is defined as ACR 2.8 mg/mmol (male) or 2.0
mg/mmol(female).
The significance of microalbuminuria test is:
1. An indicator of subclinical cardiovascular disease.
2. Marker of vascular endothelial dysfunction.
3. An important prognostic marker for kidney disease
In diabetes mellitus.
In hypertension.
4. Increasing microalbuminuria during the first 48 hours after admission
to an intensive care unit predicts elevated risk for acute respiratory
failure, multiple organ failure, and overall mortality.
16
KidneyFunctionTests
III. Special Function Tests
2. Cystatin C:
Cystatin C or Cystatin 3 (formerly gamma trace, postgammaglobulin or
neuroendocrine basic polypeptide) is mainly used as a biomarker of kidney
function.
In humans, all cells with a nucleus produce Cystatin C as a chain of 120 amino
acids. It is found in virtually all tissues and bodily fluids. It is a potent inhibitor of
lysosomal proteinases and probably one of the most important extracellular
inhibitors of cysteine proteases.
Cystatin 3 has a low molecular weight (~13.3 KD). Due to its small size it is freely
filtered by the glomerulus, and is not secreted but is fully reabsorbed and broken
down by the renal tubules. This means the primary determinate of blood Cystatin
C levels is the rate at which it is filtered at the glomerulus making it an excellent
GFR marker.
17
KidneyFunctionTests
2. Cystatin C: (0.56 to 0.98 mg/L in males)
(0.52 to 0.90 mg/L in females)
Serum levels of cystatin C are a more precise test of kidney function than serum
creatinine levels. Cystatin C levels are less dependent on age, sex, race and
muscle mass compared to creatinine.
Cystatin C is an alternative and more sensitive endogenous marker for the
estimation of GFR than serum creatinine and serum creatinine based GFR
estimations.
Cystatin C can be used as a marker of kidney function in the adjustment of
medication dosages.
Cystatin C can be measured in a random sample of serum using immunoassays
such as nephelometry or particleenhanced turbidimetry.
18
244
KidneyFunctionTests
3. Neutrophil gelatinaseassociated lipocalin (NGAL):
NGAL is a protein of a small molecular weight (25 kD), belonging to the lipocalin
superfamily initially found in activated neutrophils. It is also found in certain
epithelia, such as renal tubules, where its expression is dramatically increased in
ischemic or nephrotoxic injury.
NGAL is a promising biomarker for early detection of acute kidney injury (AKI). It is
specifically released by the damaged kidney and can be detected in both urine
and plasma.
Either alone or in combination with other biomarkers they will not only have an
impact on medical decisions in future daily clinical routine, but they will also
provide the basis for testing novel emergency therapies for a disease that is often
recognized too late.
19
KidneyFunctionTests
Clinical Application:
Creatinine is not useful for early diagnosis.
Urinary NGAL can be used as a marker for the early diagnosis of
AKI.
NGAL may be used to detect AKI early in the following cases:
1. Pediatric and adult cardiopulmonary bypass operations.
2. Percutaneous coronary interventions (PCI).
3. Critically ill patients presenting at the emergency department or in the
intensive care unit (heart failure, sepsis, multiorgan failure)
4. Renal transplantation.
5. Patients with chronic kidney disease.
20
245
MinistryofHealth
Graduates
ElectrolytesandMinerals
MineralsandElectrolytes
I Principle Elements (Major Elements= Macrominerals):
These are elements which occur in the body in large amounts and are
required in amounts greater than 100 mg / day.
They include Calcium (Ca), Phosphorus (P), Magnesium (Mg), Sodium
(Na), Potassium (K), Chloride (Cl) and Sulfur (S).
II Trace Elements (Microminerals):
These are elements which occur in the body in small amounts and are
required in amounts less than 100 mg / day.
They include Iron (Fe), Copper (Cu), Zinc (Zn), Manganese (Mn),
Iodine (I), Fluorine (F), Cobalt (Co), Chromium (Cr), Selenium (Se) and
Molybdenum (Mo).
There are other trace elements but not essential for life e.g. Cadium
(Cd), Aluminium (Al) and Lithium (Li).
2
DifferencesbetweenPrincipleElementsand
TraceElements
3
Traceelements Principalelements
Itemof
difference
Smallamount Largeamount Occurrence
<100mg/day >100mg/day Requirement
Expresseding/dl
(needstobeSIunits)
Expressedinmg/dl
(needstobeSIunits)
Plasmalevel
Iron,Copper,Zinc,Manganese,
Iodine,Fluorine,Cobalt,Chromium,
Selenium,Molybdenum,Cadium,
Aluminium,Lithium.
Calcium,Phosphorus,
Magnesium,Sodium,
Potassium,Chloride,
Sulfur.
Minerals
Electrolytes
Electrolytes are substances whose molecules
dissociate into ions when they are dissolved in
water.
They include:
4
Sodium
Sources:
Table salt (sodium chloride) is the main source of sodium.
Meats contain sodium more than vegetables.
Requirements:
The RDA of sodium is 5.0 g for adults.
Persons with family history of hypertension: 2.0 g.
Hypertensive patients: 0.2 g.
Body sodium:
About of total body sodium is present in the skeleton.
About of sodium is present in the other tissues and
body fluids where sodium is the main extracellular cation.
5
Sodium
Plasma sodium:
The normal plasma sodium level is 135 153 mmol/l.
Urine sodium level is 40220 mmol/l (24hour urine
collection)
Functions of sodium:
Maintenance of osmotic pressure and volume of
plasma and extracellular fluid.
Transmission of nerve impulses.
Contraction of muscles.
Regulation of acidbase balance.
6
246
Sodium
Alteration of plasma sodium:
A. Hypernatraemia (excess plasma sodium):
Cushing syndrome: due to excessive glucocorticoids.
Conns disease: due to excessive aldosterone.
Diabetes insipidus: due to rapid loss of water.
Drugs: as ACTH or cortisone.
B. Hyponatraemia (decreased plasma sodium):
Addisons disease: due to deficiency of aldosterone.
Renal failure: due to inhibition of sodium reabsorption.
Hypotonic dehydration: due to the treatment of water and sodium
loss by water only.
Diuretics: e.g. thiazides that block sodium reabsorption
7
Sodium
Sodium determination:
Ionselectiveelectrode(ISE).
Photometry).
AtomicAbsorptionSpectrophotometry.
Chemical(colourimetric)method.
8
Potassium
Sources:
Vegetables and fruits (especially citreous fruits).
Fish, meat and organs meat.
Requirements:
The RDA of potassium is 3.0 5.0 g.
Body potassium :
About of total body potassium is present in the
skeleton.
About of potassium is present in the other tissues and
body fluids where potassium is the main intracellular
cation.
9
Potassium
Plasma potassium:
The normal plasma potassium level is 3.5 5.0 mmol/l.
Urine potassium level is 25 125 mmol/l (24hour urine
collection).
Functions of potassium:
Maintenance of osmotic pressure and volume of
intracellular fluid.
Regulation of acidbase balance.
10
Potassium
Alteration of plasma potassium:
A. Hyperkalaemia (excess plasma potassium):
Addisons disease: due to deficiency of aldosterone.
Acidosis due to shift of K+ from intra to extracellular fluid in exchange with H+.
Crush injuries due to leakage of K+ from tissue cells.
Chronic renal failure.
Uncontrolled diabetes mellitus: because the lack of insulin prevents K+ from entering
the cells.
B. Hypokalaemia (decreased plasma potassium):
Alkalosis.
Treatment of hyperglycaemic coma by insulin without giving potassium as insulin helps
K+ to enter the cells.
Excessive vomiting and diarrhoea.
Cushing syndrome: due to excessive glucocorticoids.
Primary and secondary aldosteronism.
Diuretics.
11
Potassium
Potassium determination:
Ionselectiveelectrode(ISE).
Photometry).
AtomicAbsorptionSpectrophotometry.
Chemical(colourimetric)method.
12
Chloride
Sources:
Table salt (sodium chloride) is the main source of chloride.
Requirements:
The RDA of chloride is 5.0 g for adults.
Body Chloride:
Chloride is present with sodium in the extracellular fluid
compartment.
It has a particularly higher concentration (about 124 mmol/l) in the
cerebrospinal fluid (CSF) than in the plasma.
13
Chloride
Plasma Chloride:
The normal plasma chloride level is 94 106 mmol/l.
CSF chloride level is 120 132 mmol/l.
Urine chloride level is 110 250 mmol/l (24hour urine collection).
Functions of Chloride:
Essential for water balance, osmotic pressure and acidbase balance.
Essential role in the blood for chloride shift.
Essential for formation of HCl in the stomach.
It activates salivary and pancreatic amylase enzymes.
14
Chloride
Alteration of Plasma Chloride:
Hypo and hyperchloremia is usually associated with
hypo and hypernatremia respectively.
Hypochloraemia leads to an increase in plasma
bicarbonate as a compensatory mechanism causing
alkalosis (hypochloremic alkalosis).
Chloride determination:
Ion selective electrode (ISE).
Chemical (colourimetric) method.
15
Bicarbonate(TotalCO
2
)
Clinical significance:
1. Decreased bicarbonate:
A. With elevated pH:
Blood bicarbonate is decreased with elevated blood pH
in respiratory alkalosis as seen in the following
conditions:
Simple anxiety with increased rate and depth of breathing;
the so called hyperventilation syndrome.
Salicylates poisoning which stimulate the respiratory center.
Central nervous system lesions such as tumors located in this
part of the brain.
16
Bicarbonate(TotalCO
2
)
1. Decreased bicarbonate:
B. With decreased pH:
Blood bicarbonate is decreased with decreased blood
pH in metabolic acidosis as seen in the following
conditions:
Diabetic ketoacidosis.
Lactic acidosis.
Shock
Renal failure.
Severe diarrhea.
17
Bicarbonate(TotalCO
2
)
2. Increased bicarbonate:
A. With decreased pH:
Blood bicarbonate is increased with decreased blood pH in
respiratory acidosis as seen in the following conditions:
Lungs diseases e. g. bronchial asthma and emphysema.
Morphine poisoning.
B. With elevated pH:
Blood bicarbonate is increased with increased blood pH in
metabolic alkalosis as seen in the following conditions:
Excess intake of sodium bicarbonate as a treatment of gastric
hyperacidity e. g. in peptic ulcer.
Severe and prolonged vomiting.
18
Bicarbonate(TotalCO
2
)
Bicarbonate determination:
Ion selective electrode (ISE).
Chemical (colourimetric) method.
Calculated from HendersonHasselbalch equation:
Where pH and PCO2 (H
2
CO
3
) values can be obtained from Blood Gas Analyzer
and pKa of carbonic acid (H
2
CO
3
) is 6.1
Specimen collection:
Unhaemolyzed fresh serum or heparinized plasma is required for routine
electrolyte analysis.
Anaerobic collection in vacutainer tubes is preferred.
Fasting is not necessary although it is recommended.
19
BloodpHDisorder(AcidBaseDisturbance)
The normal blood pH is 7.40.05.
Acidbase disturbance is the change of blood pH to be either
less than 7.35 (acidosis) or more than 7.45 (alkalosis).
Acidosis is the condition in which the blood pH tends to fall
below 7.35.
Alkalosis is the condition in which the blood pH tends to rise
above 7.45.
Imbalance of blood pH may be:
A. Respiratory: [Changes in H
2
CO
3
(CO
2
) level]:
This includes respiratory acidosis and alkalosis.
B. Metabolic: [Changes in HCO
3
level]:
This includes metabolic acidosis and alkalosis.
20
Respiratory Acidosis:
It is caused by increased blood H
2
CO
3
due to failure of the
lungs to excrete CO
2
at the proper rate as in bronchial asthma
& morphine poisoning.
At first HCO
3
/H
2
CO
3
is decreased below 20:1 (HCO
3
is
normal, H
2
CO
3
is increased and pH is decreased).
This stimulates the kidneys to reabsorb more HCO
3
increasing blood HCO

3
till HCO
3
/ H
2
CO
3
becomes 20:1. This
is called compensated respiratory acidosis (HCO
3
is
increased, H
2
CO
3
is increased and pH is normal).
21
Respiratory Alkalosis:
It is caused by increased blood H
2
CO
3
due to failure of the
lungs to excrete CO
2
at the proper rate as in bronchial asthma
& morphine poisoning.
At first HCO
3
/ H
2
CO
3
is increased above 20:1 (HCO
3
is
normal, H
2
CO
3
is decreased and pH is increased).
This inhibits reabsorption of HCO
3
through the kidneys

decreasing blood HCO
3
till HCO
3
/ H
2
CO
3
becomes 20:1. This
is called compensated respiratory alkalosis (HCO
3
is
decreased, H
2
CO
3
is decreased and pH is normal).
22
MetabolicAcidosis:
It is caused by decreased blood HCO
3
due to increased
production and accumulation of acids (hydroxybutyric
acid and acetoacetic acid in diabetic ketoacidosis), failure of
excretion of acids (renal failure) and increased loss of bases
(severe diarrhea).
At first HCO
3
/H
2
CO
3
is decreased below 20:1 (HCO
3
is
decreased, H
2
CO
3
is normal and pH is decreased).
This stimulates the lungs to expirate more CO
2
decreasing
H2CO3 till HCO
3
/H
2
CO
3
becomes 20:1. This is called
compensated metabolic acidosis (HCO
3
is decreased, H
2
CO
3
is decreased and pH is normal).
BloodpHDisorder
(AcidBaseDisturbance)
23
MetabolicAcidosis:
Plasma anion gap = plasma Na
+
(plasma Cl
+ plasma HCO
3
)
The anion gap is due to unmeasured anions (e.g. proteins, SO
4
2
,
HPO
4
2
) that are present in plasma.
It is about 12 mmol/l in healthy subjects ranging from 7 12 mmol/l.
This gap can explain the cause of metabolic acidosis. It is increased in
metabolic acidosis due to increased production and accumulation of
acids (hydroxybutyric acid and acetoacetic acid in diabetic
ketoacidosis), but the gap is normal in metabolic acidosis due to
failure of excretion of acids (renal failure) and increased loss of bases
(severe diarrhea).
Urinary anion gap = (urine Na
+
+ urine K
+
) urine Cl
This gap can detect the renal cause of metabolic acidosis.

If the gap is negative this indicates that there is no renal cause of
metabolic acidosis.
BloodpHDisorder
AcidBaseDisturbance
249
MetabolicAlkalosis:
It is caused by increased blood HCO
3
due to increased
loss of acids (severe vomiting) and increased
accumulation of bases (administration of large doses of
bicarbonate in the treatment of peptic ulcer).
At first HCO
3
/H
2
CO
3
is increased above 20:1 (HCO
3
is
increased, H
2
CO
3
is normal and pH is increased).
This inhibits expiration of CO
2
through the lungs
increasing blood H
2
CO
3
till HCO
3
/H
2
CO
3
becomes 20:1.
This is called compensated metabolic alkalosis (HCO
3
is
increased, H
2
CO
3
is increased and pH is normal).
BloodpHDisorder
25
Acidaemia is uncompensated acidosis where blood pH falls
below 7.35.
Alkalaemia is uncompensated alkalosis where blood pH rises
above 7.45.
Arterial blood gases (ABG) are investigated by the followings
o Arterial blood pH.
o PO
2
.
o PCO
2
.
o Bicarbonate (total CO
2
).
BloodpHDisorder
26
Blood Calcium:
The erythrocytes contain almost no calcium.
Plasma calcium level ranges from 8.5 to 10.5 mg / dl (2.1 to
2.6 mmol / l).
Plasma calcium exists in 2 forms:
Nondiffusible (45%): This is represented by calcium bound to
plasma proteins, mainly albumin. It is physiologically inactive.
Diffusible (55%): This form of calcium may be:
Ionizable (50%): This ionizable calcium is the only
physiologically active form.
Nonionizable (5%): This is mostly in the form of citrate salt. It
is physiologically inactive.
Calcium
27
Calcium
Factors Affecting Plasma Calcium:
I Hormonal Factors: The Calcium regulating hormones are:
1. Parathyroid hormone (parathormone = PTH): It is secreted from the four parathyroid glands.
It increases the plasma calciumlevel.
2. Active form of vitamin D3 (1, 25 dihydroxycholecalciferol = calcitriol): It increases the plasma
calciumlevel.
3. Thyrocalcitonin (calcitonin): It is secreted from the parafollicularC cells of thyroid gland. It
decreases the plasma calcium level.
II NonHormonal Factors:
1. Blood pH: Ionization of calcium occurs at normal blood pH (7.4). Alkalosis decreases ionized
calcium.
2. Plasma proteins: In cases of hypoproteinaemia (as in albuminuria), the nondiffusible
calcium decreases. It was found that every one gram loss of albumin in urine leads to a
decrease of about 0.8 mg / dl in total calcium level.
3. Plasma phosphate: The solubility product (Ca X P = about 50) must be constant. If plasma
phosphate increases (as in renal failure), the plasma calcium decreases to keep Ca/P ratio
constant.
28
Calcium
Functions of Calcium
I Unionized calcium:
It enters in the structure of the skeleton.
II Ionized calcium: It is important for:
Decrease of neuromuscular excitability. So, deficiency of ionized calcium leads to
tetany.
Blood and milk clotting.
Maintenance of cell membrane permeability.
Activation of certain enzymes e.g. glycogen phosphorylase and pyruvate kinase.
Mediation of some hormone responses.
29
Calcium
Alteration of Plasma Calcium
I Hypercalcaemia: It is the increase of plasma calcium level more than 11.0
mg / dl. It is caused by:
1. Hyperparathyroidism, mainly primary (and also secondary and
tertiary).
2. Excess intake of vitamin D and/or calcium.
3. Milkalkali syndrome where hypercalcaemia is present in patients
receiving (for long time) excessive absorbable alkalies and milk for
the treatment of peptic ulcer.
4. Malignancy as in leukaemia, multiple myeloma and Pagets disease.
5. Drugs as thiazides diuretics.
6. Other causes as thyrotoxicosis and Cushings syndrome.
N.B.: In hypercalcaemia there is frequent formation of urinary tract
stones especially calcium oxalate and triple phosphate stones.
30
250
Calcium
Alteration of Plasma Calcium:
II Hypocalcaemia: It is the decrease of plasma calcium level less
than 8.0 mg / dl. It is caused by:
1. Hyporparathyroidism.
2. Alkalosis.
3. Kidney diseases.
N.B.: The decrease in ionized calcium leads to tetany.
Deficiency of calcium leads to rickets in children and
oesteomalacia in adults.
31
Phosphorus
Sources:
Milk and milk products.
Meat, organs meat and fish.
leafy vegetables and egg yolk.
Body Phosphorus:
Total body phosphorus is about 800 g.
About 80% of it is present in the skeleton.
About 20% is present in other tissues (mostly intracellular) and body fluids
Requirements:
Adults: 800 mg / day.
Children: 1200 mg / day.
Pregnant and lactating women: 1200 mg / day.
32
Phosphorus
Blood Phosphorus:
Normal plasma inorganic phosphorus is 3.0 5.0 mg/dl.
Other forms are present:
In plasma: Phospholipids.
In RBCs: Organic phosphate e.g. ATP, glucose6phosphate.
Factors Affecting Blood Phosphorus:
Parathyroid hormone decreases blood phosphorus.
Active vitamin D3 increases blood phosphorus.
Renal function: In renal failure the plasma inorganic
phosphate increases due to failure of its excretion in the
urine.
33
Phosphorus
Functions of Phosphorus:
It enters in the structure of the skeleton.
It enters in the formation of blood buffers.
It enters in the structure of the following compounds:
1. Phospholipids: e.g. lecithin, cephalins.
2. Phosphoproteins.
3. Nucleic acids: RNA and DNA.
4. Coenzymes: e.g. NAD and NADP.
5. Highenergy phosphate compounds: e.g. ATP, GTP, creatine
phosphate.
6. Cyclic AMP and cyclic GMP.
7. Carbohydrate intermediate e.g. glucose6phosphate, fructose1
phosphate.
34
Magnesium
Sources:
Green leafy vegetables, legumes, peas and nuts.
Fish, meat and organs meat.
Requirements:
The recommended daily allowance (RDA) of magnesium is 300 mg for the normal adults.
Body Magnesium:
The total body magnesium is about 21 g.
About 70% of it is present in the skeleton.
About 30% is present in the other tissues (mostly intracellular) and body fluids.
Blood Magnesium:
Plasma magnesiumis 2.0 3.0 mg / dl.
Erythrocyte magnesium is 2 3 times higher than plasma magnesium
35
Magnesium
Factors Affecting Plasma Magnesium:
Aldosterone hormone decreases plasma magnesium.
Parathyroid hormone increases plasma magnesium.
Renal function: Renal failure leads to hypermagnesaemia due to failure of excretion of
magnesiumin the urine.
Functions of Magnesium:
It enters in the formation of the skeleton.
It is important for the normal contraction of muscles.
It is important for the normal transmission of nerve impulses.
It decreases the neuromuscular excitability.
It acts as an activator of many enzymes e.g. kinases, phosphatases, phosphorylases
Alteration of Plasma Magnesium:
Hypermagnesaemia leads to muscular weakness, paralysis, somnolence and anaesthesia.
These effects can be antagonized by calcium.
Hypomagnesaemia may result from parathyroidectomy and leads to tetany which is
refractory to calcium therapy.
36
Iron
Sources:
Organs meat (liver, kidney, heart, spleen), meat and fish.
Legumes, vegetables and whole grains.
Requirements:
Adults: 10 mg / day.
Pregnant and lactating women: 30 mg/day.
37
Iron
Body Iron:
The total body iron is 3 5 g.
It is present in the body in two forms:
A. Functional forms (75%):
These are mostly in the form of haemoproteins. They are responsible for cellular
respiration. They include:
1. Haemoglobin (67%): This is the main form of iron in the body. It is the haemoprotein
present within the red blood cells.
2. Myoglobin (7.5%): This is a haemoprotein found in muscles and heart.
3. Respiratory enzymes (0.5%): These are the haemoproteins which include:
1. Respiratory cytochromes (b, c1, c, a, a3) which are electron carriers in the respiratory chain
within the mitochondria.
2. Catalase and peroxidase which are important in the detoxication of hydrogen peroxide (H
2
O
2
).
3. Tryptophan oxygenase (Pyrrolase) which is important in tryptophan metabolism.
4. Cytochrome P450 which is found in the mitochondria and microsomes of the liver. It is
important in the detoxication of xenobiotic agents (toxic drugs and foreign chemicals).
38
Iron
B. NonFunctional forms (25%):
These are nonhaeme metalloproteins. They include:
1. Transferrin: the transport form of iron in blood plasma.
2. Ferritin: the storage form of iron in the tissues. It is present in the liver,
kidney, spleen, bone marrow & intestinal mucosa.
3. Haemosiderin: found only when the body contains excess iron.
39
Iron
Blood Iron:
A. In the red blood cells:
Every gram haemoglobin contains 3 4 mg iron.
So, there is about 50 mg of iron per 100 ml blood because there is about 15 gram
haemoglobin per 100 ml blood.
B. In the plasma:
Plasma iron concentration is 60 160 g / dl.
Iron is carried by transferrin which is a glycoprotein synthesized in the liver and
runs with globulin in electrophoresis.
Transferrin may carry up to 250 400 g of iron per 100 ml blood plasma. This is
known as the total iron binding capacity (TIBC). About 30% of TIBC is saturated.
In iron deficiency anaemia, plasma iron decreases while the TIBC increases. In
liver diseases, both plasma iron and the TIBC decreases.
Plasma contains very low concentration of ferritin which is a very good index of
iron storage. It decreases in iron deficiency and increases in haemosiderosis.
40
Iron
Alteration of Plasma Iron:
A. Iron deficiency anaemia:
Causes:
Deficient intake.
Impaired absorption.
Excessive loss.
Biochemical changes:
Plasma iron is decreased.
Plasma TIBC is increased.
Plasma ferritin is decreased.
41
Iron
B. Iron overload:
Causes:
Repeated blood transfusion.
Intravenous administration of iron.
Hechromatosis (Hemosiderosis; Bronz Diabetes):
Rare hereditary disease characterized by abnormal increase of iron
absorption.
Iron is deposited in the form of hemosiderin in:
Liver: causing liver cirrhosis.
Pancreas: causing fibrosis and diabetes mellitus.
Skin: causing bronz discolouration of skin
Biochemical changes:
Plasma iron is increased.
Plasma TIBC is decreased.
Plasma ferritin is increased.
42
MinistryofHealth
Institute Graduates
ClinicalEnzymology
Definitions
Enzymes are biological catalysts of protein nature
composed of 200 or more amino acids covalently
linked in a sequence dictated by cells genetic code.
Isoenzymes are different molecular forms of the
same enzyme having the same catalytic activity but
differing in physicochemical properties.
2
PlasmaEnzymes
Most enzymes occur at higher concentration
intracellularly than in plasma.
Plasma enzymes can be classified into:
1. Functional plasma enzymes.
2. Nonfunctional plasma enzymes.
3
1.FunctionalPlasmaEnzymes
These are enzymes which act on substrates normally
present in plasma e.g. coagulation enzymes and
pseudocholinesterase.
4
2.NonFunctionalPlasmaEnzymes
These are enzymes that are synthesized in the cells where
they act.
They enter the plasma in small amounts as a result of
continuous cell aging or due to diffusion during inactivation
and catabolism or rarely by excretion into bile or urine.
They include the enzymes of intermediary metabolism e.g.
SGOT, SGPT, ALP, LDH, CPK, amylase, etc.
5
Plasma enzymes can also be classified into:
1. Diagnostic enzymes
2. Prognostic enzymes
6
253
1.DiagnosticEnzymes
These are enzymes by which one can diagnose the
disease because of the specificity of the enzyme to
the affected organ or tissue e.g.:
ALT for liver diseases.
G6PDH for favism.
CPKMB for myocardial infarction.
7
2.PrognosticEnzymes
For follow up of certain diseases and not the
diagnosis because these enzymes are present in
more than one tissue e.g.:
AST: for heart , liver and skeletal muscles.
LDH : for heart , liver and skeletal muscles.
CPK : for heart , brain and skeletal muscles.
ALP : for liver and bones.
8
The normal plasma level of enzymes reflects the
balance between:
Release of enzymes during normal cell turnover.
and
Their catabolism and excretion.
9
WhenwillPlasmaLevelofEnzymesbe
Altered?
1. If there is altered synthesis of enzymes within the
cells.
2. If there is a change in the amount of enzymes
forming tissues (by cell proliferation).
3. If there is a change in cell permeability.
4. If there is alteration in the rate of inactivation and
disposal of enzymes.
5. If there is an obstruction to normal pathway of
enzyme excretion.
10
NonspecificCausesforRaisedPlasmaEnzyme
Levels
A. Physiological:
Newborn: e.g.
Creatine phosphokinase (CPK).
Aspartate transaminase (AST).
Childhood: e.g.
Alkaline phosphatase (ALP).
Pregnancy: e.g.
ALP (in the last trimester).
CPK & AST (during & immediately after labour).
11
NonspecificCausesforRaisedPlasmaEnzyme
Levels
B. Enzyme induction by drugs:
Diphenylhydantoin & barbiturates therapy increases ALP level.
Alcohol intake increases gamma glutamyl transpeptidase (GGT).
C. Artfactual elevation:
Lactate dehydrogenase, acid phosphatase and transaminases are
elevated in haemolysed samples.
Amylase may be elevated if mouth pippetting is the rule.
Acid phosphatase is elevated if catheterization or per rectum examination
is done within one week before the investigation.
Total CPK is increased after intramuscular injection or after muscular
exercise.
12
254
ClinicallyImportantEnzymes
1. AspartateTransaminase(AST)
or
2. GlutamateOxaloacetateTransaminase(GOT)
13
ASTisaprognosisenzymeincreasedunderthe
followingconditions:
A. Physiological: Newborn .
B. Pathological: In the following diseases:
Myocardial infarction.
Liver diseases:
Viral hepatitis.
Toxic liver necrosis.
Liver cirrhosis.
Cholestatic jaundice.
Malignant infiltration of the liver.
Infectious mononucleosis.
Skeletal muscle diseases: After trauma or surgery.
Circulatory failure with shock and hypoxia.
C. Artfactual: Haemolyzed samples.
14
ClinicallyImportantEnzymes
1. AspartateTransaminase(AST)
or
2. GlutamateOxaloacetateTransaminase(GOT)
15
ALT is a diagnostic enzyme for liver diseases including:
Viral hepatitis.
Toxic liver necrosis.
Liver cirrhosis.
Cholestatic jaundice.
Liver congestion secondary to cardiac failure.
Infectious mononucleosis.
ALT is also increased in circulatory failure with shock and
hypoxia and may be slightly increased in extensive muscle
trauma.
In acute liver diseases ALT is increased first followed by an
increase in AST.
16
3.AlkalinePhosphotase(ALP)
ALP is a prognostic enzyme having five isoenzymes:
bone, liver, placenta, intestinal and Regan
isoenzymes.
It is increased in the following conditions:
A. Physiological:
1. Preterm infants.
2. Children.
3. Pregnant women in the last trimester.
17
B. Pathological: In the following diseases:
I. Bone diseases:
Osteomalacia and rickets.
Pagets disease of bone.
Primary hyperparathyroidism with bone involvement.
Secondary carcinoma deposits in bone.
Extensive osteogenic sarcoma.
Healing phase of bone fracture.
II. Liver diseases:
Cholestasis.
Hepatitis.
Liver cirrhosis.
Spaceoccupying lesions, tumours, infiltrations.
C. Induction by drugs: Barbiturates and diphenylhydantoin.
18
255
4.5Nucleotidase
5Nucleotidase is a diagnostic enzyme for liver
diseases.
Its activity is parallel to that of liver ALP.
It is indicated to exclude the bone diseases as a
cause of elevated ALP level where:
In liver diseases: high ALP and high 5Nucleotidase.
In bone diseases: high ALP and normal 5Nucleotidase.
19
5.GammaGlutamylTranspeptidase(GGT)
GGT is a diagnostic enzyme for liver diseases
increasing in the following conditions:
Liver cirrhosis.
Metastatic carcinoma.
Hepatic infiltration.
Cholestasis.
Alcoholism.
Patients on anticonvulsant therapy.
20
6.Adolase
Aldolase is a prognostic enzyme widely distributed
mainly in muscles, liver and red blood cells.
Its levels are increased in:
Myocardial infarction.
Extensive muscle trauma.
Haemolysis.
Generalized malignancy.
21
7.Amylase
Amylase is a prognostic enzyme present in both pancreatic
juice and saliva.
It is excreted in urine.
Acute pancreatitis: The enzyme rises temporary within the first 24
hours and returns to normal within 2 or 3 days as the acute attack
resolves.
Severe uraemia.
Severe diabetic ketoacidosis.
Perforated peptic ulcer.
Acute cholecystitis.
Ruptured ectopic pregnancy.
Mumps and salivary calculi.
22
8.Lipase
It is a diagnostic enzyme for pancreatic diseases
especially acute pancreatitis.
It has a similar but slower pattern of rise and fall in
pancreatitis than amylase.
Its assay is of value after 48 hours of the onset
where its peak activity is at about 48 hours and the
level usually remains high for about a week.
23
9.AcidPhosphotase
A.Prostaticacidphosphatase:
Itisadiagnosticenzymeespeciallyformetastatic
prostaticcarcinoma.
Itmayalsoincreasedinthefollowingconditions:
1. Followingrectalexamination.
2. Acuteretentionofurine.
3. Afterurinarycatheterization.
4. Chronicconstipation.
24
256
9.AcidPhosphotase
B.Totalacidphosphatase
Itisaprognosticenzymepresentmainlyintheprostateand
alsoinothersourcesincludingliver,redcells,plateletsand
bone.
Itisincreasedinthefollowingconditions:
1. Causesofelevationofprostaticacidphosphatase.
2. Gauchersdisease.
3. Thrombocytopeniawithexcessivedestructionofplatelets.
4. PagetsdiseasewithhighelevationofALP.
5. Artifactuallyinhaemolyzedsamples.
25
10.Creatine Phosphokinase(CPK)
Creatine Kinase(CK)
Total CPK is a prognostic enzyme present in cardiac muscle, brain and skeletal
muscle.
A. Physiological:
1. Newborn.
2. After labour for few days.
B. Pathological :
1. Myocardial infarction.
2. Muscular dystrophies.
3. Muscle injury.
4. After surgery.
5. Severe physical exertion.
6. Hypothyroidism.
7. Alcoholism.
8. Cerebrovascular accident and head injury.
9. Malignant hyperpyrexia.
C. Artfactual: Haemolyzed samples. 26
CPK is a dimmer enzyme composed of 2 subunits of
M & B types the combination of which results in the
formation of 3 isoenzymes:
1. CKMM: Specific for skeletal muscle disorders.
2. CKMB: Specific for heart (Myocardial infarction).
3. CKBB: Specific for brain.
27
11.LactateDehydrogenase(LDH)
LDHisaprognosticenzymewidelydistributedinheart,skeletalmuscle,
liver,kidney,lungs,brain,erythrocytesandmalignanttissues.
Itisincreasedinthefollowingconditions:
A.Pathological:
1. Myocardialinfarction.
2. Haematological disordersasperniciousanaemia,leukaemia andhaemolytic
anaemia.
3. Circulatoryfailurewithshockandhypoxia.
4. Viralhepatitis.
5. Pulmonaryembolism.
6. Infectiousmononucleosis.
7. Occasionallyincerebralandrenalinfarction.
B.Artfactual:Haemolyzed samples.
28
LDH is a tetramer composed of 4 subunits of H & M types the combination
of whish results in the formation of 5 isoenzymes:
1. LDH
1
= H
4
(HHHH)
2. LDH
2
= H
3
M (HHHM)
They are predominant in cardiac muscle (LDH
1
> LDH
2
), red blood cells and
malignant tissues (LDH
2
> LDH
1
).
In myocardial infarction, they are increased with LDH
1
/LDH
2
> 1.
These isoenzymes can be substituted by hydroxybutyryl dehydrogenase
(HBDH) which is also called cardiac LDH .
In haemolytic and pernicious anaemias, these isoenzymes are increased
with LDH
1
/LDH
2
< 1.
3. LDH
3
= H
2
M
2
(HHMM)
It is predominant in lungs and kidneys.
It is increased with LDH
2
in acute leukaemia.
It is increased in pulmonary and renal infarction.
4. LDH
4
= H M
3
(HMMM)
5. LDH
5
= M
4
(MMMM)
They are predominant in skeletal muscle and liver.
Theyareincreasedinmusclediseasesandacutehepatitis.
29
TimingofCardiacMarkersinRelationto
MyocardialInfarction
30
Cardiac Marker
Onset of Rising
(Hour)
Peak of Rising
(Hour)
Duration
of Rising
(Days)
CPKMB 3 5 8 12 1 2
TotalCPK 4 6 12 24 1.5 3
AST(GOT) 8 12 24 36 3 6
LDH 12 24 48 72 6 12
Myoglobin About one 4 8 0.5 1
Cardiactroponin I&T 3 5 24 - 48 7 - 10
257
12.Pseudocholinesterase
Twoenzymesofcholinesterasearepresentinthebody:
A.Truecholinesterase(Acetylcholinesterase):
Itisfoundinthenervoustissuesandskeletalmuscleswithlowerconcentrationin
redbloodcells.
Ithydrolyzestheexcessacetylcholinetopreventcontinuousdischargeofthe
nerveimpulseorcontinuouscontractionofthemuscleaftercompletionof
impulse.
B.Pseudocholinesterase (Plasmacholinesterase)
Itiswidelydistributedinthebodyincludingtheliver(siteofitssynthesis)and
plasma.
Ithasnoeffectonacetylcholinepresentinthenerveendingsbutitdestroysany
acetylcholinewhichmightescapetheactionofacetylcholinesteraseandreach
theblood.
Itdestroysothercholineandnoncholineesterse.g.succinylcholineusedasa
musclerelaxantduringgeneralanaesthesia.
So,itisadvisedtomeasuretheenzymeactivitybeforegeneralanaesthesia to
avoidthedangerofprolongedperiodsofapneaaftermajoroperationsin
susceptiblepersons. 31
Pseudocholinesterase activity is decreased in:
1. Hepatic parenchymal diseases (hepatitis, cirrhosis, congestion).
2. Organophosphorus poisoning:
Enzyme activity is markedly decreased before the toxic effect of organophosphorus
compounds on the central nervous system.
So, estimation of the enzyme activity is used as a monitor for patients of
organophosphorus poisoning and for workers in insecticides factories.
3. Inherited abnormal cholinesterase variants with low biological activity.
Its activity is increased in:
1. Recovery from liver damage.
2. Nephrotic syndrome.
3. Obesity, thyrotoxicosis, hypertension and alcoholism.
32
Pseudocholinesterase activity is decreased in:
1. Hepatic parenchymal diseases (hepatitis, cirrhosis, congestion).
2. Organophosphorus poisoning:
Enzyme activity is markedly decreased before the toxic effect of
organophosphorus compounds on the central nervous system.
So, estimation of the enzyme activity is used as a monitor for patients
of organophosphorus poisoning and for workers in insecticides
factories.
3. Inherited abnormal cholinesterase variants with low biological
activity.
Its activity is increased in:
1. Recovery from liver damage.
2. Nephrotic syndrome.
3. Obesity, thyrotoxicosis, hypertension and alcoholism.
33
13.Glucose6PhosphateDehydrogenase
(G6PDH)
G6PDH is the main enzyme of hexose monophosphate (HMP) shunt
pathway.
It is important for the integrity of red blood cells through the production
of reduced coenzyme II (NADPH + H+).
Its deficiency may cause haemolytic anaemia called Favism which is
precipitated by administration of certain oxidizing agents such as Fava
beans, antimalarial drugs, sulpha drugs, phenylbutazone and vitamin K
analogues.
Its synthesis is induced in these patients by the age of 9 11 years.
34
NADPH+H
+
NADP
Pentose
Phosphate
Pathway
Glutathione
Reductase
Glutathione
Peroxidase
H2O2
G-S-S-G
2 G-SH
FAD
2 H2O
Se
Glucose-6-phosphate
Dehydrogenase
258
MinistryofHealth
Institute Graduates
LiverMetabolism
RoleofLiverinMetabolism
The liver plays an important regulatory role in:
I. Carbohydrate Metabolism.
II. Lipid Metabolism.
III. Protein Metabolism.
IV. Metabolism of Foreign Organic Substances.
V. Vitamin Metabolism.
VI. Mineral Metabolism.
2
RoleofLiverinCarbohydrateMetabolism
The liver is the blood glucostat.
A. After carbohydrate meal:
The liver prevents excessive hyperglycaemia by increasing the
uptake and utilization of glucose by oxidation (glycolysis and
Krebs cycle), glycogenesis and lipogenesis.
It converts fructose and galactose into glucose preventing
their excretion in the urine.
B. During fasting:
The main source of blood glucose is the liver where
glycogenolysis and gluconeogenesis occur.
3
RoleofLiverinLipidsMetabolism
Major site for fatty acid oxidation.
Synthesis, mobilization and oxidation of triglycerides.
Site for fatty acids biosynthesis from excess glucose.
Site for synthesis of cholesterol from active acetate.
Main site for ketogenesis.
Major site for phospholipids & lipoproteins synthesis.
Site for degradation of cholesterol, phospholipids and
lipoproteins.
Only site for synthesis of bile salts.
Site of storage of fatsoluble vitamins.
Only site for detoxication of steroid hormones & drugs.
4
RoleofLiverinProteinMetabolism
Deamination of amino acids.
Formation of urea from the ammonia resulting from the
deamination of amino acids.
Gluconeogenesis from the carbon skeleton of the
deaminated amino acids.
Biosynthesis of the nonessential amino acids.
Biosynthesis and degradation of its own proteins as well as of
most of the plasma proteins.
Biosynthesis of creatine, purines and pyrimidines.
Catabolism of purines, pyrimidines and haemoglobin.
5
RoleofLiverinMetabolismofForeignOrganic
Substances
Numerous drugs are detoxicated by the liver and some are
excreted in bile.
Benzoic acid is detoxicated by conjugation with glycine to
form hippuric acid which is excreted in urine.
6
259
RoleofLiverinVitaminMetabolism
Liver has storage function for fatsoluble vitamins.
It helps absorption of the fatsoluble vitamins through the
formation of the bile salts.
It converts carotenes to vitamin A, and vitamin D
3
to 25
hydroxy vitamin D
3
.
It converts tryptophan to niacin.
It utilizes vitamin K for the synthesis of prothrombin and
blood clotting factors VII, IX and X.
7
RoleofLiverinMineralMetabolism
The liver is an important storage site for iron and copper.
It is the main route of excretion of copper.
8
BilirubinMetabolism
The average life span of the red blood cells is 120 days.
Every day, about 250 mg of bile pigments are produced by
the catabolism of about 6.25 g of haemoglobin.
The formation of the bile pigments can be divided into 3
stages:
1. In the Reticuloendothelial Cells.
2. In the Liver.
3. In the Intestines.
9
BilirubinMetabolism
10
BilirubinMetabolism
11
VandenBerghReaction
This is a reaction between bilirubin and Ehrlich diazo
(diazotized sulfanilic acid) reagent giving violet
colour.
Conjugated bilirubin reacts directly with the reagent.
So, it is called direct bilirubin.
Unconjugated bilirubin does not react with the
reagent directly except after addition of methyl
alcohol. So, it is called indirect bilirubin.
12
260
DifferencesBetweenUnconjugatedand
ConjugatedBilirubin
13
Unconjugated Bilirubin Conjugated Bilirubin
1. Present normally in plasma. 1. Present normally in bile.
2. Attached to albumin. 2. Conjugated to glucuronicacid.
3. Insolublein water. 3. Solublein water.
4. Cannot befiltered through thekidney. 4. Can befiltered through thekidney.
5. Can cross blood-brain barrier. 5. Cannot cross blood-brain barrier.
6. Gives indirect Van den Bergh reaction. 6. Gives direct Van den Bergh reaction.
Jaundice
Jaundice is the yellowish discoloration of the skin,
mucous membranes and sclera due to increased
plasma bilirubin level more than 2.0 mg/dl.
14
Jaundice
Normally: Plasma total bilirubin is 0.2 1.0 mg/dl, direct bilirubin is 0.00
0.25 mg/dl and indirect bilirubin is 0.20 0.80 mg/dl.
Hyperbilirubinaemia results when plasma bilirubin level exceeds 1.0
mg/dl.
Values between 1.0 and 2.0 mg/dl are considered latent jaundice.
Hyperbilirubinaemia may be due to increased conjugated and/or
unconjugated bilirubin.
Jaundice can be classified into:
1. Haemolytic (Prehepatic) jaundice.
2. Hepatocellular or Toxic (Hepatic) Jaundice.
3. Obstructive or cholestatic (Posthepatic) Jaundice.
15
Haemolytic Jaundice(PreHepaticJaundice)
This is due to excessive haemolysis leading to
increase in the amount of plasma unconjugated
bilirubin more than the conjugating capacity of the
liver.
Biochemical changes are:
1. Unconjugated hyperbilirubinaemia.
2. Increased urobilinogen in urine and stools.
3. No bilirubin appears in urine.
16
HepatocellularJaundice(ToxicJaundice)
(HepaticJaundice)
It is due to liver damage by cirrhosis, hepatitis and
toxins.
There is an associated obstruction of some biliary
canaliculi.
1. Unconjugated & conjugated hyperbilirubinaemia.
2. Trace amount of urobilinogen in urine & stools.
3. Bilirubin appears in urine.
17
ObstructiveJaundice(Cholestatic Jaundice)
(PostHepaticJaundice)
Cholestasis may be due to obstruction of biliary tree
by gall stone in common bile duct, cancer head
pancreas or carcinoma of biliary tree.
1. Conjugated hyperbilirubinaemia.
2. Urobilinogen is absent in urine and stools.
3. Bilirubin appears in urine.
18
261
CausesofJaundice
19
BiochemicalChangesinNormals andthe3
TypesofJaundice
20
Condition
Plasma Urine
Foecal
Stercobilinogen
(mg/day)
T.Bilirubin
(mg/dl)
D.Bilirubin
(mg/dl)
Bilirubin
Urobilinogen
(mg/day)
1. Normals
2.Haemolytic
jaundice
3.Hepatic
jaundice
4.Obstructive
jaundice
0.2 1.0
Increased
Increased
Increased
0.0 0.25
Normal
Increased
Increased
Negative
Negative
Positive
Positive
0 3
Increased
Decreased
Normal
Decreased
30 300
Increased
Decreased
Normal
Decreased
PhysiologicalNeonatalJaundice
This is a transient condition occurring in some newborn infants especially
if they are premature due to immaturity of UDP glucuronyltansferase
enzyme and accelerated haemolysis of RBCs.
This leads to unconjugated hyperbilirubinaemia and jaundice which lasts
2 3 days in fullterm infants and 1 2 weeks in premature infants.
If plasma indirect bilirubin exceeds the capacity of plasma albumin (20
25 mg/dl), free bilirubin can cross bloodbrain barrier causing kernicterus
(toxic encephalopathy) which can lead to mental retardation.
Neonatal jaundice is treated by Phenobarbital and by exposure of the
infant to visible light (phototherapy).
21
CongenitalHyperbilirubinaemia
Gilberts Disease:
Asymptomatic unconjugated hyperbilirubinaemia due to a defect in the uptake of
bilirubin by the liver cells and a mild deficiency of UDP glucuronyltransferase
enzyme.
CriglerNajjar Syndrome:
Severe unconjugated hyperbilirubinaemia due to marked reduction of UDP
glucuronyltransferase.
DubinJohnson syndrome:
Conjugated hyperbilirubinaemia due to a defect in the hepatic secretion of direct
bilirubin into the bile.
Rotors syndrome:
Conjugated hyperbilirubinaemia with normal liver histology.
Its cause has not been identified, but it may be due to a defect in transport by
hepatocytes for organic anions, including bilirubin.
22
23
BileAcidsandBileSalts
II. Secondary Bile Acids:
They are synthesized in the intestine from primary bile acids.
They include:
1. Deoxycholic acid.
2. Lithocholic acid.
Greater proportions of these bile acids are reabsorbed from
the intestine to the liver again and then are reexcreted in the
bile forming what is called enterohepatic circulation of bile
acids.
The nonabsorbed bile acids are excreted in foeces.
24
262
Gallstones
Gallstones are solid concretions that are most commonly
formed in the gallbladder and occasionally in the bile ducts.
There are three major types of gallstones:
1. Cholesterol gallstones:
They can be examined and detected by the LiebermannBurchard reaction.
2. Pigmented gallstones:
The pigment is mainly bilirubin which is present as calcium bilirubinate.
They can be examined and detected by using Ehrlich diazo reagent.
3. Mixed gallstones:
They may contain a mixture of cholesterol, bilirubin, calcium phosphate,
calcium carbonate and mucoproteins.
25
LiverFunctionTests
26
I. SyntheticFunctionTests:
1) Plasma total protein: (6.5 8.0 g/dl).
All plasma proteins are synthesized in the liver except globulin.
2) Plasma albumin: (3.8 5.0 g/dl).
Albumin is synthesized in the liver.
Hypoalbuminaemia occurs in acute and chronic liver diseases.
3) Albumin / globulin ratio (A/G): (1 2/1).
Plasma albumin
A/G =
Plasma total protein (Plasma albumin + Fibrinogen)
Serum albumin
A/G =
Serum total protein Serum albumin
4) Prothrombin time: (Concentration: 70 120%).
Measurement of Prothrombin concentration and its response to vitamin
K is a useful test of liver function.
LiverFunctionTests
II. Excretory Function Tests:
1. Plasma total bilirubin: (0.2 1.0 mg/dl).
It is increased in all types of jaundice.
2. Plasma direct bilirubin: (0.0 0.25 mg/dl).
It is increased in obstructive and hepatocellular jaundice.
3. Alkaline phosphatase:
It is normally increased in children, and pregnant women
in last months.
It is increased in obstructive jaundice and osteolytic
bone diseases.
27
LiverFunctionTests
III. Tests Depending on Integrity of Liver cells (Liver Enzymes):
The following enzymes are increased in acute hepatocellular damage as
in viral hepatitis:
1. Plasma alanine transaminase (ALT)
= Plasma glutamate pyruvate transaminase (GPT)
2. Plasma aspartate transaminase (AST)
= Plasma glutamate oxaloacetate transaminase (GOT)
3. Plasma gamma glutamyl transpeptidase (GGT)
It is induced by alcoholic intake and by hypnotics.
4. Other plasma enzymes:
These include 5`nucleotidase, aldolase and lactate dehydrogenase (LDH)
especially LDH
5
.
28
263
MinistryofHealth
Institute Graduates
CardiacBiomarkers
2
HEART
TheHeart
The heart is an efficient and durable pump.
It is a muscular organ responsible for moving blood through
blood vessel to all parts of the body.
Work of the heart is generated by the alternating contraction
and relaxation of heart muscle fibres.
These muscle fibres are composed of cardiacspecific
contractile proteins (actin and myosin) and regulatory
proteins (troponin and tropomyocin).
They also contain proteins (such as myoglobin) and enzymes
(such as creatine kinase, lactate dehydrogenase and
aspartate transaminase) that are vital for energy use.
3
SourcesofEnergyfortheHeart
Energy is liberated from various substances that are used in the heart as
fuels by several pathways:
1. EmbdenMeyerhof glycolytic pathway.
2. Fatty acid oxidation.
3. Krebs citric acid cycle.
The heart uses free fatty acids as its predominant fuel. It also consumes
significant quantities of glucose and lactate, as well as lesser amounts of
pyruvate, ketone bodies and amino acids.
Most of the energy for cardiac function is obtained from the breakdown
of metabolites through the citric acid cycle and the oxidative
phosphorylation
4
StorageofEnergyinCardiacMuscle
The heart requires an effective storage method to
maintain a reserve of ATP.
This is achieved through the synthesis of creatine
phosphate, which acts as an available store for rapid
regeneration of ATP on need.
Mg
2+
5
EncounteredMedicalTerms
Atherosclerosis.
Acute coronary syndrome.
Acute myocardial infarction.
Deep venous thrombosis.
Pulmonary circulation.
Pulmonary embolism.
Heart failure.
6
264
Artherosclerosis
Atherosclerosis is the
deposition of plaques
containing cholesterol
and lipids on the intima
(innermost layer of the
walls) of large and
mediumsized arteries.
7
CoronaryAtherosclerosis
Coronary atherosclerosis is an inflammatory disease
characterized by the accumulation of white blood
cells, cell debris, fatty substances (cholesterol and
fatty acids), calcium, and fibrous tissue (plaque or
atheromas) on the walls of the coronary arteries
that supply the heart muscle.
8
CoronaryStenosis
As plaque slowly increases in size over many years,
the artery narrows in places (stenosis), and blood
flow to the heart is reduced.
9
CoronaryStenosis
Cholesterolcontaining plaques are highly
dangerous even without narrowing of the vessel
because the fibrous cap can be softened and
rupture suddenly during acute heavy exercise or
activity. This can cause bleeding from the blood
vessel wall, resulting in blood clot formation that
may obstruct the vessel.
10
MyocardialIschaemia
The stenosis of coronary arteries may become so
significant that the blood supply is inadequate to
meet the needs of the heart (myocardial ischaemia),
and the affected part of the heart muscle no longer
functions normally.
Myocardial ischaemia typically results in chest pain
(angina pectoris), but may also cause no symptoms
(silent ischaemia). Total blockage of a coronary
artery results in a heart attack (myocardial
infarction).
11
AcuteCoronarySyndrome
Acute coronary syndrome is a spectrum of
conditions involving chest discomfort or other
symptoms caused by lack of oxygen to the heart
muscle (the myocardium) due to sudden, reduced
blood flow to the heart (insufficient blood supply to
the heart muscle that results from coronary artery
disease; CHD).
Acute coronary syndrome is treatable if diagnosed
quickly.
12
265
Acute myocardial infarction is the myocardial tissue
death (necrosis) due to prolonged ischaemia that is
usually caused by arteriosclerosis with narrowing of
the coronary arteries, leading to thrombosis (clot
formation).
13
Diagramofamyocardialinfarction
LCA: Left coronary artery
RCA: Right coronary artery
1. Branch of the left coronary artery
2. Tip of the anterior wall of the heart (an apical infarct).
14
DeepVenousThrombosis
Deep venous thrombosis (DVT) is clotting of blood in
a deep vein of an extremity (usually calf or thigh) or
the pelvis.
It is a form of thrombophlebitis (inflammation of a
vein with clot formation).
DVT results from conditions that impair venous
return, lead to endothelial injury or dysfunction, or
cause hypercoagulability).
DVT is the primary cause of pulmonary embolism.
15
PulmonaryCirculation
Deep venous thrombosis (DVT) is clotting of blood in
a deep vein of an extremity (usually calf or thigh) or
the pelvis.
It is a form of thrombophlebitis (inflammation of a
vein with clot formation).
DVT results from conditions that impair venous
return, lead to endothelial injury or dysfunction, or
cause hypercoagulability).
DVT is the primary cause of pulmonary embolism.
16
PulmonaryCirculation
Pulmonary circulation is the portion of the cardiovascular
system which carries venous nonoxygenated blood from the
right ventricle of the heart, to the lungs, and then returns
oxygenated blood back to the left atrium of the heart.
17
PulmonaryEmbolism
Pulmonary embolism is the obstruction of the
pulmonary artery or a branch of it leading to the
lungs by a blood clot, usually from the leg, or foreign
material causing sudden closure of the vessel.
18
266
HeartFailure
Heart failure is a clinical syndrome characterized by systemic
perfusion inadequacy to meet the body's metabolic demands
as a result of impaired cardiac pump function.
This may be further subdivided into systolic or diastolic heart
failure.
In systolic heart failure, there is reduced cardiac contractility,
whereas in diastolic heart failure there is impaired cardiac
relaxation and abnormal ventricular filling.
Heart failure may be due to failure of the right or left or both
ventricles.
19
CardiacMarkers
These markers help in the early diagnosis of acute
myocardial infarction (AMI) resulting from coronary
heart disease (CHD) in addition to the clinical picture
of the patient and electrocardiography (ECG).
When myocytes become necrotic, they loose their
membrane integrity, & intracellular macromolecules
diffuse into the cardiac interstitial space & ultimately
into the cardiac microvasculature and lymphatics,
then these macromolecules are detectable in the
peripheral circulation.
20
CardiacMarkers
21
IdealCardiacMarker
Diagnostically it has:
1. High sensitivity (Detection of AMI positive cases).
2. High specificity (Absent in nonmyocardial injury).
3. Rapidly release at a detectable concentration.
4. Correlates efficiently with extent of the infarction.
5. Persists in blood for valuable time (Prolonged life)
Analytically it has:
1. High sensitivity (Low detectable limit).
2. High specificity (Less interferences).
3. Easy, inexpensive and tested rapidly (Short TAT).
22
ImportanceofCardiacMarkers
1. To confirm the diagnosis of AMI when diagnosis by
ECG is unclear (no STsegment elevation).
2. To distinguish patients with unstable angina from
those with nonQ wave AMI.
3. To provide prognostic information: as a non
invasive assessment of the likehood that the
patient has undergone successful reperfusion when
thrombolytic therapy is administered.
23
ClassificationofCardiacBiomarkers
I. Traditional cardiac markers:
A. Cardiac enzymes:
1. Creatine kinase (CK) = Creatine phosphokinase (CPK).
2. Aspartate transaminase (AST).
3. Lactate dehydrogenase (LDH).
4. Hydroxy butyrate dehydrogenase (HBDH).
B. Cardiac proteins:
1. Myoglobin.
2. Cardiac Tropinin I & T
24
II. Recent cardiac markers:
A. Markers for AMI:
1. Cardiac Tropinin I & T.
2. CKMB mass.
3. Myoglobin.
B. Marker for heart failure:
1. Bnatriuretic peptides (BNP & Pro BNP).
C. Marker for pulmonary embolism:
1. Ddimer.
25
III. Future cardiac markers:
1. Ischaemia Modified Albumin (IMA).
2. Heart typeFatty Acid Binding Protein (HFABP).
3. Glycogen PhosphorylaseBB (GPBB).
4. Copeptin.
26
Creatine Kinase(CK)
Creatine Phosphokinase(CPK)
It is also called total CK where it is found in most tissues, but
in high concentration in skeletal muscle, cardiac muscle and
brain. So, it is a prognostic but not diagnostic enzyme.
It plays an important role in production of energy for muscle
on need.
Creatine Kinase
Creatine + ATP Creatine Phosphate + ADP
Mg
2+
Its level in males is more than that in females because of the
difference in muscle bulk.
27
CreatineKinase(CK)
Its level is increased in:
1. Muscular diseases e.g. muscular dystrophy &polymyositis,
and muscle fatigue as after severe muscular exercise.
2. AMI where the enzyme starts to increase 4 6 hours after
the chest pain, then reaches its peak 12 24 hours after the
attack and lastly returns to its normal level 36 72 hours
after the onset of infarction.
3. Cerebrovascular accidents and severe neurogenic shock.
28
CreatineKinase(CK)
CK is a dimmer composed of two subunits: M (muscle)
and/or B (brain). The combination of these subunits
results in the formation of 3 isoenzymes:
1. CKMM: Specific for skeletal muscle.
2. CKBB: In brain, bladder, placenta, prostate, lungs and
thyroid glands.
3. CKMB: About 45% in cardiac muscle and < 2% in skeletal
muscle. Its level starts to increase 3 5 hours after AMI,
then reaches its peak 8 12 hours after chest pain and
lastly returns to its normal level 24 48 hours after the
attack.
29
CreatineKinase(CK)
For CK assay in patients with AMI, the blood sample
should not be aspirated within the first 3 hours or
after 3 days of the attack.
During the follow up of these patients by total CK
assay, the patients must not take analgesics or
sedatives by intramuscular route to avoid any
increase in CKMM that leads to misdiagnosis of
superimposed infarction.
30
268
CKMB
mass
For CK assay in patients with AMI, the blood sample
should not be aspirated within the first 3 hours or
after 3 days of the attack.
During the follow up of these patients by total CK
assay, the patients must not take analgesics or
sedatives by intramuscular route to avoid any
increase in CKMM that leads to misdiagnosis of
superimposed infarction.
31
CKMB
mass
32
The mass assay for CKMB measures the amount of CKMB
present in human serum or plasma for confirmation of acute
myocardial infarction. The mass CKMB assay gives more
specific patient results than traditional measurement of
enzyme activity assays.
CKMB
mass
relative index (%RI) is calculated as follows:
%RI = [CKMb
mass
(g/L) Total CK activity (U/L)] X 100
Increased RI suggests myocardial origin.
RI > 3 6 % with increased total CK activity suggests
myocardial necrosis.
RI helps to ruleout patients with skeletal muscle injury.
AspartateTransaminase(AST)
It is also called glutamate oxaloacetate transaminase
(GOT).
It is also a liver enzyme. So, it is a prognostic
enzyme, but not a diagnostic one.
Its level starts to increase 8 12 hours after
infarction, then reaches its peak 24 36 hours after
onset of chest pain and lastly returns to its normal
level 3 6 days after the infarction.
33
LactateDehydrogenase(LDH)
LDH is a reversible hydrogen transfer enzyme that
catalyzes the reduction of lactate to pyruvate and
vice versa using NAD as a hydrogen carrier.
LDH
Lactate + NAD Pyruvate + NADH
+
H
+
It is also called total LDH where it is found in skeletal
muscle, liver, heart, kidney and red blood cells.
34
35
It is a tetramer composed of 4 subunits which
are of two types; H (heart) and M (muscle). The
combination of these subunits results in the
formation of five isoenzymes:
1) LDH
1
(HHHH)
2) LDH
2
(HHHM)
3) LDH
3
(HHMM)
4) LDH
4
(HMMM)
5) LDH
5
(MMMM)
269
37
3) LDH
3
(HHMM):
It is predominant in lungs and kidneys.
It is increased with LDH
2
in leukaemia.
It is increased in pulmonary and renal infarction.
4) LDH
4
(HMMM):
5) LDH
5
(MMMM):
LDH
4
and LDH
5
are predominant in skeletal muscle
and liver.
They are increased in muscle diseases and acute
hepatitis.
Blood sample for assay should be in plain tube
because plasma samples may contain platelets that
are rich in LDH.
During LDH assay, the blood sample must be free
from haemolysis because red blood cells contain
high concentration of this enzyme.
EDTA, oxalate and borates are interfering substances
in its assay.
38
Myoglobin
A. Myoglobin is an oxygenbinding haemoprotein of cardiac and skeletal
muscle with a molecular weight of 17,800 daltons.
B. Myoglobin consists of a single protein chain with 153 amino acids and
one heme group that stores oxygen in the muscle cells.
39
Myoglobin
Unlike haemoglobin, myoglobin does not exhibit
cooperative binding of oxygen. Instead, the binding
of oxygen by myoglobin is unaffected by the oxygen
tension in the surrounding tissue.
Its low molecular weight and cytoplasmic location
account for its early appearance in the circulation
following muscle injury.
Serum myoglobin is increased after trauma to either
skeletal or cardiac muscle as in crush injuries or AMI
respectively.
40
Myoglobin
The methods of serum myoglobin determination are unable to distinguish
the tissue of origin. So, even minor skeletal muscle injury may result in
elevated serum myoglobin concentration which may lead to misdiagnosis
of myocardial infarction.
Myoglobin is the most sensitive marker in the early phase of a heart
attack, when other cardiac markers may often still be normal. An acute
myocardiac infarction (AMI) can be ruled out if the myoglobin level does
not rise within 4 6 hours after the onset of the symptoms.
Myoglobin is most useful when combined with an ECG. Elevated
myoglobin and ST segment changes on an ECG are very indicative of an
AMI.
41
Myoglobin
Myoglobin is an early marker of myocardial necrosis.
It is often used as a negative marker for acute
myocardial infarction (AMI) since two consecutive
results below the cutoff, along with other clinical
information, could be used to rule out a diagnosis of
AMI.
Its level is increased as early as one to two hours
after AMI with a peak at 4 8 hours and then is
cleared and returns to its normal level 12 24 hours
after infarction.
42
270
CardiacTropininI&T
Troponin is one of the minor protein components of the muscle.
It is a regulatory protein of contractile proteins of the myofibril.
It is a complex of 3 protein subunits: tropinin C (calciumbinding
component) troponin I (inhibitory component) and troponin T
(trobomyosinbinding component).
43
CardiacTropininI&T
It is found in both skeletal and heart muscles. However,
cardiacspecific troponinI (cTnI) and cardiacspecific
troponinT (cTnT) have been identified.
In contrast to cTnI & cTnT, troponinC is identical in both
cardial and skeletal muscle. So, troponin C is not useful as a
cardiac marker.
Due to their absolute coronary specificity and high sensitivity,
cTnI & cTnT are the preferred biomarker for diagnosing
cardiac muscle damage.
They serve for confirmation, even if the acute event is about
2 weeks in the past.
44
CardiacTropininI&T
Cardiac specificity of cTnI & cTnT eliminates a false diagnosis
of AMI in patients with increased CKMB after skeletal muscle
injury.
cTnI or cTnT has significant prognostic usefulness in unstable
angina patients. Patients presenting with unstable angina
who had concentrations above the normal level of this
troponin had 5 10 times greater risk of MI or death than
patients with normal troponin levels.
The levels of cardiac cTnI & cTnT start to rise 3 5 hours after
chest pain with a peak at 24 48 hours. The levels can
remain elevated up to 7 10 days for cTnI or up to 10 15
days for cTnT after AMI.
45
TimingofCardiacMarkersinRelationto
MyocardialInfarction
46
Durationof
rising(Days)
Peakofrising
(Hours)
Onsetofrising
(Hours)
Cardiacmarker
1 2 8 12 3 5 CPKMB
1.5 3 12 24 4 6 TotalCPK
3 6 24 36 8 12 AST(GOT)
5 10 48 72 12 24 LDH
0.5 1 4 8 1 2 MYOGLOBIN
7 10 24 48 3 5 CardiactroponinI
10 15 24 48 3 5 CardiactroponinT
NatriureticPeptides
Natriuretic peptide is one of the peptides that causes natriuresis.
The natriuretic peptides are produced by the heart and vasculature:
Atype natriuretic peptide is secreted largely by the atrial myocardium
in response to dilatation.
Btype natriuretic peptide is manufactured mainly by the ventricular
myocardium.
Ctype natriuretic peptide is produced by endothelial cells that line
the blood vessels.
The natriuretic peptides serve to maintain intravascular homeostasis
through their diuretic, natriuretic & vasodilator properties.
47
NatriureticPeptides
48
271
BNatriureticPeptides
Bnatriuretic peptide is a small peptide (32 amino acids)
secreted by heart myocytes for regulation of blood pressure
and fluid balance.
This peptide is synthesized by ventricular cells and stored as
ProBNP (108 amino acids).
Proteolysis of ProBNP results in:
1. Active BNP (halflife 20 minutes) containing 32 amino
acids (77 108). It is the biologically active part.
2. Nterminal fragment designated NTProBNP (halflife 120
minutes) that contains 76 amino acids (1 76). It is
biologically inactive.
49
50
ReleaseofBNPandNTproBNP
Bnatriuretic peptide is useful in the diagnosis of heart
failure. The finding of a low level of this peptide tends to
exclude heart failure.
There is excellent clinical and statistical correlation between
assays for BNP and NTproBNP.
Both BNP and NTproBNP are sensitive, diagnostic markers
for heart failure.
Plasma concentrations of both BNP and NTproBNP are
significantly increased in patients with asymptomatic and
symptomatic left ventricular dysfunction.
51
Due to the longer halflife in the circulation, NTproBNP levels
in blood plasma are generally 610 times higher than BNP.
The better stability and wider dynamic range for NTproBNP
may provide an advantage.
However, in the clinical setting, their overall diagnostic and
prognostic abilities are comparable and both BNP and NT
proBNP have been shown to be extremely helpful in the
diagnosis and management of patients with heart failure.
Changes in NTproBNP concentration can be used to evaluate
the success of treatment in patients with left
ventriculardysfunction.
52
DDimer
Ddimers are specific degradation
products of crosslinked fibrin that
are released when the endogenous
fibrinolytic system attacks the fibrin
matrix of fresh venous
thromboemboli.
It is so named because it contains
two crosslinked D fragments of the
fibrinogen protein
53
DDimer
The Ddimer concentration is an indicator for the fibrinolytic activity of
plasmin in the vascular system.
A higher concentration of Ddimer indicates increased coagulation and
fibrinolysis activity.
Ddimer is a specific marker of degradation of fibrin clot and an indirect
marker of clot formation
DDimer is elevated in several clinical conditions including deep venous
thrombosis (DVT), pulmonary embolism (PE) and disseminated
intravascular coagulation (DIC ).
Acute deep veinous thrombosis and pulmonary embolism can be ruled
out within shortest time and high accuracy with the Ddimer assay.
272
IschaemicModifiedAlbumin(IMA)
IMA is a novel marker of ischaemia.
It is produced when circulating plasma albumin comes in
contact with ischaemic heart tissues.
During ischaemia, Nterminus of albumin is altered, probably
through a series of chemical reactions involving free radical
damagealtered albumin, forming Ischaemic Modified
Albumin
IMA is unable to bind metals, such as cobalt, at the N
terminus.
It is produced continually during ischaemia leading to rapid
increase of its blood concentration that remains elevated
during the ischaemic event
55
Ischaemic patients have proportionately more IMA than non
ischaemic ones.
IMA has twice the sensitivity of cardiac troponin for
diagnosing patient with AMI. It has value as rule out AMI.
Negative IMA can be used to predict subsequent negative
troponin.
Combination of negative IMA and troponin and non
diagnostic ECG yields a negative predictive value of 99%.
IMA has specificity of 45 65%.
56
IMA level starts to increase 6 10 minutes after the
ischaemic cardiac event, then reaches its peak about 6 hours
after the attack and lastly returns to its baseline about 12
hours after the event.
IMA may increase falsely in:
1. Some cancers.
2. Acute infections.
3. End stage renal failure.
4. Liver cirrhosis.
5. Brain ischaemia.
57
HearttypeFattyAcidBindingProtein(HFABP)
Fatty acid binding proteins (FABPs) are members of
cytosolic protein family.
FABP is relatively tissue specific. They are most
abundantly found in heart (HFABP), liver (LFABP)
and intestine (IFABP).
HFABP is a new cardiac marker.
It is released into the circulation shortly after the
onset of ischaemia where it achieves its diagnostic
level before 3 hours and returns to the normal level
12 24 hours of the attack.
58
HearttypeFattyAcidBindingProtein(HFABP)
HFABP can be considered as a diagnostic marker of
acute coronary syndrome due to:
High myocardial content (more than 10 folds of
skeletal muscle content)
Present mainly in the cytosol
Low molecular weight ( about 15 Kdalton)
Relative specificity
Early appearance in plasma and urine after AMI onset
59
GlycogenPhosphorylaseBB(GPBB)
Glycogen phosphorylase is the main enzyme in the
glycogenolysis process (second source of blood
glucose; the fuel of cardiac muscle).
GPBB is one of glycogen phosphorylase isoenzymes.
This isoform exists in heart and brain.
Other isoenzymes are found in liver (GPLL) and
muscle (GPMM).
Due to an intact bloodbrain barrier, this marker can
be regarded as a cardiac specific.
60
273
GlycogenPhosphorylaseBB(GPBB)
GPBB is a sensitive marker for the AMI diagnosis.
It has also been shown that GPBB is increased in a
considerable proportion of AMI patients within 3 4
hours from onset of chest pain.
Level of GPBB is increased early in patients with
unstable angina.
61
Copeptin
Copeptin is glycopeptide formed of the Cterminal fragment (39 amino
acids) of provasopressin; the precursor of vasopressin hormone.
As a marker of endogenous stress, Copeptin is increased immediately
after the onset of acute myocardial infarction and then steadily
decreases.
Copeptin can be used in conjunction with cardiac troponin to improve the
speed of diagnosing or ruling out myocardial infarction.
Copeptin predicts prognosis in patients with heart failure.
Moreover, copeptin has been found to be a prognostically relevant
biomarker in a variety of illnesses such as sepsis, shock, pneumonia and
acute exacerbation of COPD.
62
RelationofInfarctSizetoCardiacMarkersand
ECG
63
274
MinistryofHealth
Institute Graduates
Lipids
SourcesofLipids
Dietary lipids
Triglycerides: Chief dietary lipids.
Cholesterol & phospholipids: Small amounts.
Fatsoluble vitamins & carotenoids: Trace amounts.
2
DigestionandAbsorption
The end products of triacylglycerols digestion are:
2monoacylglycerol + two fatty acids (72%)
glycerol + three fatty acids (22%)
1monoacylglycerol + two fatty acids (6%)
The end products of phospholipids digestion are:
Glycerol.
Two fatty acids.
Phosphoric acid.
Nitrogenous bases (choline, serine, ethanolamine).
Cholesterol itself undergoes no digestion and is absorbed as such.
Cholesterol esters are hydrolyzed by pancreatic cholesterol esterase into
cholesterol and fatty acids.
3
With the aid of bile salts, fatty acids and monoglycerides are dispersed into
smaller particles known as micelles which enter the intestinal mucosal cells where
fat digestion may be completed by intestinal lipase liberating glycerol and fatty
acids.
Shortchain fatty acids and glycerol are absorbed through portal circulation to the
liver.
Longchain fatty acids are utilized for resynthesis of triglycerides within mucosal
cells.
The droplets of resynthesized triglycerides are then coated with a layer of protein
and phospholipids, forming chylomicrons.
Chylomicrons are then carried by lymphatics to the thoracic duct which transports
them to general circulation.
Free cholesterol is esterified within the mucosal cells forming cholesterol esters
which are carried by lymphatics to enter the thoracic duct to the general
circulation.
4
Steatorrhea
Steatorrhea is a disease due to defects in digestion and/or
absorption of lipids.
It is the appearance of excessive amounts of lipids (<5
gm/day) in stools.
Normal fat content of stools is >5 gm/day.
Steatorrhea may be caused by one or more of the following:
Deficiency of pancreatic lipase.
Deficiency of bile salts.
Deficiency of healthy intestinal mucosa.
5
FateofAbsorbedLipids
Uptake by tissues.
Utilization by tissues.
A. Oxidation of fatty acids.
There are three pathways:
1. Oxidation (Knoobs theory).
2. Alpha oxidation.
3. Omega oxidation.
B. Conversion to glucose.
C. Formation of tissue fat.
Storage.
Secretion.
6
275
Oxidation
The major pathway for oxidation of fatty acids.
Carnitine is necessary for Oxidation to be started
Importance of Oxidation:
1. Source of energy.
2. Production of acetylCoA.
3. Ketone bodies formation.
7
AlphaOxidation
A minor pathway for fatty acid oxidation.
In position of methyl FA e.g. phytanic acid.
Mainly in the brain & also in liver tissue.
No energy production.
Refsums disease:
1. Failure of oxidation.
2. Accumulation of large amounts of phytanic acid in the brain,
liver and blood.
3. Polyneuropathy, deafness and blindness occur at young age.
8
OmegaOxidation
At carbon (terminal CH3) of Fatty acid.
Formation of dicarboxylic acids.
Necessary for the structure of cell membrane of the
brain cells.
9
DifferencesbetweenTissueFatandDepotFat
10
Depotfat Tissuefat Itemofdifference
Fatcellsofadiposetissue
Everycelle.g.
cellmembranese.g.myelin
sheath.
Site
Mainlytriglycerideswithsmall
amountsofcholesteroland
phospholipids
Cholesterol,phospholipids,
glycollipids withlittle
triglycerides
Composition
Affectedbynutritionalstate
(variableelement)
Notaffectedbythe
nutritionalstate
) (constantelement
Stability
Yes No Sourceofenergy
Insulatortoheat
Protection
Support
Membranepermeability
Respiratoryenzymes
Insulatortonerveimpulse
Functions
PlasmaLipids
Cholesterol:
Cholesterol is an animal esterol.
All tissues synthesize their own cholesterol.
AcetylCoA is the source of all carbon atoms in cholesterol.
HMGCoA reductase is the key enzyme in the biosynthesis of cholesterol.
Plasma cholesterol is of both intrinsic and extrinsic origin.
Liver is the only source of blood cholesterol.
Plasma total cholesterol concentration is 150 250 mg/dl.
In plasma: free cholesterol 30% cholesterol ester 70%.
In plasma: LDLcholesterol 60% LDLcholesterol 40%.
Functions of cholesterol:
Formation of lipoproteins and cell membranes.
Precursor of:
1. Bile salts.
2. Steroid hormones.
3. Vitamin D3.
11
PlasmaLipids
Hypercholesterolaemia:
It is the increase of blood cholesterol level.
It is due to:
Dietary e.g. diet rich in fat and carbohydrates.
Hypothyroidism.
Diabetes mellitus.
Nephrosis.
Obstructive jaundice.
Familial hyperlipoproteinaemia
12
276
PlasmaLipids
Hypocholesterolaemia:
It is the decrease of blood cholesterol level
It is due to:
1. Dietary:
2. Starvation.
Low dietary cholesterol and carbohydrates.
Liver diseases: Hepatocellular damage.
Severe anaemia.
Hyperthyroidism.
Infection e.g. tuberculosis.
13
PlasmaLipids
Triacylglycerols (triglycerides):
Plasma triglycerides are of two origins:
1. Intrinsic origin:
Synthesized in the liver.
Secreted into the plasma as very low density lipoproteins (VLDL) which represent endogenous
triglycerides.
2. Extrinsic origin:
Absorbed from dietary sources.
Transported into the intestinal lymph and then into the plasma in the form of chylomicrons
that represent exogenous or dietary triglycerides.
In the postabsorptive state (12 14 hours):
Source of plasma triglycerides is the endogenous one.
Exogenous triglycerides of chylomicrons are cleared from the circulation by the clearing factor
(lipoprotein lipase enzyme).
Plasma triglycerides concentration is 50 150 mg/dl.
14
PlasmaLipids
Phospholipids:
The major phospholipids in plasma are lecithin and sphingomyelin.
Phospholipids are mainly synthesized in the mucosa of the small intestine
and in the liver.
They circulate in the plasma as a component of HDL.
They are used as important constituents of all cells particularly those of
the nervous system.
The normal plasma phospholipids level is 150 250 mg/dl.
15
PlasmaLipids
Free fatty acids (FFA):
They are also called nonesterified fatty acids (NEFA).
FFA are carried mainly bound to plasma albumin.
The normal plasma FFA level is10 30 mg/dl.
Steroid hormones, fatsoluble vitamins and carotenoids:
They are present in minute amounts.
They are called derived lipids because they are associated with lipids in
nature and related to them in properties and metabolism.
They are transported in plasma carried on specific carrier protein for
each.
16
Lipoproteins
Lipids are relatively insoluble in water but they are carried in
the body fluids as soluble protein complexes known as
lipoproteins.
The lipoprotein particles contain the polar (water soluble)
coat of protein and phospholipids at their surface, whereas
the nonpolar (water insoluble) molecules such as cholesterol
esters and triglycerides are present at the core of the
lipoprotein molecules.
This structure makes it possible for these complex particles to
transport waterinsoluble lipids in plasma.
17
Lipoproteins
The protein components of lipoproteins are known as apolipoproteins that are
divided into 5 groups known as apolipoproteins: ApoA (apo AI, apo AII and apo
AIV), ApoB (apo B48 and apo B100), ApoC (apo CI, apo CII and Apo CIII),
ApoD (apo D), and E (apo E
2
, apo E
3
and apo E
4
).
Apolipoproteina (apoa) is the protein moiety of the plasma human lipoprotein
a (Lpa), whose concentration is highly correlated with coronary artery disease.
The protein moiety of this lipoprotein consists of apoa and apo B100, linked by
one or more disulfide bonds.
They are transported in plasma in the form of lipoproteins that can be
separated by ultracentrifugation or electrophoresis into chylomicron, VLDL (pre
lipoprotein), LDL (lipoprotein), HDL (lipoprotein), and FFA & albumin.
18
Lipoproteins
1. Chylomicrons:
They transport mainly the absorbed (exogenous) triglycerides with small amounts of
cholesterol and phospholipids.
The lowest density of lipoproteins, and contain very little protein.
They are synthesized in the intestinal mucosa and reach the systemic circulation via thoracic
duct.
Their apolipoproteins are C, B and E.
2. Very lowdensity lipoproteins (VLDL):
They are also called prebeta (pre) lipoproteins on separation by electrophoresis.
They are mainly formed in the liver and to a lesser extent by the intestinal mucosa.
They are responsible mainly for the transport of endogenous triglycerides with a small
quantity of cholesterol from the liver to the cells.
Their apolipoproteins are C (55% = apo CI 5%, apo CII 20%, apo CIII 30%), B (35%) and E
(10%).
19
Lipoproteins
3. Lowdensity lipoproteins (LDL) = Betalipoproteins:
Beta () lipoproteins on separation by electrophoresis.
They are formed in the liver from VLDL through the formation of intermediate
density lipoproteins (IDL) by the removal of more triglycerides and
apolipoproteins, before being secreted into the plasma.
About 60% of total cholesterol with smaller amounts of phospholipids and
triglycerides are carried on them.
Transport cholesterol from the liver to the peripheral tissues.
Their apolipoproteins are mostly apo B (96%), with only small amounts of apo C
(2%) and apo A (1%).
LDL is considered as a positive risk factor for atherosclerosis and ischaemic heart
diseases, i.e. if LDL is increased the risk will be increased.
N.B.: Intermediate density lipoproteins (IDL) are usually transient
Intermediates in the metabolism of VLDL to LDL.
20
Lipoproteins
4. Highdensity lipoproteins (HDL):
Alpha () lipoproteins on separation by electrophoresis.
The smallest lipoprotein particles but are the densest because they contain a
large amount of proteins.
Their lipids are chiefly phospholipids with smaller amounts of cholesterol (40% of
total cholesterol) and triglycerides.
They transport cholesterol from peripheral tissues to the liver to be metabolized
and excreted.
Their apolipoproteins are mostly apo AI (70%) and apo AII (20%), with only small
amounts of apo C (10%).
HDL is considered as a negative risk factor, i.e. if HDL is increased it will protect
against atherosclerosis and ischaemic heart diseases
5. Free fatty acids and albumin:
Free fatty acids are transported in plasma bound to albumin.
21
Hypolipoproteinaemias
Decreased plasma lipoproteins.
The following familial diseases are known:
Tangier disease (lipoprotein deficiency):
It is due to very low plasma apo AI which results from increased rate of catabolism of such
apolipoprotein.
Only traces of HDL are present in plasma.
Cholesterol esters accumulate in the tissue.
A betalipoproteinaemia:
There is complete absence of apoB.
VLDL and LDL are absent from the plasma.
Plasma cholesterol and triglycerides are very low.
Hypobetalipoproteinaemia:
Synthesis of apoB is decreased but VLDL and LDL, although reduced, are present in plasma.
Plasma cholesterol is decreased but not as markedly as in abetalipoproteinaemia.
22
Hyperlipoproteinaemias
They are disorders of metabolism in which one or more of
plasma lipoproteins are increased.
They may be primary (inherited) or secondary (to diseases
such as diabetes mellitus, obesity, nephrotic syndrome and
hypothyroidism).
Hyperlipoproteinaemias can be classified as follows:
A. TypeI hyperlipoproteinaemia:
This is also known as hyperchylomicronaemia or exogenous
hypertriglyceridaemia.
It is due to deficiency of lipoprotein lipase enzyme leading to
accumulation of chylomicrons.
There is no increased risk of atherosclerosis.
The principle of treatment is to reduce the level of circulating
chylomicrons by reducing dietary fats.
23
B.TypeIIhyperlipoproteinaemia:
1) TypeII
a
hyperlipoproteinaemia (hyperlipoproteinaemia):
ItischaracterizedbyincreasedLDL,plasmaisclearandplasmacholesteroliselevated.
2) TypeII
b
hyperlipoproteinaemia (hyperandhyperpre
lipoproteinaemia):
IncreaseinbothLDLandVLDL,plasmamaybeturbid,andbothcholesteroland
triglyceridesareelevated.
Veryhighlevelofplasmacholesterol(500 1000mg/dl).
DefectiveLDLreceptorsinperipheraltissues.
Xanthelasma (depositsofcholesterolaroundtheeyes) andxanthomas (depositsof
cholesterolinskinandtendons).
Severeatherosclerosisanddeathfromcoronaryarterydiseasearecommon.
Dietarytherapyisimportantbydecreasingcholesterolandsaturatedfatrichdiet,and
increasingitspolyunsaturatedfat.
24
278
C. TypeIII hyperlipoproteinaemia:
Broad or floating hyperlipoproteinaemia.
It is similar to typeII
b
hyperlipoproteinaemia.
It is due to defect in IDL catabolism.
D. TypeIV hyperlipoproteinaemia:
Hyperpre lipoproteinaemia or endogenous hypertriglyceridaemia.
The most frequent one of hyperlipoproteinaemias.
Increase in VLDL without other lipoprotein abnormalities.
Hypertriglyceridaemia with hypercholesterolaemia and glucose
intolerance are common.
25
E.TypeVhyperlipoproteinaemia:
Hyperchylomicronaemiawithhyperprebetalipoproteinaemia.
Elevationofchylomicron&VLDLcausingHypertriglyceridaemiaand
hypercholesterolaemia.
Frequentxanthomasbutincidenceofatherosclerosisisnotrisky.
Increasedincidenceofglucoseintoleranceandhyperuricemia.
26
CholesterolandAtherosclerosis
Atherosclerosis is the deposition of cholesteryl ester and
other lipids in the connective tissue of the arterial wall.
LDL/HDL cholesterol ratio helps in predicting atherosclerosis
where:
Increased LDL/HDL ratio predisposes to atherosclerosis.
Decreased LDL/HDL ratio gives protection against atherosclerosis.
Atherosclerosis may lead to coronary heart disease and
myocardial infarction.
27
Lipogenesis
Synthesis of triacylglycerols from carbohydrates.
Site:
Liver, adipose tissue and lactating mammary gland.
Steps:
1. Biosynthesis of active glycerol:
Glycerol is synthesized from intermediates of glucose oxidation through
glycolysis.
2. Biosynthesis of fatty acids and their activation:
Extramitochondrial system (De novo synthesis of fatty acid).
Microsomal system.
Mitochondrial system.
3. Combination of active glycerol and three fatty acids to form
triacylglycerols.
28
Lipolysis
Hydrolysis of stored triacylglycerols in adipose tissue
into glycerol & free fatty acids.
29
KetoneBodies
Ketone bodies are substances which are normally formed in small amounts
including:
Acetone.
Acetoacetic acid
Hydroxybutyric acid
Normally, the plasma concentration of ketone bodies does not exceed 2
mg/dl and its urinary excretion is less than 10 mg/day.
Ketogenesis:
Formation ketone bodies from active acetate of fat origin in the
mitochondria of the liver.
Ketolysis:
Complete oxidation of ketone bodies to CO
2
and H
2
O in the mitochondria
of extrahepatic tissues.
30
279
KetoneBodies
Ketosis:
Increased ketone bodies in the blood (ketonaemia) and in the urine
(ketonuria).
It occurs in states of decreased carbohydrates utilization e.g. starvation,
low carbohydrateshigh fat diet and severe uncontrolled diabetes
mellitus.
If ketosis is not treated, the buffer system (mainly bicarbonate) is
depleted leading to acidosis i.e. ketoacidosis (decreased blood pH) which
may lead to coma & death in severe & advanced cases.
31
BileAcidsandSalts
Bile acids are the excretory products of cholesterol.
I. Primary Bile Acids:
They are synthesized in the liver from cholesterol.
1. Cholic acid.
2. Chenodeoxycholic acid.
They are conjugated with glycine (75%) or taurine (25%) to form conjugated bile acids
which are secreted in bile as their sodiumor potassium salts.
Bile salts are:
1. Na (K) glycocholate.
2. Na (K) taurocholate.
3. Na (K) glycochenodeoxycholate.
4. Na (K) taurochenodeoxycholate.
Bile salts are important for the digestion &absorption of fats because of their
ability to lower the surface tension leading to emulsification of fat.
In obstructive jaundice, bile salts are increased in blood leading to itching and
bradycardia.
32
BileAcidsandSalts
II. Secondary Bile Acids:
They are synthesized in the intestine from primary bile acids by deconjugation and
7dehydroxylation.
They include:
Deoxycholic acid.
Lithocholic acid.
Greater proportions of these bile acids are reabsorbed from the intestine to the
liver again and then are reexcreted in the bile forming enterohepatic circulation
of bile acids.
The nonreabsorbed bile acids are excreted in foeces.
33
RoleofLiverinLipidMetabolism
Fatty acid oxidation when the physiological conditions of the body need
lipid oxidation.
Synthesis, mobilization and oxidation of triglycerides.
Fatty acids biosynthesis from excess glucose.
Synthesis of cholesterol from active acetate.
Ketogenesis.
Phospholipids and lipoproteins synthesis.
Degradation of cholesterol, phospholipids and lipoproteins.
Synthesis of bile salts facilitating the digestion and absorption of fats.
Storage of most fatsoluble vitamins.
Detoxication of steroid hormones and drugs by conjugating them with
sulfate and/or glucuronic acid.
34
FattyLiver
Fatty liver is the accumulation of excessive amounts of lipids (mainly
triglycerides)in the liver.
The lipid content of the normal liver is about 4% of which only about
are triglycerides.
In severe cases of fatty liver, the lipid content may reach up to 40% of the
liver weight.
In prolonged conditions, liver cells die and become fibrosed leading to
liver cirrhosis and fibrosis with impaired liver function.
Causes:
1. Overmobilization of fat from extrahepatic tissue to the liver.
2. During high carbohydrate diet.
3. Undermobilization of fat from the liver to the blood.
35
LipotropicFactors
Lipotropic factors are the factors which help the mobilization of
triacylglycerols from the liver.
Deficiency of these factors leads to undermobilization of fat from the liver
to the blood with the development of fatty liver.
Lipotropic factors include:
A. Substances important for the biosynthesis of phospholipids:
Essential fatty acids.
Inositol.
Choline
Amino acids including methionine and serine.
Vitamins including vitamin B12 and folic acid.
B. Substances important for the biosynthesis of proteins:
Proteins of high biological value providing essential amino acids.
36
MinistryofHealth
Graduates
Special Tests in Clinical Chemistry
LectureOutline
ProteinElectrophoresis
SpecialProteins
HumanImmunoglobulins(IgG,IgA
andIgM)
Apolipoproteins
Haptoglobulin
1AcidGlycoprotein
Cystatin C
UrinaryMicroalbumin
2Microglobulin
1Microglobulin
2Macroglobulin
Prealbumin
NonroutineinvestigationsinClinical
Chemistry:
Glucose6Phosphate
Dehydrogenase(G6PDH)
Urinary17Ketosteroids
Urinary5HydroxyIndole Acetic
Acid(5HIAA)
UrinaryVanilyl Mandelic Acid(VM)
UrinaryPorphyrins and
Porphobilinogens
Neutrophilgelatinaseassociated
lipocalin (NGAL)
Nephrolithiasis(UrinaryCalculi)
Gallstone
2
ProteinElectrophoresis
Electrophoresisisthemigrationofchargedparticles
inaliquidmediumundertheinfluenceofanelectric
field.
Achargedparticleplacedinanelectricalfield
migratestowardstheanode(+)orcathode(),
dependingonthenetchargecarriedbytheparticle.
Therateofmigrationinaporousmediumvaries
withitsnetchargeandthestrengthoftheelectrical
field.
3
ProteinElectrophoresis(continued)
UsingProteinElectrophoresis,5maingroupsofproteinscanbe
distinguished:
Albumin.
Alfa1globulin.
Alfa1antitrypsin
Alfa1glycoprotein
Alfalipoproteins
Alfa2globulin.
Alfa2macroglobulin
Haptoglobulin
Ceruloplasmin
Betaglobulin.
Betalipoproteins
Transferrin
Severalcomponentsofthecomplementsystem.
Gammaglobulin.
4
5
SpecialProteins
I.HumanImmunoglobulins(IgG,IgAandIgM)
II.Apolipoproteins
III.Haptoglobulin
IV:1AcidGlycoprotein
V:Cystatin C
VI:UrinaryMicroalbumin
VII:2Microglobulin
VIII:1Microglobulin
IX:2Macroglobulin
X:Prealbumin
281
I:HumanImmunoglobulins(IgG,IgAandIgM):
Immunoglobulinsareformedbyplasmacellsasahumoralimmune
responsetocontactoftheimmunesystemwithantigens.
Theprimaryreactionaftertheinitialcontactistheformationof
antibodiesoftheIgM classfollowedlaterbyIgG andalsoIgAantibodies.
Quantitativedeterminationoftheimmunoglobulinscanprovide
importantinformationonthehumoralimmunestatus.
Decreasedserumimmunoglobulinconcentrationsoccurin:
Primaryimmunodeficiencyconditions
Secondaryimmuneinsufficienciese.g.inadvancedmalignanttumours,
lymphaticleukaemia,multiplemyelomaandWaldenstroms disease.
Increasedserumimmunoglobulinconcentrations occurin:
Polyclonaloroligoclonal Igproliferation,e.g.inhepaticdiseases(hepatitis,
livercirrhosis),acuteandchronicinfections,autoimmunediseasesaswellas
inthecordbloodofneonateswithintrauterineandperinatalinfections.
Monoclonalimmunoglobulinproliferationsintheserumarefounde.g.in
plasmacytomas,Waldenstroms diseaseandheavychaindisease.
Localimmunereactionsresultinelevatedimmunoglobulinlevels,particularly
IgG,inthecerebrospinalfluid.
ElevatedurinaryconcentrationsofIgG arefoundinpatientswithnon
selectiveglomerularproteinuria.
7
I:HumanImmunoglobulins(IgG,IgAandIgM)
HumanIgG Subclasses1 4
ThehumanIgG antibodiesarecomposedofthefoursubclassesIgG1,IgG2,IgG
3,IgG4.
ThedifferencesbetweentheIgG subclassesarereflectedindifferent,biologically
importantfunctionssuchasantigenrecognition,complementactivationandcell
surfacereceptorbinding.
DiminishedIgG1concentrationsoccursin:
Generalimmunodeficiency
Infectionsoftheupperairwaysandinbronchopulmonary infections.
DiminishedIgG1andIgG2concentrationsoccursin:
Nephrotic syndrome,butparticularlyinminimalchangenephritis.
Virusrelatedinfectionsoftheurinarytract.
Patientswithchronicbronchopulmonary diseasesandbronchiectases
ChangesintheconcentrationsofIgG subclasseshavealsobeenreportedin
patientswithautoimmunediseases,neurologicalsyndromesandHIVinfection.
8
II:Apolipoproteins
Apolipoprotein AI:
Apolipoprotein AIisthemainproteincomponentofhigh
densitylipoprotein(HDL)andaccountsforapproximately
65%ofthetotalproteincontentofHDL.
ApoAIactivateslecithincholesterolacyltransferase which
catalyses theesterificationofcholesterol.
Theresultingesterifiedcholesterolcanthenbetransported
totheliver,metabolizedandexcreted.
Personswithatheroscleroticvascularchangesfrequently
exhibitdecreasedlevelsofApoAI.
DecreasedconcentrationsofApoAIalsooccurin
dyslipoproteinaemias,acutehepatitis,hepaticcirrhosisand
ininsulintreateddiabetics.
9
II:Apolipoproteins
Apolipoprotein AII:
Apolipoprotein AIIisastructuralproteinofHDLwhichhasphospholipidbindingproperties.
ApoAIIconsistsoftwoidenticalproteinchainslinkedbyadisulfidebridge,eachchain
containing77aminoacids.
Thedimeric moleculehasamolecularweightof17440daltons.Geneticallyrelated
structuralvariantsofApoAIIhavenotbeenreported.
ThehumanplasmacontainsdifferentisoformsofApoAIIwithsimilarmolecularweightsand
immunologicalpropertiesbutwithdifferentisoelectricpoints.
ThesynthesissiteofApoAIIistheliver.
ThepercentageconcentrationsofApoAIIintheserumlipoproteinsare:chylomicrons
(traces),VLDL(traces),LDL(traces),HDL2(10%),HDL3(23%).
ThehalflifeofApoAIIinthebloodamountsto4 5days.
Studiesperformedinpatientswithcoronaryheartdiseaseormyocardialinfarctionswere
unabletoestablishaclearcorrelationbetweentheplasmaconcentrationsofApoAIIand
coronaryrisk.
Insomecentres ApoAIIismeasuredinordertoobtaininformationontheHDL2andHDL3
fractionsfromtheratioofApoAItoApoAII.
DiminishedApoAIIconcentrationshavebeenfoundinTangierdisease(averyrareApoAI
defect),inpatientswithahighconsumptionofcigarettesandinhepaticinsufficiency.
ElevatedApoAIIconcentrationshavebeenfoundincasesofhighalcoholconsumption. 10
II:Apolipoproteins
Apolipoprotein B:
Apolipoprotein Bisthemainproteincomponentoflow
densitylipoprotein(LDL)andaccountsforapproximately
95%ofthetotalproteincontentofLDL.
ApoBisnecessaryforthereactionwithLDLreceptorsin
theliverandoncellwallsandisthusinvolvedin
transportingcholesterolfromthelivertothevesselcell.
ElevatedlevelsofApoBarefrequentlyfoundinpatients
withatheroscleroticvascularchangesandarearisk
factorforatherosclerosis.
11
II:Apolipoproteins
Apolipoprotein E
Apolipoprotein Ehasamolecularweightof34000
daltons (299aminoacids)andissynthesizedinthe
liver.
Approx.10 20%ofthetotalproteininVLDL
consistsofApoE.
ElevatedApoEconcentrationsindicateraisedlevels
ofthehighlyatherogenic degradationproductsof
chylomicronsandVLDL("remnants").
282
III:Haptoglobin
Haptoglobin bindshemoglobinwhichisreleasedduring
erythrocytelysis.Thehaptoglobin/hemoglobincomplex
israpidlyeliminatedfromthebloodstream.
Increasedreleaseofhemoglobinduetointravascular
hemolysisresultsinareductioninthehaptoglobin
concentrations,andduringseverehemolysis,to
completeconsumptionofthehaptoglobin.
Inchildrenhaptoglobin haslowerphysiologicalserum
concentrationsandthereforeisnotsuitedforhemolysis
testing.
Haptoglobin isanacutephaseproteinwhichcan
developveryhighserumlevelsduringinflammatory
conditions.
13
1acidglycoproteinisaplasmaglycoproteinwithacarbohydratecontentof
approx.40%.
Inhealthypersonsdifferencesinitsserumlevelscanbefoundwithinthe
referenceinterval.Inparticular,comparedtomen,lowerconcentrationsoccurin
womenofchildbearingage,onoralcontraceptivesandduringpregnancy.
Asanacutephaseprotein,theserumlevelsof1acidglycoproteinareelevated
duringinfectionsaswellasacuteandchronicinflammatoryprocesses(e.g.
Crohn's disease).Inthesecasesahighlysensitiveassessmentoftheconditionof
thepatientcanbeobtainedbypreparingaprognosticindexof1acid
glycoproteinandotherparameterssuchasCRP.
Patientswithinjuries,burnsortumours exhibitelevatedserumconcentrations.
Patientswithchronicrenalfailurearefoundtohavehighserumconcentrationsof
1acidglycoprotein,withnomajordifferencereportedbetweendialyzedand
nondialyzedpatients.
Diminishedserumconcentrationsduetorestrictedproductionof1acid
glycoproteinarefoundinpatientswithchronicliverdiseases.Lowserum
concentrationsduetoincreasedexcretionoftheproteinareassociatedwith
nephrotic syndrome.
14
V:CystatinC
Cystatin Corcystatin 3isacysteineproteinaseinhibitormainlyusedasabiomarkerof
kidneyfunction.
Inhumans,allcellswithanucleusproducecystatin Casachainof120aminoacids.Itis
foundinvirtuallyalltissuesandbodilyfluids.Itisapotentinhibitoroflysosomal proteinases
andprobablyoneofthemostimportantextracellularinhibitorsofcysteineproteases.
Cystatin 3hasalowmolecularweight(~13.3KD).
Duetoitssmallsizeitisfreelyfilteredbytheglomerulus,andisnotsecretedbutisfullyreabsorbed
andbrokendownbytherenaltubules.ThismeanstheprimarydeterminateofbloodCystatin C
levelsistherateatwhichitisfilteredattheglomerulusmakingitanexcellentGFRmarker.
Serumlevelsofcystatin Careamoreprecisetestofkidneyfunctionthanserumcreatinine
levels.Cystatin Clevelsarelessdependentonage,sex,raceandmusclemasscomparedto
creatinine.
Cystatin CisanalternativeandmoresensitiveendogenousmarkerfortheestimationofGFR
thanserumcreatinineandserumcreatininebasedGFRestimations.
Cystatin Ccanbeusedasamarkerofkidneyfunctionintheadjustmentofmedication
dosages.
Cystatin Ccanbemeasuredinarandomsampleofserumusingimmunoassayssuchas
nephelometry orparticleenhancedturbidimetry.
15
VI:UrinaryMicroalbumin
Normally,albuminisnotpresentinurinebecauseitisfilteredfromthebloodstreambythe
kidneys.
Microalbuminuria occurswhenthereisanabnormallyhighpermeabilityforalbumininthe
renalglomerulus.
Microalbuminuria cannotbedetectedbyurinedipstickmethodsbutthereisspecific
Microalbumin urinetesttodeterminethepresenceofthealbumininurine.
Microalbuminuria isdiagnosedfromelevatedconcentrations(30to300mg/L)onatleast
twooccasions.Analbuminlevelabovethesevaluesiscalled"macroalbuminuria",orjust
albuminuria.
Tocompensateforvariationsinurineconcentrationinspotchecksamples,the
albumin/creatinineratio(ACR)iscalculated.
Microalbuminuria isdefinedasACR2.8mg/mmol (male)or2.0mg/mmol(female).
Thesignificanceofmicroalbuminuria testis:
Anindicatorofsubclinicalcardiovasculardisease.
Markerofvascularendothelialdysfunction.
Animportantprognosticmarkerforkidneydisease
Indiabetesmellitus.
Inhypertension.
Increasingmicroalbuminuria duringthefirst48hoursafteradmissiontoanintensivecareunit
predictselevatedriskforacuterespiratoryfailure,multipleorganfailure,andoverallmortality. 16
VII:2Microglobulin
2microglobulinhasamolecularweightof11,800daltons and
occursonallnucleatedcellsasacomponentoftheHLAcomplex.
Itisconstantlyreleasedintothebloodinsmallquantities.Asitis
freelyfilteredandreabsorbedinthekidneytheserumlevelsfound
inhealthypersonsremainataconsistentlylowlevelwhereasthe
urineisfoundtocontainalmostno2microglobulin.
Anincreaseinthereleaseof2microglobulin(duetoincreased
activityoftheimmunesystem,celldeath)ordiminished
elimination(duetoglomerularrenaldamage)leadstoariseinthe
serumconcentration.
Theserumconcentrationof2microglobulinisthusasensitive
markerfortheglomerularfiltrationcapacityofthekidney.
ChronicinflammationsandautoimmunediseasessuchasSLE,
rheumatoidarthritisandSjgrens syndromearealsoassociated
withelevatedlevels.
17
VIII:1Microglobulin
Theclinicalrelevanceofthe1microglobulinassayrelatestothe
identificationoftubularproteinurias.
1microglobulin,alsoreferredtoasproteinHC,isalowmolecular
weightglycoprotein(33KD),thefreeproportionofwhichis
quantitativelyfilteredthroughtheglomeruli.Aswithotherplasma
proteinswhicharefilteredthroughtheglomeruli,reabsorptionand
catabolismtakeplaceintheproximaltubules.
Detectionofelevatedurinaryconcentrationsof1 microglobulin
canbeindicativeoftubulardamageasmayoccurinthecontextof
nephritides,advanceddiabeticnephropathy,afterexposureto
heavymetalsorafteradministrationofnephrotoxicmedicaments.
Detectionofelevatedurinaryconcentrationsof1microglobulin
patientswithurinarytractinfectionsisindicativeofrenal
involvement.
18
283
IX:2Macroglobulin
2macroglobulinisaproteinaseinhibitorwhichexertsaparticulareffecton
endopeptidases.
Ittransportshormonesandenzymes,exhibitseffectorandinhibitorfunctionsin
thedevelopmentofthelymphaticsystemandinhibitscomponentsofthe
complementsystemandhaemostasis system.
Thereferencevaluesareslightlyhigherinwomenthaninmen.
Inhyperfibrinolytic states,aftermajorsurgery,insepticaemia andseverehepatic
insufficiencythemeasuredlevelsof2macroglobulinareoftenlow.
Patientswithacutepancreatitisexhibitlowserumconcentrationswhichcorrelate
withtheseverityofthedisease.
Acutemyocardialinfarctionpatientswithlow2macroglobulinconcentrations
haveasignificantlybetterprognosis.
Theassayof2macroglobulinisofmajorsignificanceforthedifferential
diagnosisofnephrotic syndrome.
Here,anelevated2macroglobulin/albuminratioisindicativeofpostrenal
haematuria.
Inpatientswithlivercirrhosisanddiabetesthelevelsof2macroglobulinare
foundtobeelevated.
19
X:Prealbumin
Prealbumin actsasabindingproteinforthyroxin,and
RBParethetransportproteinforretinol(vitaminA).
Prealbumin andretinolbindingprptein (RBP)are
synthesizedintheliver.
Theserumconcentrationsofbothproteinsreflectthe
synthesiscapacityoftheliverandaremarkedly
diminishedinmalnutritionandotherconditions.
Duetotheirshorthalflivesofapprox.twodaysand
twelvehoursrespectivelyprealbumin andRBPmaybe
suitableformonitoringthenutritionalstatusand
efficacyofparenteralnutrition.
20
Glucose6PhosphateDehydrogenase(G6PDH)
G6PDHisthemainenzymeofhexose
monophosphate(HMP)shuntpathway.
21
(continued)
Importantfortheintegrityofredbloodcellsthrough
theproductionofreducedcoenzymeII(NADPH+
H
+
).MostoftheinterestofG6PDHfocusesonits
roleintheerythrocyte.Here,itfunctionsto
maintainNADPHinitsreducedform.Anadequate
concentrationofNADPHisrequiredtoregenerate
sulfhydrylcontainingproteinssuchasglutathione
formtheoxidizedtothereducedstate.Glutathione
inthereducedform,inturn,protectshemoglobin
fromoxidationbyagentsthatmaybepresentinthe
cell.
22
(continued)
G6PDHDeficiency
Maycausehaemolytic anaemia calledFavism
precipitatedbyadministrationofcertainoxidizingagentssuchasFavabeans,antimalarialdrugs,
sulpha drugs,phenylbutazone andvitaminKanalogues.
DeficiencyresultsinaninadequatesupplyofNADPH
Inabilitytomaintainreducedglutathionelevels.
Whenerythrocytesareexposedtooxidizingagents,hemolysisoccursbecauseofoxidationof
hemoglobinandtodamageofthecellmembrane.
Deficiencyisaninheritedsexlinkedtrait.
Fullexpressionoftraitoccursinhemizygous males wherethesingleXchromosomecarriesthe
mutantgeneandinhomozygousfemaleswherebothsexchromosomes(XX)carryamutantgene.
Intermediateexpressionisfoundinheterozygousfemaleswhereexpressionisvariable.
Femaleheterozygoteshavetwopopulationsofredcells:onewithnormalandtheotherwith
deficientenzymeactivity.Theproportionofthetwopopulationsindifferentheterozygotes
resultsinG6PDHactivitieswhichmayvaryfromalmostnormaltothosefoundfor
hemizygotes
Synthesisisinducedattheageof9 11years.
Thedisordercanresultinseveraldifferentclinicalmanifestations,eg druginducedhemolyticanemia.
Whenexposedtoanoxidantdrugsuchasprimaquine,anantimalarialdrug,affectedindividuals
experienceahemolyticepisode.
ResearchsuggeststhatthedeficiencyconferssomeprotectionagainstFalciparummalaria.
Maylessentheseverityofmalarialinfectionsinyoungchildrenandinfants. 23
Theyarebreakdownproductsofadrenalandrogensin
bothmalesandfemales.
Thesesteroidsincludeandrostenedione,androsterone,
estrone,anddehydroepiandrosterone.
Thereferencerangeofurinary17ketosteroidsis820
mg/dayinmalesand612mg/dayinfemales.
Increasesinlevelsof24hoururinary17ketosteroidsare
associatedwithadrenaltumours,cushing syndrome,
ovariancancer,testicularcancerandpolycysticovarian
syndrome.
DecreasedlevelsareassociatedwithAddisondisease,
hypopituitarism,myxoedema,nephrosis.
24
Urinary5HydroxyIndole AceticAcid(5HIAA)
5HIAAistheexcretorymetaboliteofserotonin.
Itismostfrequentlyperformedforthediagnosisof
carcinoidtumorsoftheenterochromaffincellsof
thesmallintestine,whichreleaselargeamountsof
serotonin.
Valuesmorethan25 mg/dayarestrongevidencefor
carcinoid.Thenormalrangeis2to6mg/day.
25
UrinaryVanilylMandelicAcid(VM)
Vanilyl Mandelic acid(VMA)isthemetaboliteofcatecholaminemeasuredinurine
ofpatientssufferingfrompheochromocytoma;abenignadrenaltumor.
MeasurementofVMAisimportantasbecauseevensmalladrenaltumorsecrete
largeamountofcatecholamine.
Catecholaminesecretionisusuallyparoxysmal,sothemeasurementin24hour
urinesampleismoreusefulthansingleplasmameasurementforscreening.
Noparticularrestrictioninthedietprecedingtheurinecollectionisrequired,as
theblanksamplesubtractsdietaryvanillin,whichmaygivefalselyhighvalues.
SomedrugsinfluencetheVMAurinaryexcretion:
Increasedvaluesresultfromadministrationofinsulin,reserpine,epinephrine,
norepinephrine
Decreasedvaluesresultfromadministrationofmorphine,pentobarbital,
chloropromazine,iproniazid.
TheVMA/creatinineratioinurineallowsperformingthetestonasingle
micturitionandmaygivequitepreciseresults,withincertainlimits,forscreening
purposes.
However,24hoururineVMAispreferredanditisrequiredwhenthe
VMA/creatinineratioiscloseorlightlybeyondnormalvalueslimit.
26
UrinaryPorphyrinsandPorphobilinogens
Theseareintermediatesinhaemoglobin synthesis.They
areexcretedinpatientsurineofporphyrias whichare
groupofdiseases,resultingfromdeficiencyofoneor
moreoftheenzymesinvolvedinheme synthesis.
Urineporphyrins areusefulfortheevaluationof
cutaneousphotosensitivitytoexcludeporphyriacutanea
tarda.
Urineporphobilinogen isusefulfortheevaluationof
neurologicand/orpsychiatricsymptomstoexclude
acuteporphyrias suchasacuteintermittentporphyria.
27
Neutrophilgelatinaseassociatedlipocalin
(NGAL)
NGALisaproteinofasmallmolecularweight(25kD),
belongingtothelipocalin superfamilyinitiallyfoundin
activatedneutrophils.Itisalsofoundincertainepithelia,
suchasrenaltubules,whereitsexpressionisdramatically
increasedinischemicornephrotoxicinjury.
NGALisapromisingbiomarkerforearlydetectionofacute
kidneyinjury(AKI).Itisspecificallyreleasedbythedamaged
kidneyandcanbedetectedinbothurineandplasma.
Eitheraloneorincombinationwithotherbiomarkersthey
willnotonlyhaveanimpactonmedicaldecisionsinfuture
dailyclinicalroutine,buttheywillalsoprovidethebasisfor
testingnovelemergencytherapiesforadiseasethatisoften
recognizedtoolate.
28
Neutrophilgelatinaseassociatedlipocalin
(NGAL) ClinicalApplication
Creatinineisnotusefulforearlydiagnosis.
UrinaryNGALcanbeusedasamarkerfortheearlydiagnosis
ofAKI.
NGALmaybeusedtodetectAKIearlyinthefollowingcases:
Pediatricandadultcardiopulmonarybypassoperations.
Percutaneouscoronaryinterventions(PCI).
Criticallyillpatientspresentingattheemergency
departmentorintheintensivecareunit(heartfailure,
sepsis,multiorganfailure)
Renaltransplantation.
Patientswithchronickidneydisease.
29
Conditioncharacterizedbythepresenceofrenalcalculi(stones).
Duetonutritional,environmentalorgeneticfactors.
Urinarycalculioccurinkidneys,theuretersorthebladder.Theymaybe:
Singlestoneswhichmaybecomposedofanyof:calciumoxalate,uric
acid,calciumcarbonate,calciumphosphateormagnesium
ammoniumphosphate(triplephosphate).
Mixedstoneswhichmaybecomposedoftwoormoreofthe
mentionedconstituents.
Cystine orxanthinestoneswhicharerareandfoundintheinherited
metabolicabnormalities:cystinuria orxanthinuria respectively.
Bothqualitativeandquantitativeanalysesofthechemicalconstituentsof
kidneystonesmaybeusefulinestablishingtheaetiology andinplanning
adequatetherapy.
Radiologicalexaminationsarerequiredtoexplorethedegreeofintrarenal
calcificationandpapillarydamage.
30
Gallstone
Gallstonesaresolidconcretionsthataremostcommonlyformedin
thegallbladderandoccasionallyinthebileducts.
Therearethreemajortypesofgallstones:
1. Cholesterolgallstones:
TheycanbeexaminedanddetectedbytheLiebermannBurchard
reaction.
2. Pigmentedgallstones:
Thepigmentismainlybilirubinwhichispresentascalciumbilirubinate.
TheycanbeexaminedanddetectedbyusingEhrlichdiazo reagent.
3. Mixedgallstones:
Theymaycontainamixtureofcholesterol,bilirubin,calciumphosphate,
calciumcarbonateandmucoproteins.
31
Summary
Electrophoresisisthemigrationofchargedparticlesinaliquidmediumundertheinfluence
ofanelectricfield.UsingProteinElectrophoresis,5maingroupsofproteinscanbe
distinguished.
SpecialProteins
I.HumanImmunoglobulins(IgG,IgAandIgM)
II.Apolipoproteins
III.Haptoglobulin
V:Cystatin C
NonroutineinvestigationsaredoneinClinicalChemistry,thatinclude:
Urinary5HydroxyIndole AceticAcid(5HIAA)
UrinaryVanilyl Mandelic Acid(VM)
UrinaryPorphyrins andPorphobilinogens
Neutrophilgelatinaseassociatedlipocalin (NGAL)
Gallstone 32
VI:Urinary
Microalbumin
VII:2Microglobulin
VIII:1Microglobulin
IX:2Macroglobulin
X:Prealbumin
286
MinistryofHealth
Graduates
BodyFluids
BodyFluids
Body fluids include the following:
1. Blood.
2. Urine.
3. Cerebrospinal fluid (CSF).
4. Milk.
5. Seminal fluid.
6. Pleural Fluid.
7. Pericardial.
8. Peritoneal (Ascetic) fluid.
9. Synovial fluid.
10. Amniotic fluid.
2
BodyFluids
Biochemically, the following body fluids are discussed
in this lecture:
1. Urine.
3. Pleural Fluid.
4. Pericardial.
5. Peritoneal (Ascetic) fluid.
6. Synovial fluid.
7. Amniotic fluid.
3
Urine
Urine is a typically sterile fluid secreted and then excreted through a
process called micturition (urination) by the urinary system which is the
main excretory system in the human body especially for excreting water
soluble chemicals from the body.
These chemicals can be detected and analyzed by urinalysis.
Certain diseases can result in pathogencontaminated urine.
Clinical significance of urine examination:
1. Diagnosis and management of renal or urinary tract diseases.
2. Detection of metabolic or systemic diseases not directly related to the
kidney.
4
Urine
Collection of urine samples:
Single specimens of urine (random or morning samples) are used for ward
examinations and qualitative tests.
Doublevoided specimen is the urine excreted during a time period after
complete emptying of the bladder by 15 30 min.
24 hoursurine collections are preferred for quantitative tests due to the diurnal
variation in the excretion of some substances.
The 24 hourscollection is as follows:
At a suitable time (e.g. 8 :00am), the patient empties his bladder and the urine is discarded.
All urine passed during the following 24 hours is saved in specific container.
At the same time of the next morning, the patient empties his bladder and the urine is added
to the collected one.
N.B.: Creatinine level of the urine sample can be used as a rough check on the
reliability of the collection to exclude adulteration of the urine sample..
5
Urine
Storage of urine samples:
It is satisfactory in most cases to use specimens collected in
cool, clean containers.
Urine sample can be stored for about one week in the
refrigerator at 2 8C.
Urine samples can be stored for many months at 20C
without any addition.
Concentrated hydrochloric acid (HCl), thymol or chloroform
can be used for urine storage.
Acid should not be used for storage of proteins, creatinine
and steroids determination.
6
287
PhysicochemicalExaminationbyUrineStrips
Urine strips are dry reagent strips which are plastic supports containing one or more
chemically impregnated test sites on an absorbent pad.
When the strips come in contact with urine, the reagents are activated and a chemical
reaction occurs.
The chemical reaction is a specific colour change, which may be:
Observed visually and compared with a special colour chart.
Or
Measured electronically (usually by reflectance) by an instrument designed to be used with the reagent
strips.
The intensity of the colour formed is proportional to the amount of substance present in the
specimen when observed at a specific time.
Some test areas are used as:
Screening tests which are reported as positive or negative.
Or
Semiquantitative estimation of the amount of certain substance present in the specimen. These are
reported in a plus system or in international values (e.g. mg/dl, g/dl, mmol/l).
7
PhysicochemicalExaminationbyUrineStrips
Urine strips contain one or more of the following tests:
pH: 5 7
Specific Gravity: 1015 1025
Ascorbic Acid: Negative
Nitrites: Negative
White blood cells: Negative
Blood (Intact red blood cells and haemoglobin): Negative
Protein/Albumin: Negative
Glucose: Negative
Ketones/Acetone: Negative
Bilirubin: Negative
Urobilinogen: Trace
8
UrineChemicalInvestigations
1. Proteins:
o Normally, protein in urine is up to 150 mg/day.
o Abnormal gamma globulin (BenceJoones protein) can be detected in the urine of
multiple myeloma patients.
2. Albumin (Microalbumin):
o It is diagnosed from elevated concentrations (30 to 300 mg/L) on at least two
occasions.
o An albumin level above these values is called "macroalbuminuria", or just
albuminuria.
o Values below 30 mg/L indicate negative microalbuminuria.
3. Nonprotein nitrogenous (NPN) compounds:
o These compounds include urea (20 40 g/day), uric acid (About 1.0 g/day), creatine
(0.0 0.2 g/day), creatinine (About 1.5 g/day), ammonia (About 0.7 g/day) and
amino acids (150 200 mg/day).
9
4. Creatinine clearance:
o It is the practical and most convenient method of obtaining an
acceptable accurate estimation of glomerular filtration rate (GFR).
o It ranges from 90 to 130 ml/min in males and from 80 to 120 ml/min
in females.
5. Minerals and electrolytes:
o They include calcium, phosphorus, sodium, potassium, chloride and
bicarbonate.
6. Osmolality:
o It indicates the number of particles of solute per unit of solution
and reflects the relative degree of concentration or dilution of a
urine specimen.
o The normal adult with a normal fluid intake will produce a urine of
about 300900 mOsm/kg water.
10
o It is a promising biomarker for early detection of acute kidney injury.
o It is specifically released by the damaged kidney and can be detected in
both urine and plasma.
8. Vanilyl Mandelic Acid (VMA):
o VMA is one of catecholamines metabolites.
o Urinary excretion of VMA is a monitor of adrenal medullary function, and is
increased in pheochromocytoma (benign medullary tumour).
9. 5Hydroxy Indole Acetic Acid (5HIAA):
o 5HIAA is the excretory metabolite of serotonin.
o It is most frequently performed for the diagnosis of carcinoid tumors of the
enterochromaffin cells of the small intestine, which release large amounts of
serotonin.
o Values more than 25 mg/day are strong evidence for carcinoid. The normal
range is 2 to 6 mg/day.
11
10. 17Ketosteroids:
o They are breakdown products of adrenal androgens in both males
and females.
o These steroids include androstenedione, androsterone, estrone, and
dehydroepiandrosterone.
o The reference range of urinary 17ketosteroids is 820 mg/day in
males and 612 mg/day in females.
o Increases in levels of 24hour urinary 17ketosteroids are associated
with adrenal tumours, cushing syndrome, ovarian cancer, testicular
cancer and polycystic ovarian syndrome.
o Decreased levels are associated with Addison disease,
hypopituitarism, myxoedema, nephrosis.
12
288
11. Porphyrins and Porphobilinogens:
o These are intermediates in haemoglobin synthesis. They are excreted in patients
urine of porphyrias which are group of diseases, resulting from deficiency of one
or more of the enzymes involved in heme synthesis.
o Urine porphyrins are useful for the evaluation of cutaneous photosensitivity to
exclude porphyria cutanea tarda.
o Urine porphobilinogen is useful for the evaluation of neurologic and/or
psychiatric symptoms to exclude acute porphyrias such as acute intermittent
porphyria.
12. Drugs and Toxicological Tests:
o These tests are performed to monitor the therapeutic level of the drugs and to
detect any toxicological doses.
o There are also urinary screening tests for certain addictive drugs
13
CerebrospinalFluid(CSF)
Cerebrospinal fluid (CSF) is a fluid contained within the membranous coverings of
the spinal cord and brain within the space known as the subarachnoid space.
Most CSF in formed by the choroid plexuses of the brain ventricles. It is produced
by ultrafiltration and active secretion of the blood plasma.
CSF is constantly produced (250 750 ml / day), but it is also constantly
reabsorbed from the subarachnoid space into the blood stream, hence the
volume remains constraint.
Functions:
It provides a fluid cushion to protect the brain and spinal card.
It carries nutrients and removes waste products.
It maintains the pressure inside the head and around the spinal cord.
It is the pathway whereby hypothalamus releasing factors are transported to the cells of the
median eminence.
It maintains central nervous system ionic homeostasis.
14
CerebrospinalFluid
CharacteristicsofCSF:
Volume:50 150ml.
pH:7.4
Aspect:Clear
Color:Colourless
15
CSFsupernatantcolor Associateddiseases/disorders
Pink RBClysis/hemoglobinbreakdownproducts
Yellow
RBClysis/hemoglobinbreakdownproducts
Hyperbilirubinemia
CSFprotein>150mg/dL (1.5g/L)
Orange
RBClysis/hemoglobinbreakdownproducts
Hypervitaminosis A(carotenoids)
Yellowgreen Hyperbilirubinemia (biliverdin)
Brown Meningeal metastaticmelanoma
CerebrospinalFluid
Chemical Composition:
Glucose : 50 70 mg/dl.
Proteins: 15 45 mg/dl.
Lipids: Cholesrerol (about 0.4 mg/dl).
NPN: as urea (20 40 mg/dl).
Minerals: Na
+
, K
++
, Mg
++
, and Phosphorus.
16
SpecimenCollection
Cerebrospinal fluid may be obtained by lumbar, cisternal or lateral cervical puncture or
through ventricular cannulas or shunts.
Up to 20 ml of CSF can be safely removed from an adult although this amount is not usually
required. Clinician should provide clinical history to the laboratory. The puncture site (i.e.,
lumbar, cisternal, etc.) should be noted since cytologic and chemical parameters vary at
different sites.
3 4 ml of CSF are allowed to drip into 3 plain tubes; the first tube should be used for
chemical tests, the second for microbiological tests, and the third for microscopic and
cytological examination.
Highly bloody sample for cell count should be collected in EDTA tube to prevent formation of
clot.
Glass tubes should be avoided since cell adhesion to glass affects the cell count.
Specimen should be delivered to the laboratory and processed immediately to minimize
cellular degradation, which begins within 1 hour of collection.
Refrigeration is contraindicated for culture specimens because organisms like Haemophilus
influenzae and Neisseria meningitidis will not survive.
17
CSFChemicalInvestigations
1. Proteins (total and specific):
Over 80% of CSF Protein content is derived from the plasma, in concentrations less than 1%
of their blood level.
An increased CSF protein serves as a useful but nonspecific indicator of disease. Increased
CSF protein (more than 45 mg/dl) may be caused by:
Increased permeability of the bloodbrain barrier.
Decreased reorption at the arachnoid villi.
Mechanical obstruction of CSF flow due to spinal block above the site of puncture.
Increase in intrathecal immunoglobulin (IgG) synthesis as in multiple sclerosis and in conditions associated
with lymphocytic and plasmacytic infiltrate of the central nervous system (CNS).
Low lumber CSF total protein levels (less than 15 mg/dl) occur in:
Normally in children between 6 months and 2 years.
Following removal of large volume of CSF.
CSF Leakage by trauma or lumber puncture.
Increased intracranial pressure.
Hyperthyroidism.
Protein electrophoresis of concentrated normal CSF reveals two distinct differences from
serum: prominent transthyretin band & extra transferrin band
18
289
2. Glucose :
CSF glucose results should be compared with plasma levels for clinical interpretation. So, the
necessity for simultaneous plasma glucose should also be considered. This is best obtained
24 hours before lumbar puncture because of the delay in plasmaCSF equilibrium. The
normal CSF /plasma glucose ratio varies from 0.30.9.
CSF values below 40 mg/dl (2.2 mmol/L) or ratios below 0.3 are considered to be abnormal.
Low glucose level is a characteristic finding of:
Bacterial, tuberculous and fungal meningitis.
Some cases of viral meningoencephalitis also have low glucose levels, but generally not to the degree seen
in bacterial meningitis.
Meningeal involvement by a malignant tumor, sarcoidosis, cysticercosis, trichinosis, amoeba (Naegleria),
and acute syphilitic meningitis, intrathecal administration of radioiodinated serum albumin, subarachnoid
hemorrhage, symptomatic hypoglycemia and rheumatoid meningitis may also produce low CSF glucose
levels.
CSF glucose levels normalize before protein levels and cell counts during recovery from
meningitis, making it a useful parameter in assessing response to treatment.
Increased CSF glucose is of no clinical significance. It reflects increased blood glucose level.
19
3. Lactate:
CSF lactate concentration is largely independent of blood glucose levels.
The primary source of CSF lactate is CNS anaerobic metabolism.
Any condition associated with tissue hypoxia of the CNS may cause increase in
CSF lactate as in:
Traumatic brain injury.
Cerebral oedema.
Intracranial haemorrhage.
Cerebral infarct.
Hydrocephalus.
Brain abscess.
Cerebral ischaemia.
Metastatic neoplasm.
Meningitis.
20
4. Chloride :
CSF chloride (Cl) reflects blood electrolyte only, but in tuberculous meningitis, a
decrease of 25% may exceed the serum Cl decrease.
It is not useful in diagnosis of tuberculous meningitis.
5. Ammonia and Glutamine:

Ammonia levels in CSF are about one third that of arterial blood ammonia.
There is relation between increased CSF ammonia and hepatic encephalopathy.
Elevated ammonia levels lead to high CSF glutamine levels. Accordingly,
increased CSF glutamine reflects increased brain ammonia.
Elevated CSF glutamine occurs in hepatic encephalopathy, septic
encephalopathy, & encephalopathy secondary to respiratory failure.
21
6. Enzymes:
Lactate dehydrogenase (LDH):
LDH is useful in differentiating a traumatic tap from intracranial
hemorrhage since a current traumatic tap with intact RBCs does not
significantly elevate the LDH level.
LDH activity is also significantly higher in:
Bacterial meningitis.
Patients with CNS leukemia, lymphoma, metastatic carcinoma, bacterial meningitis, and subarachnoid
hemorrhage.
Creatine kinase (CK):
Assay of total CK activity in CSF has only limited value in clinical diagnosis
because any changes obtained are irregular & often nonspecific.
CSF elevations can be found in:
Epileptic patients.
Patients with brain tumour, cerebral infarcts cerebral haemorrhage or any cerebral damage.
CSF CKBB increases after certain types of neurological injury.
22
6. Enzymes (continued):
Aspartate transaminase (AST) :
Changes in AST are irregular and generally of limited diagnostic value.
AST elevations occur in :
Cases of large brain infarct during the first 10 days.
About 40% of CNS tumour.
Cerebrovascular accidents.
Cholinesterase :
Cholinesterase activity is very low in CSF.
Its activity is increased in :
Brain tumours and brain abscess.
Hydrocephalus.
Meningitis.
Multiple sclerosis.
23
AdultLumbarCSFReferenceValues
24
Analyte ReferenceInterval
Protein 15 45mg/dl
Glucose 50 70mg/dl
Lactate 10 22mg/dl
Chloride 115 130mmol/L
Ammonia 10 35g/dl
Glutamine 6 15mg/dl
LDH ~10%ofserumvalue
CK 0 5U/L
AST 7 49U/L
Cholinesterase 13 21U/L
290
PleuralFluid
The pleural cavity is a potential space lined by mesothelium of the
visceral and parietal pleura.
The pleural cavity normally contains a small amount of fluid that
facilitates movement of the two membranes against each other.
Pleural fluid may be transudate or exudate.
Turbid pleural effusion may be due to septic or nonseptic inflammation,
tuberculosis, rheumatoid disease or rheumatic fever.
Hemorrhagic effusions suggest malignancy, pulmonary infarct and
trauma.
Chylous (milky) fluid is due to trauma or obstruction to the thoracic duct.
25
PleuralFluid
Thoracocentesis is indicated for any undiagnosed pleural effusion or for
therapeutic purposes in patients with massive symptomatic effusions.
This collection/handling and often under testing or inappropriate testing
is more common than with other body fluids.
Specimen is usually divided into three serially collected sterile tubes: tube
1 for chemical investigations; tube 2 for microbiologic examination; and
tube 3 for total and differential cell count.
Highly bloody sample, for cell count, is better to be collected in EDTA tube
to prevent clot formation.
26
ChemicalInvestigationsonPleuralFluid
1. Protein:
o Proteins are of value in differential diagnosis of transudates (protein < 3 g/dl) or exudates
(protein > 3 g/dl).
o Pleural fluid to serum proteins ratio of < 5 is with transudates while if > 5 is with exudates.
2. Glucose:
o Pleural fluid to serum glucose ratio under 0.5 may be found in tuberculosis, SLE, empyema
and rheumatoid pleurisy.
o Low pleural fluid glucose may also be present in malignancy, tuberculosis, nonpurulent
bacterial infections, lupus pleuritis, and esophageal rupture.
3. Lactate:
o Pleural fluid levels of lactate can be useful in the rapid diagnosis of infectious pleuritis.
o Levels are significantly higher in bacterial and tuberculous pleural infections than other
pleural effusions.
o Values more than 90 mg/dl have a positive predictive value for infectious pleuritis of 94%
and negative predictive value of 100%.
27
4. Lactate Dehydrogenase (LDH) :
o High LDH level is associated with pneumonic effusion, rheumatoid pleurisy and
some malignant effusions.
5. Lipids :
o Some serous effusions appear to be chylous (i.e., a milky appearance) but are not
(pseudochylous)
o Lipid measurements are also helpful in identifying chylous effusions. Thus,
pleural fluid triglyceride levels above 110 mg/dl indicate a chylous effusion.
Nonchylous and pseudochylous effusions generally have triglyceride levels below
50 mg/dl.
o A total cholesterol value < 55 mg/dl are found in transudates, and > 55 mg/dl in
exudates.
6. Tumour Markers :
o Combined CEA and CA125 have a sensitivity of 75 100% and specificity of 95%
for detection of malignant effusions due to carcinoma of lung, heart,
gastrointestinal tract and ovary.
28
LaboratoryCriteriaforPleuralFluidExudate
29
Pleuralfluid/serumproteinratio 0.50
Pleuralfluid/serumLDHratio 0.60
PleuralfluidLDH
upperlimitofnormalserum
LDH
Pleuralfluidcholesterol >45mg/dl
Pleuralfluid/serumcholesterol
ratio
0.30
Serumpleuralfluidalbumin
gradient
1.2g/dl
Pleuralfluid/serumbilirubin
ratio
0.60
PericardialFluid
10 50 ml of fluid is normally present in the pericardial space. Normal pericardial
fluid is pale yellow and clear.
Increased fluid in the pericardial cavity may be caused by inflammation, tumor or
hemorrhage.
Pericardial effusions are most often caused by viral infection.
They may also develop as a result of bacterial, tuberculous or fungal infections,
HIV infection, autoimmune disorders, renal failure, myocardial infarction,
mediastinal injury, tumor, hemorrhage, due to the effects of various drugs or it
may be idiopathic.
Large effusions (>350 ml) are most often caused by malignancy or uremia or are
idiopathic.
Infection or malignancy typically produces turbid effusions, whereas effusions due
to uremia are usually clear and straw colored. These and several other disorders
may produce hemorrhagic effusions.
A milky appearance suggests the presence of a chylous or pseudochylous
effusion.
30
291
PericardialFluid
Specimen Collection
Fluid is obtained either by pericardiotomy following limited thoracotomy,
or by pericardiocentesis.
Specimen is usually divided into three serially collected sterile plain
tubes: the first one is for chemical investigations; the second for
microbiologic examination; and the third one for total and differential cell
count.
Bloodlike fluid obtained by pericardiocentesis might represent a
hemorrhagic effusion or inadvertent aspiration of blood from the heart.
For cell count, fluid is better to be collected in EDTA tube to prevent clot
formation.
31
ClinicalInvestigationsonPericardialFluid
1. Protein:
o Pericardial effusions are either transudates (protein < 3 g/dl) or
exudates (protein > 3 g/dl).
2. Glucose:
o It is decreased to less than 40 mg/dl in effusions due to bacterial or
tuberculous infections, rheumatoid disease and malignancy.
3. Lactate Dehydrogenase (LDH):
o A pericardial fluid LDH level more than 300 U/L is of significance to
differentiate pericardial exudates from transudates.
4. Lipids:
o Separation of true chylous from pseudochylous effusions may be
facilitated by triglyceride and cholesterol measurements as well as
lipoprotein electrophoresis for chylomicrons.
32
PeritonealFluid
Up to 50 ml of fluid is normally present in this mesothelial lined potential space
that represents the peritoneal cavity.
Ascites is the pathologic accumulation of excess fluid in the peritoneal cavity.
Abnormal fluid may be collected in the peritoneal cavity in certain pathological
conditions.
Diagnostic paracentesis in performed in most patients with new ascitis, or if there
is a change in the clinical picture of a patient with ascitis. A minimum of 30 ml is
needed for complete evaluation.
Diagnostic peritoneal lavage (DPL) is no longer used routinely in the evaluation of
abdominal trauma.
Specimen is usually divided into three serially collected sterile tubes: tube 1 for
chemical investigations; tube 2 for microbiologic examination; and tube 3 (EDTA
tube) for total and differential cell count.
33
ChemicalInvestigationsonPeritonealFluid
1. Protein :
o Peritoneal effusions are either transudates (protein < 3 g/dl) or exudates
(protein > 3 g/dl).
o Transudates occur in cases of congestive heart failure, hepatic cirrhosis and
hypoproteinemia as in nephritic syndrome.
o Exudates are frequent in infections due to tuberculosis, primary bacterial
peritonitis, and secondary peritonitis e.g. in appendicitis and mesothelioma,
trauma, pancreatitis and bile peritonitis e.g. ruptured gall bladder.
o The serumascites albumin gradient, defined as the serum albumin
concentration minus the ascitic fluid albumin concentration, is widely
considered as the most reliable method to differentiate peritoneal
transudates from exudates.
o Ascites caused by portal hypertension has a gradient of at least 1.1 g/dl
(transudate) whereas ascites produced by other causes has a gradient less
than 1.1 g/dl (exudate).
34
2. Glucose :
o An ascetic fluid to serum glucose ratio < 1.0 may occur in patient with bacterial peritonitis.
3. Lactate:
o Ascitic fluid lactate has been used with pH measurements to differentiate bacterial from
uncomplicated ascites.
o Sensitivity and specificity are approximately 90% using a cutoff value of about 62%.
Malignant and tuberculous ascites are associated with elevated lactate.
4. Amylase:
o Enzyme activity is elevated in patients with acute pancreatitis, pancreatic trauma or
pancreatic pseudocyst. The ratio of ascitic fluid to serum amylase is over 2.0.
5. Alkaline Phosphatase:
o This measurement may be helpful in the differentiation of primary bacterial peritonitis from
secondary bacterial peritonitis due to bowel perforation.
35
o Enzyme activity is elevated in patients with spontaneous bacterial peritonitis and
malignant effusions.
7. Creatinine and Urea:
o Measurement of creatinine and urea nitrogen is useful to differentiate between peritoneal
fluid and urine.
o Elevated peritoneal fluid urea nitrogen and creatinine, in association with elevated serum
urea but normal serum creatinine suggest urinary bladder rupture.
8. Bilirubin:
o Ascitic fluid/serum bilirubin ratio over 1.0 suggests a ruptured gallbladder.
9. Cholesterol:
o The ascitic fluid cholesterol has been used for differential diagnosis of uncomplicated
ascites versus ascites caused by malignancy.
o A cutoff value of above 1.2 mmol/l provides a good sensitivity, specificity, positive and
negative predictive value, and overall diagnostic accuracy for differentiating malignant
from nonmalignant.
36
292
SynovialFluid
Synovium refers to the tissue lining synovial tendon sheaths, bursae, and
joints.
It is composed of one to three cell layers that form a discontinuous
surface overlying fatty, fibrous or periosteal joint tissue.
Synovial fluid is ultrafiltrate of blood plasma combined with hyaluronic
acid produced by the synovial cells. Small ions and molecules (e.g., Na
+
,
K
+
, glucose, urea, etc.) readily pass into the joint space and are therefore
similar in concentration to plasma while large molecules are absent or
present in trace amounts.
Resorption of synovial molecules is by the lymphatics and is not size
dependent.
Synovial fluid acts as a lubricant and provides nutrients for the avascular
articular cartilage.
37
SynovialFluid
Joint fluid aspiration (arthrocentesis) should be performed by
an experienced operator using good sterile technique.
The specimen should ideally be separated into three parts: 3
10 ml into a sterile heparinized tube or syringe for
microbiological studies, 25 ml in an anticoagulant tube
(sodium heparin) for microscopic examination; and about 5
ml in a plain tube for chemical analysis.
38
ClinicalInvestigationsonSynovialFluid
1. Protein:
o The mean normal protein concentration is about is 1.0 3.0 g/dl.
o With increasing inflammation, larger proteins (e.g., fibrinogen) enter the synovial
space. Spontaneous clot formation may be detected in nonanticoagulated
specimen tubes.
2. Glucose:
o Proper interpretation of synovial fluid glucose values requires comparison with
serum levels. The serumsynovia difference is less than 10 mg/dl in normal and
many noninflammatory conditions.
o In septic arthritis, this difference ranges from 20 60 mg/dl.
o LDH is elevated in rheumatoisd arthritis, gout, failed arthroplasties, and
infectious arthritis.
39
ChemicalInvestigationsonSynovialFluid
4. Uric Acid:
o The mean normal protein concentration is about is 1.0 3.0 g/dl.
o With increasing inflammation, larger proteins (e.g., fibrinogen) enter the synovial
space. Spontaneous clot formation may be detected in nonanticoagulated specimen
tubes.
5. Lipids:
o In contrast to plasma, normal synovial fluid contains extremely low concentrations of
lipids.
o Synovial fluid lipid abnormalities include:
o Rare cholesterolrich pseudochylous effusions associated with chronic rheumatoid
arthritis.
o Lipid droplets associated with trauma.
o Rare chylous effusions seen in association with rheumatoid arthritis, systemic lupus
erythematosus, filariasis, pancreatitis and trauma.
N.B.: In case of sticky sample, dilution of 1:1 should be done with normal saline and
the result will be multiplied by 2.
40
ChemicalInvestigationsonSynovialFluid
ReferenceIntervalsforSynovialFluidConstituents
41
Constituent Synovialfluid Plasma
Total protein
1 3g/dl 6 8g/dl
Albumin 55 70% 50 65%
Gammaglobulin 10 14% 12 22%
Hyaluronic acid 0.3 0.4g/dl
Glucose 70 110mg/dl 70 110mg/dl
Uric acid 2 8mg/dl 2 8mg/dl
Lactate 9 29mg/dl 9 29mg/dl
AmnioticFluid
Amniotic fluid is the protective liquid contained by the amniotic sac of a pregnant female.
It is clear pale yellow fluid with pH of about 7.2 and a specific gravity of 1.006 1.008.
At very early stages the amniotic fluid is secreted by the amniotic cells, later most of it is
derived from the maternal tissue fluid by diffusion across the amniochorionic membrane
and from the placenta.
The composition of the amniotic fluid changes with gestation age. In early pregnancy it is
similar to maternal and fetal serum.
9899% of the amniotic fluid is water.
A large number of dissolved substances such as creatinine, urea, bile pigments, renin,
glucose, fructose, proteins (albumin and globulin), lipids, hormones (estrogen and
progestrone), enzymes, minerals (Na
+
, K
+
, Cl
).
Some undissolved materials (such as some fetal epithelial cells) are suspended in it.
During the second half of gestation its osmolarity decreases and is close to dilute fetal urine
with added phospholipids and other substances from fetal lung and other metabolites.
42
293
AmnioticFluid
Amniotic fluid is collected by amniocentesis which is the
aspiration of a small amount of amniotic fluid from the sac
around the baby.
This is usually performed at 16 weeks in pregnancy.
A fine needle is inserted under ultrasound guidance through
the mothers' abdomen into a pool of amniotic fluid.
43
ChemicalInvestigationsonAmnioticFluid
1. Lecithine/sphingomyelin ratio:
o This test predicts foetal lung maturity more reliably than it predicts immaturity.
o As the lung matures, the concentration of phospholipids (especially lecithin) increases
since lecithin is the major lung surfactant.
o This test is done to assess the maturation of the fetal lungs, a ratio 3/1 indicates mature
lungs and a ratio less than 3/1 indicates immature lungs.
2. Bilirubin :
o It indicates the degree of fetal red blood cell destruction, where abnormally high levels
could indicate serious cases such as mother fetal blood incompatibility.
3. Acetylcholinesterase (AChE):
o It is a useful adjunct in the diagnosis of neural tube defects. This enzyme is relatively
specific for neural tissue.
44
ChemicalInvestigationsonAmnioticFluid
4. Alphafetoprotein (AFP):
o High levels of AFP in the amniotic fluid indicate the presence of a severe neural tube defect
whereas low levels of alphafetoproteins may indicate chromosomal abnormalities.
o It is more accurate than that in maternal serum screening.
o It has been measured as early as 8 weeks. It cannot be measured around 14 weeks because
amniotic fluid AFP at that time may lead misdiagnosis of neural tube defects at that
gestational age.
o Increased amniotic fluid AFP should be tested for foetal haemoglobin which is a sensitive
marker of foetal blood contamination.
5. Chromosomal analysis of the cells:
o This is usually performed during the second trimester, after increasing the number of foetal
cells by culture.
o This analysis is done to achieve early diagnosis of both numerical and structural disorders.
45
294
Ministry of Health
Blood Bank
295

Blood Bank

Donor Recruitment & Retention
- Slide 39 Importance of regular voluntary non remunerate blood donation
With the rapid increase in the number of people with transfusion-transmissible infections, and
in particular the human immunodeficiency virus (HIV)
Whatever your job, your role in this process is extremely important and you need to develop the
knowledge and skills to ensure that blood donation is safe, both for donors themselves and for
the recipients of their blood.
- Slide 58 and 59 Donor Complaints
Give some examples of donor complaints and explain how the quality system should deal with
them. Emphasise the quality system aspect NOT the complaint
The BTS must ensure that there are standard procedures for dealing with donor complaints no
matter how trivial. All staff need to be trained to the standard procedures.
The procedures should ensure that all complaints are investigated and where necessary,
corrective and preventive action is taken.
Hear, empathise, apologise, take responsibility for your actions
- Slide 61 Positive Outcomes
Discuss how donors can contribute to the quality cycle
Donors can often have insight into how they would like to be treated. Listen to any suggestions.
When introducing new campaigns, awards and practices, one way of soliciting donor
suggestions is to ask them to fill in a short questionnaire about the BTS activities.

Blood Grouping Discrepancies
- Slide 33 1. Mixed-field Agglutination
Mixed cell populations resulting from massive transfusion of another blood group such as an B
individual receiving "O" red blood cell donor units since the transfusion center did not have
enough B donor units.
Bone marrow transplant patients may have both some of their original type of cells and the type
of the bone marrow transplant.
Weak subgroups of A3 traditionally give a mixed field reaction.
Rarely the condition called chimerism due to intrauterine exchange of erythrocyte precursors
between twins or 2 fertilized eggs fuse into one individual.
You should try to find cause of mixed field agglutination before setting up blood to transfuse so
be sure to check the patient's transfusion records and clinical history. If it appears to be a weak
subgroup performed the tests discussed under Unexpected Anti-A
- Slide 38 Acquired B
Galactosamine results from the deacetylating reaction, resembling D-galactose (found in Group
B individuals). This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but still
technically it is extra). Patients should receive Group A units. Acquired B usually goes away
when the condition resolves.

Antibody Identification
- Slide 31 Guidelines
DTR delayed transfusion reaction (donor cells are sensitized with patients antibody)

Transfusion Transmitted Diseases
- Slide 5 Types of Blood Borne Pathogens
Human Immunodeficiency Virus (HIV): viruses or bacteria that are carried in blood and cause
disease in people.
- Slide 8 Hepatitis A (HAV)
HAV- May not have symptom

- Slide 15 Hepatitis C (HCV)
These persons are at risk for developing cirrhosis and liver cancer.
- Slide 16 Hepatitis C Virus Geographic Distribution
HCV was discovered in 1989 and was soon recognized as the primary cause of post-transfusion
non-A, non-B hepatitis.
- Slide 17 Hepatitis C Virus Mode of Transmission
HCV is transmitted primarily through large or repeated percutaneous (i.e., passage through the
skin) exposures to infectious blood, such as Injection drug use (currently the most common
means of HCV transmission in the United States); Receipt of donated blood, blood products, and
organs (once a common means of transmission but now rare in the United States since blood
screening became available in 1992); Needlestick injuries in healthcare settings; Birth to an
HCV-infected mother
HCV can also be spread infrequently through sex with an HCV-infected person (an inefficient
means of transmission); Sharing personal items contaminated with infectious blood, such as
razors or toothbrushes (also inefficient vectors of transmission); Other healthcare procedures
that involve invasive procedures, such as injections (usually recognized in the context of
outbreaks)
- Slide 24 Human Immuno-deficiency Virus (HIV)
Drugs are available to reduce the viral load of the HIV virus and can prevent cross infection with
other people.
- Slide 26 - 27 Transmission of HIV and Incubation Period of HIV
HIV was discovered in 1989 and was soon recognized as the primary cause of post-transfusion
non-A, non-B hepatitis.
- Slide 50 Compliance Control Methods
All human blood and certain human bodily fluids are treated as known to be infectious for HIV,
HBV, and other BBPs.
Change PPE between patients and wash hands each time after removal of glove.
- Slide 51 Compliance Control Methods
Change gloves between tasks and procedures on the same patient and after contact with
material that may contain a high concentration of germs.
Change gloves if they become damaged or torn.
Wash hands or use alcohol gel immediately after removing gloves.
Always change gloves between patients.
- Slide 53-54 Compliance Control Methods
Dont take food and drink in work areas.
Take care to minimize splashing of all materials.
Cover any open cuts, scrapes, rashes and broken skin.
Dont touch anything thats contaminated, such as sharps or body fluids.

Screening & Confirmatory
- Slide 35 Thick blood film
The blood smear must be thick enough to just be able to make out the print on a newspaper (or
similar) when the slide is placed on top of that piece of paper.

NAT Types
- Slide 13 Window Period
Window Period: Infection to Detection
This information comes from Model Testing based on Sero Conversion Panels. Note the
significant differences in NAT versus Ab or Ag for HIV and HCV
HBV takes longer to replicate and therefore the difference is not quite as dramatic, yet it is
significant.
297

- Slide 19 - Published Yield Cases - Demonstrated NAT Yield in Developing Countries (Ekiaby et
al. 2010)
Kehua Manual extraction, single-lex using ABI 7300
- Slide 27 Mechanisms for OBI occurrence
Need to define OBI for students

298
MinistryofHealth
Criteria of Donor Selection and
Deferral
Institute Graduates
Theproperdonorselectionisthefirstlineofdefense
againstthetransfusiontransmitteddiseases.
Theprimaryresponsibilityofabloodbankistoattract
blooddonorsandtoensuretheirreturnforcontinuous
donation.
Bloodsubstitutesaremeanttobeusedonashortterm
basisandarenotmeanttobereplacementforblood.
Volunteerblooddonorsaretheonlyprecioussourcefor
providingthisgiftoflife.
Careshouldbegiventoensuresafetyofbothblood
donorandrecipient.
Introduction
2
Therearecertaincriticalstepstoestablishblooddonor
suitabilityforcollectionofasafeunitofbloodor
hemapheresis procedure
DonorRegistration.
Physicalexamination.
MedicalHistory.
DeferralPolicy
StepsofDonorSelection
3
Donor registration makes it possible to trace a unit from the
beginning of the donation process to component preparation
and testing to the distribution of the unit to its final destination.
DonorRegistration
4
1. Dateofdonation.
2. Name:first,middle,last.
3. Ageordateofbirth
4. Nationality
5. Sex
6. Typeofdonation:Voluntary,Directed,Autologous,
Replacement,Apheresisorother.
7. Address:residenceorbusiness
8. TelephoneNo.
9. DonorIDNo.:Type,Dateofissue&placeofissue.
DonorRegistration
5
Thepurposeofthelimitedphysicalexaminationisto
determineifthedonorappearstobeingoodhealth.
Ifthereisevidencethatdonorisundertheinfluence
ofdrugsoralcohol,orifthedonorshowsobvious
signsofacoldorofexcessivenervousness,thenthe
donorshouldbedeferred.
Shouldbedonebyqualifiedbloodbankphysician,or
qualifiedwelltrainednurseorinterviewerunderthe
supervisionofthebloodbankphysician.
PhysicalExamination
6
299
Age: 1865years
Weight: >50kg.
Temperature: 37.5C(99,5F)
BloodPressure:
Systolic:100 180mmHg
Diastolic:60 100mmHg
Pulse:
50 100beat/minute,Regularity,Force,Deficits.
PhysicalExamination
7
HemoglobinorHematocrit:
Method:
Acceptedlevels:
RoutineandApheresisHb:12.5 18gm/dl&Hct:38 50%
Autologous:Hb:11gm/dl.Hct:33%
ArmInspection
ApheresisDonors:
Plat.Count:200x10
9
/Lfordoubleproduct.
150x10
9
/Lforsingleproduct.
WBCs:<12
9
/L
TotalProtein:>60g/L
IgG:>6.8g/L
IgA:>0.5g/L
IgM:>0.3g/L
PhysicalExamination
8
Informationprovidedtothedonor:Educational
informationaboutbloodfunction,blooddonation,
andtransfusiontransmitteddiseaseandspecially
HIV.
Itisessentialthatthedonorunderstandthe
informationpresentedandbeabletomakean
informeddecisiontodonate.
MedicalHistory
9
Thedonorshouldfeelcomfortableduringthe
interviewprocessaswellasduringthedonation
process.
Theprospectivedonormaybeaskedthequestions
orally,ormaycompletehisownquestionnaire.Ifthe
donorcompletesaquestionnaire,hemustbe
reviewedwithaqualifiedinterviewerbeforehis
acceptanceasadonor.
MedicalHistory
10
Ensuretheprivacyduringtheblooddonorinterview.
Verbalprivacyallowstheprospectivedonorand
interviewertodiscussconfidentialmedicalhistory
whichmayaffectthedonor'ssuitability .
MedicalHistory
11
1. Inthepast8weekshaveyoudonatedbloodorits
components?
Frequencyofdonation:routine,automated2unitPRBCs,
autologous,apheresis:
Wholeblood:every8weeks,notmorethan5time/year
Automated2unitsPRBCs:every16weeks
Apheresisdonation:every48hours,notmorethan2
times/week,or24times/year.
Autologousdonation:everyweek,nosoonerthan3daysbefore
surgery.
2. Haveyoueverbeenrejectedasablooddonor?Why?
3. Areyoufeelingwellandhealthytoday?
MedicalHistoryQuestionnaire
12
300
4. HaveyoueverhadcontactwithAIDSpatients?
5. HaveyoubeenoutsidetheKingdomforanytime
inthepasttwelvemonths?
6. Doyouknowthat,ifyouhaveAIDSvirus,youcan
transmititevenwithnegativeAIDStest?
13
7. Inthepastoneweekhaveyouhadanydental
surgery?
8. Inthepast12monthshaveyouhadanysurgery
orsevereillness?
9. HaveyoueverbeenI.V.druguser,orused
intranasalcocaine?
10. Haveyouhadgrowthhormone,oreverinjected
withbeefinsulinsince1980?
14
11. Haveyouoroneofyourfamilymembersever
hadMadCowdisease?
12. Haveyoueverhadbrainsurgeryforduramater
transplantorresideinU.Kfor6months?
13. Forfemaledonors:duringthepast6week,have
youbeenpregnantordeliveredababy?
15
14. Inthepast12months:
1. Haveyouoryourspousereceivedbloodororgantransplant?
2. Haveyoubeengivenrabiesshots?
3. Haveyoubeendialysisunitnurse,rapevictim,inaprisonora
patientinmentalhospital?
4. Haveyouhadatattoo,acupuncture,earpiercingorneedlesticks?
5. HaveyouhadcontactwithhepatitisBpatient,orreceivedhepatitis
Bimmunoglobulin.
6. Haveyouhadsexwithsomeonewhohashemophilia AorBorhas
takenmoneyordrugsforsex?
15. Duringthepast4weekshaveyouhad:vaccination,
Acutane,Proscar,Propecia,orProzacmedications.
16. Haveyouhad:Asprin,Soriatane orotherMedications.
16
17. Doyousufferorhaveyousufferedfrom:
SyphilisorGonorrhea
HepatitisorJaundice
Diabetes/Insulin
Aidssymptoms:
o Prolongedfeverordiarrhea
o Enlargedlymphnodes
o Unexplainedweightloss
T.B.,Asthmaallergyoranyseverelungdisease
Epilepsy,cancer,bleedingabnormalities
Brucellosis,Babesiosis,Leshmanasis orshagas disease
Stroke
17
Malaria:
o Treatment.
o Traveltoendemicarea
o Citizenofendemicarea
SARS:
o Patient
o Traveltoendemicareaduringlastmonth
o Incontactwithapatientduringlastmonth
18. ForAutologousdonation:
Medication,illness,cardiovascularfitnessand
bacteremia
NotnecessarytoaskaboutTTD
18
301
Donorsshouldbedeferredpermanentlyifthey
sufferfromoriftheyhave,had:
1. Bleedingabnormalities/Bloodclots
2. Cancer
3. Chaga'sdisease
4. Diabetes/Insulin
5. Epilepsy
6. Heartdisease/chestpain
7. Hepatitis
DeferralPolicy
19
8. SevereKidneydisease
9. VisceralLeishmaniasis
10. SevereLungdisease
11. SARS
12. PositiveHIV,serology(AIDSPatients)
13. T.B
14. FamilymemberwithCreutzfeldtJacobsdisease
15. I.V.drugusersorusedintranasalcocaine
16. Stroke
DeferralPolicy(continued)
20
DeferralPolicy(continued)
18. DuramattertransplantorresideinUKfor6
months.
19. SymptomsofAIDS
Prolongedfeverordiarrhea.
Enlargedlymphnodes
Unexplainedweightloss(morethan5kg)
Nightsweats
Persistentcough
Whitespotsinmouth
2
1
Donorsshouldbedeferredfor3yearsiftheyhave:
oBeenfromcountrieswithendemicmalaria.
oHadbeendiagnosedandtreatedfromMalaria.
oSoriatane medication,becauseofitslongacting
teratogenic effect.
oHadbeendiagnosedandtreatedfrom
Brucellosis.
DeferralPolicy
22
Donorsshouldbedeferredfor1year(12months)if
theysufferfromortheyhavehad:
oHimselforhisspousereceivedbloodororgan
transplant.
oRabiesshots
oBeenanurseforkidneydialysisunit.
oBeenincarceratedinaprisonmorethan72
hours
DeferralPolicy
23
Beenapatientinamentalhospital
Hasatattoo
Acupuncture
Earornosepiercing
Needlestick
Stabwound
IncontactwithAIDSpatient
Bodyfluidsplashtomucousmembrane
Gonorrhea,aftertreatment
Syphilis,aftertreatment
Incontactwithhepatitispatient,orreceiveAntiHB
immuneglobulins
DeferralPolicy
24
302
Beentreatedwithantimalarialtreatmentas
prophylaxis
TraveledtoMalariaendemicareawithoutsymptoms
Animalbite
Iftheyhavehadanysurgeryorsevereillness
HavehadsexwithsomeonewhohashemophiliaA
orB
DeferralPolicy
25
Femaledonorsshouldbedeferredfor6weeksifthey
havebeenpregnantordeliveredababy.
Donorsshouldbedeferredfor4weeksiftheyhavehad:
Lowhemoglobin(lessthan12.5g/dl)
Highpulserate(morethan100beat/minute)
Lowpulserate(lessthan50beat/minute)
Highbloodpressure(morethan180mmHgforsystoleandor
morethan100mmHgfordiastole)
Vaccination,traveltoendemicareaorincontactwithSARS
patient.
AccutaneMedicationforAcne.
Proscar mediationforprostate.
Propecia orprozac medications.
DeferralPolicy
26
Donorsshouldbedeferredforoneweekiftheyhavehad:
o MildFever
o Fluorcommoncold
o Sorethroat
o Dentalextraction
o Antibiotics
DeferralPolicy
27
Donorsshouldbedeferredfor72hoursiftheyhavehad:
o Aspirinoranyaspirincontainingmedication,ifweintendtoseparateplatelet
concentrate.
DeferralPolicy
28
Iread,understood&answeredaccuratelyallthe
abovequestionstothebestofmyknowledge.I
herebygrantpermissiontothebloodbanktodraw
oneunitofwholebloodortoperformapheresis
procedure&useitthewayitmaydeemdesirable.
Signature:
DonationConsent
29
All donors must be given the opportunity to
indicate confidentially whether their blood is
safe for transfusion, or not.
The CUE may be accomplished during or after
the donation process, allowing the donor
another chance to indicate whether the unit
is suitable for transfusion.
ConfidentialUnitExclusion(CUE)
30
303
Writtenconsentfromthepatient/donor
Incaseofminors,parents/guardianscangive
consent
Age:nobaranypersonwhocanbearthephysio
dynamicchangescanbeaccepted
Weight:Noweightrestrictioncancollect8ml/Kg.
Bodyweight
Hb.:Donorshouldnotbelessthan11gm.%
CriteriaforAutologousDonor
31
CriteriaforPlateletPheresisDonor
Donorshouldmeetalltheacceptablecriteriafor
routinewholeblooddonationhowever:
Ageofthedonor18to50years.
Weightofthedonor>than5560kg.
TTIResults nonreactive
Thepreprocedureplateletcountshouldbemore
than150,000percubicmm.
3
2
CriteriaforPlateletpheresisDonor
Donorshouldnothavetakenaspirinoranyother
plateletinhibitorinlast72hours.
Thedonorshouldnotbefastingpriortothe
procedure,howevershouldrefrainoily/spicyfood,
alsocitrusfruitsorjuices.
Donorshouldhaveaprominentandeasilyaccessible
centralanticubital veininatleastoneofthearm.
33
Theminimumtimegapbetweentwoblood
donationsshouldbe12weeks/3months
Wholeblooddonationmustbedeferredforatleast
72hoursafterplateletpheresis
Incaseofreinfusionfailure,donorshouldnot
donatewholebloodfor12weeks
DonationInterval
34
Approvalmustbeinwritingbybloodbank
physicianbeforedonation
Accordinglythedonorwillbesentfor
phlebotomy.
ApprovalofDonorSuitability
35
1. AmericanAssociationofbloodbank Technical
Manual,14
th
edition,Bestheda,MD,2002
2. StandardsforBloodTransfusionservices,AABB,
22
nd
edition,Bestheda,MD,2003
References
36
304
MinistryofHealth
Phlebotomy (Collection of Blood)
Institute Graduates
Characteristicsofbloodcontainer:
Sterile
Pyrogenfree
approvedbyFDAorCE
Thevenipuncturesitemustbeaseptic.
Bloodandanticoagulantmustbemixedcontinuallyduring
theprocedureofbloodcollection,usingcalibratedblood
mixer.
Afterthedonationiscompleted,thedonorrecoverywillbe
observedandmonitored.
Refreshments(Juice&Biscuit)willbegiven.
Thedonorwillreceivethepostphlebotomycareinstructions
priortobeingallowedtoleavethedonationroom.
Principle
2
1. Allphlebotomystaffmustdondisposableglovesandgowns
priortotouchingthepatient/donor.
2. Priortophlebotomywriteunitnumberanddonornameon
themaincollectionbagandalltransferbags,donormedical
historyform,threeredtoptubes,andthickfilmformalaria.
3. Placethebloodcollectionsetonthebloodmixerscale,and
threaddonortubingonthebloodmixerscale.
4. Inspectarmforsuitablevein(usuallyintheantecubital
fossa.)
5. Applytourniquet,identifysuitablevein,andreleaseit.
Procedure
3
5. Preparedonorarm:
Scrub4cmareainalldirectionsfromintendedsitewith2%PVP
iodinesolutionfor30seconds(ifadonorssensitivetoiodine,use
alcoholswab.)
Apply10%PVPiodineswabstick,startatthecenterwith
concentricspiraloutwardfor30seconds.
Coverareawithsterile4x4gauze,anddonottouchtheskin.
Procedure
4
6. Applytourniquetagain
7. Removetheneedlecover.(16gaugeneedle),andperform
phlebotomy,byinsertingtheneedlewithitsbevelupward
instraightsteadymotioninthevein.
8. Tapeneedleinplaceonarmwithadhesivestrips.
9. Releasetourniquet
10. Switchonbloodmixer.
11. Havedonoropenandclosefist(squeezingfoamballevery
1012seconds.)
Procedure
5
11. When585gm(469ml)ofbloodhasbeencollected,the
devicewillautomaticallystopthebloodflow,andalarm
willsound(completedrawwithin1015minutes;to
separateallbloodcomponentscollectiontimemustnot
exceed10minutes,ifitreach15minuteswecanseparate
onlyPRBCsanddiscardplateletrichplasma,andifit
exceed15minuteswestopdonationdiscardblooddueto
slowbleed)
12. Applyhemostat(thisneedstobeexplainedastowhatitis
?Aclamp)totubingnearvenipunctureandmakeatight
knotfrompreviouslypreparedlooseknotjustdistalto
inlineneedleandhemostat.
Procedure
305
13. Cuttubingbyscissorbetweenthetightknotandhemostat
andseparatethebloodcollectionset.
14. Obtainbloodsamplesbyunclampthehemostat,fillthe
tubeswithbloodandreclampagain.
15. Releasethetourniquet,(shouldntthetourniquethave
beenreleasedatthebeginning????)withdrawtheneedle,
andapplypressurewithagauzepad,andhavedonorraise
arm.
16. Discardneedleassemblyintoasharpscontainer.
Procedure
7
17. Recordtimestartedandfinishedandunitweight.
18. Sealtubingnexttotheknot,stripdonortubingthreetimes
andheatsealintosegmentswithclearreadablenumbers
uptothemainbag.
19. Removethefirstsegmentandlabelitwithunitnumber
andplaceitintodailysegmenttray.
20. Putallbloodcollectionsetwithrubberbandandsendto
componentpreparation.
Procedure
8
21. Placeredtoptubesinrackforsendingtodonorprocessing
andinfectiousdiseasescreening.
22. Completethedonormedicalhistoryform.
23. Assessdonationsite.Ifsatisfactory,applyBandAid.
24. Allowdonortositupandstaywithhim.
25. Providedonorwithjuiceandcookiesandobservehim.
26. Allowdonortoleaveafter10minutesrest,andinabsence
ofanyadversereaction.
Procedure
9
Recordadversereactions
Recordanyexception
Doublephlebotomy(explainthisfurther)
Incompletebleed
Slowbleed(morethan10minutes)
Arterialpuncture
Contamination
Overweightunit(>522g)+wt ofset
Lowvolumeunit(<316g)
ReportingResults
10
Unitstakemorethan10minutesnotsuitableforpreparation
ofcomponents.onlyWBorPRBCS)
Incaseofcardiacarrestcallemergencyroomteaminthe
hospital.
Inmobilesitecardiacarrestcasescallambulance,orPoliceor
Fire
Contaminationofvenipuncturesite,repeatentireprocedure.
Doublephlebotomy,usedifferentsiteandset.
Unitsfromdonorsingestedaspirinwithinlast3days,not
suitableforplateletpreparation.
ProcedureNotes
11
306
MinistryofHealth
Donor Adverse Reactions
Institute Graduates
Blood donors normally tolerate the donation very well, but
occasionally adverse reactions of variable severity may occur
before, during or at the end of the collection.
The adverse reactions that occur in donors can be divided
into local reactions and systemic reactions.
2
Introduction
Definition:sideeffect;anundesirableorallergic
responsethathappensduringthedonationprocess.
Causes:
Psychologicalfactors.
Physicalreasons.
3
AdverseReactions
Seeingofhis/herownblood
Anxietyoveradonorscondition
Firsttimedonating
Fear
Witnessingofanotherdonorsreaction
Group/individualexcitement
4
PsychologicalFactors
Hyperventilation
Shiftofbodyfluids
Lackofsleep
Decreasedfluidintake(dehydration)
Physicalactivity
Lackofnutrition
5
PhysicalReactions
Firsttimedonors>Repeateddonors
Femaledonors>Maledonors
Youngerdonors(under20)> Olderdonors
Lowbodyweight>Highbodyweight
NodifferenceinRace
307
1. Arminjury
i. Vessel injury
ii. Nerveinjury
iii. Localallergy
2. Vasovagalevent
i. Vasovagalreaction
ii. Vasovagalsyncope
3. Hyperventilation
4. Epilepticcrisis
5. Cardiovascularevent
i. Angina
ii. Myocardialinfarction
iii. Stroke
6. Allergicreaction
i. Mildallergicreaction
ii. Anaphylacticreaction
7. Haemolyticreaction
8. Airembolism
9. Citratetoxicity
10. Chillsand/orrigors
11. Hypotension
TypesofDonorReactions
7
A. Haematoma(bruise)
B. Arterialpuncture
C. Arteriovenous fistula
D. Thrombophlebitis(superficial)
E. Deepvenousthrombosis
8
1.ArmInjury i.VesselInjury
Definition:Anabnormal,localizedcollectionof
bloodundertheskin.
Signsandsymptoms:
Colourchangeintheskin(bruise,ifnoothersigns)
Swelling
Painortendernessatthevenipuncturesite
9
A.Hematoma(bruise)
Howtotakecare:
Removetourniquet&pulltheline.
Apologize&Reassureeverythingisappropriate.
Applypressuretosite&elevatearm.
ApplyiceX15min.&periodicallythroughouttheday.
Ifpaincontinues,maytakeTylenol.
10
A.Hematoma(bruise)
Definition:Accidentalpunctureofanartery.
Signsandsymptoms:
Highbloodflowrate(bloodunit<4minutes).
Brightredcolourofthecollectedblood.
Pulsatingneedle.
11
B.ArterialPuncture
Howtotakecare:
Pulllineimmediately&addpressurefor10minutes.
Apologizetothedonor.
Wrapwithflexiblebandage.
DoNOTRestickDonor!
308
Definition:Formationofachannelbetweenavein&
anarteryfollowinglacerationofthevesselsbythe
penetratingneedle.
Signsandsymptoms:
Bruise,haematoma,stiffness,swelling&paininthe
arm.
Distalveinsdilatedandpulsate.
Pulsatingmasswithacontinuousmurmurandpalpable
thrill.
13
C.ArteriovenousFistula
Definition:Formationofabloodclotinthepunctured
superficialveinassociatedwithaninflammatory
reactionofthevein.
Signsandsymptoms:
Tendernessandhardnessofthevein.
Rednessoftheoverlyingskin.
Howtotakecare:
Requiremedicalevaluation.
14
D.Thrombophlebitis(superficial)
Definition:Formationofabloodclotinadeepvein
withverylittlereactionintheveinwall.
Signsandsymptoms:
Maybeasymptomatic.
Swelling&Discolorationofthearm.
Antecubital tenderness.
Increasingarmpain.
Venousdistentionofthearm.
15
E.DeepVenousThrombosis
Definition:Injuryofanervebytheneedleorbya
haematoma.
Signsandsymptoms:
Sensorychanges(numbness,tingling).
Excessive/burning/radiatingpaininthearm.
Lossofarmorhandstrength.
16
1.ArmInjury ii.NerveInjury
Definition:Allergicreactiontoanadhesivetapeor
skinpreparationsolution
Signsandsymptoms:
Erythema,pruritus
Howtotakecare:
Treatmentofsymptomsiswithantihistaminesandthe
countertopicalsteroids.
17
1.ArmInjury iii.LocalInjury
Definition:Afeelingofdiscomfortjustbefore,during,or
shortlyafterblooddonation.
Signsandsymptoms:
Pallor
Weakness
Dizziness
Sweating
Anxiety
Nausea
Vomiting
Hypotension
Bradycardia
18
2.VasovagalEvent i.VasovagalReaction
Definition:Donorunconsciousforashortperiodof
time.Cantrememberallwhathappened.Asyncope
mayoccurafterthedonorleftthecollectionsite
Signsandsymptoms:
Symptomsofavasovagalreaction.
Lossofconsciousness.
Incontinence&Convulsions.
19
VasovagalEvent ii.VasovagalSyncope
Howtotakecare:
Removeneedleimmediately.
Reassurethedonor.
Loosenclothing,monitor&recordvitalsigns.
Coldpackbehindtheneck,bendknees&elevatefeet.
Administeraromaticspiritsofammoniabyinhalation.
20
VasovagalEvent
Definition:
Fasteranddeeperbreathingresultinginexhalingexcessive
amountsofcarbondioxide.
Thiscausesadecreasedbloodcarbondioxideleveland
increasedpHlevel.
Bothcancausecerebrovascularconstriction.
Signsandsymptoms:
Paresthesias/tingling,twitching
Anxiety,sensationofsuffocation
Howtotakecare:
Askthedonortobreatheintopaperbag.
21
3.Hyperventilation
Definition:Suddenattackoflossofconsciousnessor
awarenessassociatedwithabnormalmovementsor
convulsion
Signsandsymptoms:
Suddenonset.
Lossofconsciousness.
Tonicconvulsivemovements.
Upturningeyes
Howtotakecare:
Turnthedonoronhissidetoprotecttheairway.
Useapaddedtonguebladetopreventinjurytothetongue.
Preventfallsandinjurytothedonor
Instructthedonortoavoidfurtherdonations
22
4.EpilepticCrisis
i. Angina
ii. Myocardialinfarction
iii. Stroke
Howtotakecare:
Veryrareincident
D.D.betweenvasovagalreaction&cardiacshock
Callhospitalemergency
StartCPRincardiacarrest
23
5.CardiovascularEvent
Definition:Anallergicreactiontoasubstancethatis
transfusedtothedonorduringanaphaeresis
procedure.
Signsandsymptoms:
Pruritus,rash,urticaria.
24
6.MildAllergicReaction
310
Definition:Immediateseverehypersensitivityreaction.
Signsandsymptoms:
Erythema,urticaria,laryngeal,pharyngeal&facialoedema.
Bronchospasm,respiratorydistress.
Hypotension,shock.
Howtotakecare:
Stopprocedure.
IVAntiallergic.
IVDecadron.
IVCalcium.
25
6.AnaphylacticReaction
Definition:Return ofhaemolysedblood
(mechanical)orhaemolyticreactioninthedonor
followingaccidentalinfusionofahypotonic
solutionduringanaphaeresisprocedure
Signsandsymptoms:
Haemoglobinuria,haemolysedplasma
Renaldysfunction
Hypotension,DICandfever
26
7.HaemolyticReaction
Definition:Fastinfusionofalargeairbubbleintoa
donorduringanaphaeresisprocedure.
Signsandsymptoms:
Abruptonset
Cough
Dyspnoea
Cyanosis
Hypotension
Cardiacarrhythmia
27
8.AirEmbolism
Howtotakecare:
TurnonLeftside
Lowerhead&elevatelegs.
Callforemergencydepartment.
28
8.AirEmbolism
Definition:Citrateinfusionduringanaphaeresis
procedurecausingreducedfreecalciumandassociated
symptoms.Subsideswithreductionofthebloodflow.
Signsandsymptoms:
Paresthesia/tingling
Nausea,vomiting
Arrhythmia
Howtotakecare:
Warmblankets.
AphaeresisstafftodecreaseInlet checkCitrateMonitor
Monitorandreassuredonor
Oral/IVcalcium
29
9.CitrateToxicity
Definition:Returnofcoldbloodduringan
aphaeresisproceduremaycauseacoldfeeling.
Signsandsymptoms:
Chills,rigor.
Howtotakecare:
Warmblankets,
Monitordonor
Reassuredonor
30
10.Chillsand/orRigor
311
Definition:Decreasebloodpressure.
Signsandsymptoms:
Lightheadedness
Increasepulserate
Shallowrespiration
Perspiration
Howtotakecare:
Lowerhead
Raisefeet
Givefluidsbolus
31
11.Hypotension
Althoughthenumberofdonorswhodeveloped
disturbancesbefore,duringorattheendofblood
donationswasverylow,itisneverthelessdesirable
toreduceriskstoaminimum.
Asetofadvicesisprovidedforpreventingproblems.
32
StepsforPrevention
Shortenthewaitingtimes.
Allowthedonoralightbreakfast,excludingsugar,milk&
milkproducts.
Ensureacomfortableroomtemperatureandhumidity.
Engageparticularlyanxiousdonorsinconversation,in
ordertodistracttheirattentionfromwhatishappening.
33
StepsforPrevention
Avoidtraumaticneedleinsertionwithinvasiveand
painfulmaneuvers.
Identifythebestvenousaccess,byinspectingboth
forearms.
Ifthefirstvenipunctureisfailed,allowthedonorto
restandreassurehimorher,beforeattemptinga
newone.
34
StepsforPrevention
Invitedonorstowearcomfortableclothes,avoiding
tighttiesandbelts.
Donotletthedonorsdrinkveryhotorverycold
drinksduringtherecoveryphase.
Donotallowdonorstoeatsolidfoodsduringthe
donation.
35
StepsforPrevention
Reactswiftlytotheinitialsymptomofpallor,by
puttingthesubjectintheTrendelenburgposition.
Donotletthedonorleavethedonorsitetoo
quickly.
Continuousadequatelytrainingfortheblood
donationstaff.
36
StepsforPrevention
312
MinistryofHealth
Donor Recruitment and Retained
Strategies
Institute Graduates
A. Government
B. MinistryofHealth
C. TheDonors
D. BloodBanks
E. Community
Whoisresponsibleforensuringanadequate
bloodsupply?
2
A. Doctors
B. Nurses
C. Bloodbanktechnicalstaff
D. Publicofficials
E. Marketingprofessionals
WhoisBestQualifiedtoPerformtheFunction
ofDonorRecruitment?
3
1. Absenceofprofessionaldonorrecruiters.
2. Limitingrecruitmenttonationallevel.
3. Misconceptionsregardingblooddonation.
4. Lackofunderstandingofdonormotivation.
ProblemsinDonorRecruitment
4
5. Poorgoalsettingandwastingofresources.
6. Lackofsustainededucationalandrecruitment
efforts.
7. Poordonortreatment.
8. Donorconfusionwhenrecruitedby
competingbloodcenter.
ProblemsinDonorRecruitment
5
Motivatingpotentialdonorstodonate
andorganizethemtoprovideadequate
sustainableandsafebloodsupplyallyear
around.
TheChallenge
6
313
Donorshallbevolunteersandshallnotbe
paid.
Itiseasiertointroduceyoungpeopletoblood
donationthantheirparents.
Groupleadershipisthebestwaytorecruit
newdonors.
GuidingPrinciples
7
Regulardonorsaresaferthannew
donors.
Donorrecruitmentmustbeasteady
yearroundactivity.
Donationbypublicleaderscanseta
valuableexample.
GuidingPrinciples
8
I. Fieldstudy
II. Assessingneedsandsettinggoals
III. Teamwork
IV. Motivatingdonors
V. EducationalProgram
VI. Selectingsafedonors
DonorRecruitment
9
VII. BloodDonationPlan
VIII. CounselingandDonorcare
IX. Retainingdonors
X. Recordmaintenance
XI. Monitoringandevaluation
DonorRecruitment
10
I.FieldStudy
11
Assessandidentifythestrengthsandweaknessesof
theBloodBankintermsoftheavailabilityoftrained
manpowerandexpertise,publicrelationsandthe
absorptivecapacityandfinancialresources.
Identifyopportunitiesandobstaclesinthe
surroundingenvironmentandthestudyofculture
andlifestylebehaviorsamongarandomsampleof
donorsandnon blooddonors.
FieldStudy
314
Determinethelistofhospitalslocatedinthevicinity
ofthebloodbank,anddetermineitsneedtoblood.
Alertstrengthsandworktoimprovetheweaknesses
andexploitthechancesofsuccessandtrytoavoid
obstaclesorforeigncopewithit.
FieldStudy
13
II.SettingGoals
14
Oneofthemostimportantandmost
difficultstepsineffectivedonor
recruitment.
Goalsmustbesetathigherlevelthan
theapparentneedforblood.
SettingGoals
15
Somedonorsarerefusedeithertemporarilyorpermanently.
Somedonorsdontcompletethedonation.
Someunitsofbloodhadpositiveserologyresults.
Bloodunitsthatareexposedtodamageorcontamination.
Unitsofblood,whichexpirewithoutuse.
Asastrategicstockforemergencyresponse.
ReasonsofIncreasingTheTargetThanThe
ActualRequirement
16
1. TotalPopulation:2%
2. AcuteHospitalBeds:
o PrimaryUnits:5to7/Bed.
o SpecializedHospitals:25to30/Bed.
o Surgery:10 12/bed/year.
3. MedicalFacilitiesinthearea.
4. AnnualBloodUsage:Past,Present&Future
identifiedwithanannualincreaseofabout10%.
ApproachtoSettingCollectionGoals
17
III.TeamWork
18
315
Teamworkistheconceptofthepeopleworking
togethercooperativelyasateaminordertoaccomplish
thesamegoal.
Thegoodunderstandingofteamworkandteambuilding
acriticalforeveryworksuccess.
TeamWork
19
Choose the members of the team (doctors,
administrators, nurses, motivators and
coordinators campaigns, workers) depends on
the size of the program and the nature of the
work to be carried out.
Put the job description with identifying tasks and
responsibilities for each of them and the
issuance of ways of working standard and train
to become a high degree of knowledge and skill.
TheTeam
20
Theremustbeamechanismforcooperationand
communicationbetweenthedifferentdepartments
oftheCenterandthesupervisorontherecruitment
program.
Workrelationshipsseemtohaverules:
Acceptfairshareofwork.
Cooperateinsharingresources.
Helpeachotherwhenasked.
Mutualtrust
TeamRelationship
21
Members have a clear goal.
The focus is on achieving results.
There is a plan for achieving the goal.
Members have effective interpersonal skills.
They know each other well and have good
relationships.
The team has the support of management.
The team is open to new ideas.
There is periodic self-assessment.
There are sufficient resources to support the team
work.
WhatareCharacteristicsofEffectiveTeams
22
IV.MotivatingDonors
23
1. Peopledontgivebloodunlessthey
areaskedtodothat.
2. Peoplearenotnaturallymotivatedto
donatetheirblood.
3. Therearemorethanenoughpotential
blooddonors.
4. Theremanymisconceptionabout
blooddonationinpublic.
IV.MotivatingDonors
316
1. Awareness:Essentialbutnot
sufficienttocausepeopleactuallyto
giveblood.
2. Interest:Developsovertimethrough
discussionandreconsideration.
StagesofMotivation
25
3. Desire:Usuallycomethrough
personalorpubliccrisis.Thisa
normalhumanreactionbutnot
sufficientforsustainedbloodsupply.
4. Action:Thisisthetargetofthe
motivationreconsiderationprocess.
StagesofMotivation
26
V.EducationalProgram
27
Mosteffectivemethodofrecruitingdonors
Talkonneedofblood,shortageofblood,easeof
donationandmythaboutblooddonation,possibly
illustratedbyfilmsisveryeffective
TheSpeakers/Recruitersmusthavethepersuasive
powertoappealtothehumanitarianfeelingsofthe
audience.
Timeshouldbeavailableintheendoftalkforthe
audiencetoaskquestionsandtogiveprecise
answers
OralCommunication
28
Brochures,Postersandinformativeleafletsare
variousformsofcommunication
Materialmustcatchtheeyeandbeeasyto
understand
Publicitymaterialseg Posters,television,
advertisements,cartoonsetc,shouldbepreparedby
professionals
GreetingCardsonBirthday,MarriageAnniversaries,
NewYearsdayorotherauspiciousdayscarrying
motivationalslogansforvoluntarydonationarealso
effective
PrintedCommunication
29
Educationamongtheyoungisusefultoremove
superstitionandmythconnectedwithblood
donation.
Itisimportanttointroducethesubjectofblood
donationintoSchoolsaspartofScienceandcivic
studies.
TheyoungarepotentialDonors.
EducationalInstitutions
30
Participationofmediaisthemosteffectivewaytomobilise
voluntaryblooddonation.
Abilitytoreachlargenumbersofpeopleismostimportant
advantage.
Mediaseemstobeexpensive?
Althoughnewspapers,radioandtelevisionmaychargefor
advertisementsorregularpublicservicebroadcasts.
RoleofMedia
31
Journalistsarealwayslookingforgoodstories
whichdoesntinvolveanycost,foregg:interview
ofpersonwithhisfamilysharinghisexperience
howbloodsaveshislifeandpersonallythanking
thedonorsforthesame.
Involvementofmediagivesaemotionaltouchto
publictowardsblooddonation.
RoleofMedia
32
We can encourage internet service providers and
search engines to promote blood donation on their
home pages on World Blood Donor Day and other
special events.
Or an FM radio station to broadcast date and
location of blood donation camp.
RoleofMedia
33
Request mobile telephone companies to send
SMS/text messages to all subscribers urging them to
become blood donors.
Utilising entertainment industry to add storylines
on blood donation in their television programmes,
plays and movies.
RoleofMedia
34
VI.SelectingSafeDonors
35
VoluntaryNonRemuneratedBloodDonorsarethe
lowriskdonorsfor:
Safeblood.
SustainablebloodSupply.
ReplacementDonorsnotapreferredsystem:
RisksofprofessionalDonorsingarbof
replacementdonors.
Riskofhiddeninformation.
BloodDonors
36
318
Isdefinedas:
"Avoluntarynonremuneratedblooddonorwho
donatesatleastonceperyearormoreaccordingto
nationalstandards fortwoconsecutiveyears.
Regularvoluntarynonremuneratedblood
donor
37
Donateforaltruisticreasons.
Entrusttheirblooddonationtobeusedaccordingto
theneed.
Arenotunderpressuretogiveblood.
Arecommittedtogivebloodwhenrequired.
Providethefoundationofasafeandsustainable
bloodsupply.
Characteristicsofregularvoluntary
nonremuneratedblooddonors
38
Bloodsafety.
Sustainabilityandavailabilityofblood.
Avoidexploitationandcommercializationofthe
humanbody,thepoor&vulnerable.
Altruismandsocialsolidarity.
Importanceofregularvoluntarynon
remuneratedblooddonation
39
Thisoccursincountrieswhere:
bloodsuppliesarescarce.
thereisnocultureofvoluntaryblooddonation.
Thereisnotrustinbloodbankingfacilities.
Wherefamilyreplacementdonorsareforcedintodonating
blood,theyarelesslikelytobetruthfulabouttheirhealth
statusorhighriskbehavior.
ThisresultsinahigherincidenceandprevalenceofTTIs
comparedtoasystembasedonvoluntaryblooddonation
Characteristicsofpaid&familyreplacement
donors
40
Theuseofincentivesdifferfromcountryto
country.
Dependingoneachpersonshierarchyofneedse.g.
culturalbeliefs,personalvalues,economicfactors.
Theuseofincentivesshouldnever:
Compromisethesafetyofthebloodsupply.
Beconditionalonthepersonactuallygivingblood.
Incentives&RisktoBloodSafety
41
VII.BloodDonationPlan
42
319
Recruitmentprocessshouldbe
basedonscientificstudiesnot
assumptions.
Localculture&religiousbeliefsmust
beunderstoodtomotivatepeople
appropriately.
BloodDonationPlan
43
Trainsomeofouryoungactiveemployeetoworkas
recruiters.
Increasethenumberofblooddrives.
Forminformationdatabaseforarrangetheprocessof
donationanddonorsrecall.
DeveloptheAppropriateScreeningQuestionsForm.
TargetLowRiskGroups.
PlanofAction
44
Actasaimportanttargetpopulationdespitetherisks
associatedwiththem.
Theyunderstandtheimportanceofthetimelyavailabilityof
bloodbecauseoftheirdirectexperienceoftheurgentneed
ofdonors.
Shiftingfamily/replacementdonationsystemtovoluntary
blooddonationsystemrequiresachangeinpracticeby
hospitalsitself.
Encourageandeducatehospitalandhealthcentrestaffto
discussvoluntaryblooddonationwiththem.
Convertreplacementdonorstovoluntary
blooddonors
45
Theyalsoconstituteanimportanttargetdonor
population.
Thesedonorsarealreadyawarded
Aboutdonationprocess
Needofblood
Andalreadymotivated.
Reactivatingformerdonorscanbemoreeasythan
recruitingnewdonors.
Theysimplyneedareminderandencouragementto
return.
Donordatabaseordonorregisterisavitaltoolfor
recallingpreviouslyactivedonors.
RecallInfrequentorTemporaryDeferred
Donors
46
VIII.Counseling&
DonorCare
47
Provideacounsellingsessionbeforeeveryblood
donation
Predonationinterview/sessionshouldbe:
Private
Confidential
Notinterrupted
2 waycommunication
PredonationCounselling
48
Tomakeapreliminaryassessmentofdonorstateof
health.
Explaininadvanceeverystepinvolvedinblood
donation.
Obtaininformedconsentforblooddonationand
testing.
ToincreaseawarenessabouttestingforTTIsandthe
possibleconsequencesofpositiveresults.
ObjectivesofPreDonationCounselling
49
Toencourageselfdeferralofpeoplewhomayhave
ahistoryofriskbehaviour
Todiscouragepeoplefromusingthebloodservice
asacentreforvoluntarycounsellingandtesting
Emphasisetheneedtomaintainahealthylifestyle
Dispelmythsandmisconceptions
ObjectivesofPreDonationCounselling(cont)
50
Post DonationCounsellingisofferedtoaperson
whohasdonatedbloodandhaslearnttheresultsof;
HIV,HepatitisB&C,Syphilisandhasbeentold
diagnosisofthesediseasesbythecounselor.
Post DonationCounselling
51
Convenientlocation,
operatinghoursand
accessibility.
Welladvertisedwithhigh
visibility.
Staffshouldbealways
smart,cleaninappearance
andmaintainahigh
standardofpersonal
hygiene.
Generalrulesofdonorcare
52
Staffshouldbewell
trainedandqualifiedon
venepuncture.
Donorbedcomfortable&
cleansheets.
Comfortableposition.
Personalattention.
Explanationofprocedures
&technology.
Duringblooddonation
53
Ensurequalityandsafetystandards.
Maximisinganeducationopportunity.
Observingandlisteningtodonor.
Manageuneventfuldonations.
Providepostdonationadvice.
Duringblooddonation(cont)
54
321
Appropriaterestingtime.
Providecomplimentaryrefreshments.
Appropriatelytrainedstaff/orvolunteers
Goodobservationskillsfordonorsreactions.
Postdonation
55
Managereactionsprofessionally
Encouragedonorfeedback
Thankthedonor,remindhimofnextdonationand
invitehimtobringafriend
Postdonation(cont)
56
Encouraged,welcomedandacknowledgede.g.
suggestionbox,donorsurveys,donorcomplaint
form.
Positiveandnegativefeedbackisdealtwithand
recorded.
Respondtoalldonorfeedback.
Donorfeedback
57
Toensuredonorsatisfactionandretentionany
donorcomplaintsmustbetakenseriously:
complaintsmaybeaboutanythingthattheyhave
experiencedintheircontactwiththeBTS.
complaintsmayrangefromtrivialtoserious.
DonorComplaints
58
Standardproceduresfor
handlingcomplaints.
Trainedstafffordealing
withcomplaints.
Measurestoensurethat
errorsthatareidentified
arecorrectedand
preventedfromrecurring.
DonorComplaints
59
Complaintsshouldbedealtwithprofessionally.
Donorsmustfeelthattheircomplainthasbeen
listenedtoandunderstood,andwillbeinvestigated.
Complaintsmustbeinvestigated.
Feedbackshouldbegiventothedonor
immediateifappropriate
laterinwriting
Allcomplaintsmustbeloggedandtheinvestigations
andoutcomesrecorded
DealingwithComplaints
60
322
Donorcomplaintsmaybeusefulinidentifyingareas
ofpoorpractice poorquality
Thedonorsarethebestpeopletoassesshowthey
areactuallytreatedatadonorclinic
TheBTSmustalwaysbeopentocriticismandbe
willingtolearnfromit
Positiveresponsestocomplaintsmaymakeiteasier
toretainthedonor
PositiveOutcomes
61
FormsofDonorRecognition
Letterofappreciation
Greetingscards/Thankyouletters
Certificate
Badge
Memento
Annualfelicitationbywayofconvocation
Donorscard
Specialawardfor10/25/50/100timedonation
Recognitionandawardsactasanincentivetoexisting
donorsandmotivatepotentialdonorstobecomeregular
voluntarydonors.
DonorRecognition
62
Recognitionplaysamajorroleindonorrecruitment
strategies.
Recognitionofvoluntaryblooddonormeanshonour
withoutanymonetaryvalue.
Recognitionofdonorshelpsas:
Automaticincentive.
Socialprestige.
Motivationtopaneldonors.
Spursdonorstogreaterparticipation.
Feelingappreciatedandhonoured.
Formsstrongercommitment.
Helpinretentionofdonors.
RecognizeDonorsContribution
63
Voluntarydonorsarepriceless.
Qualitydonorcareisabasicdonorright.
Mobiledonorclinicsaremoreconvenienttodonor
population.
Donormustbewellinformed,safe&appreciated.
Emphasizetheimportanceofdonorsatisfactionasthefirst
stepindonorretention.
Asatisfiedandcommitteddonoristhebestambassadorof
recruitingotherdonors.
KeyPoints
64
IX.RetainingDonors
65
Itisdifficulttorecruitnewdonors.
Itcostsmoneytorecruitnewdonors.
Itcostsmoretotakeonedonationonly.
Itissafertouseregulardonorblood.
WhyDonorRetention?
66
323
Thefirstimportantmessagetothebloodcollection
teamistoconsiderthevoluntarydonorasapartner
inthesupplyofbloodforthecommunity
Donorsaretheownersoftheserviceandnotonly
customers.
Identifydonorsexpectations.
Howtodoitstandards
67
Identifydonorsneeds.
Viewsvalued,respectedandactedupon.
Warmwelcome.
Ensuredonorisappreciated,thankedand
encouragedtoreturn.
Howtodoitstandards(cont.)
68
Alwaysaddressthedonorbyhisname.
Alwaysmakeeyecontact.
Explainthedonationprocess.
Explainwhathappensnext.
Ensureprivacyanddiscretion.
Answerquestions/querieshonestlyandtactfully.
Talkupthesystembeitsadvocate.
Bepositiveduringthedonationprocess.
Whattodo?
69
Talktothedonorduringbloodcollection
Chattothedonoriftheywant.
Askdonorsregularlyiftheyarewell.
Assessaccordingtoeyecontact.
Escortdonortotherefreshmentarea.
Makethemfeelwelcomeandvalued.
Whattodo?(cont.)
70
Abandonthedonor evenforashortperiod.
Talkjargonwhenexplaining.
Talkoverwithothercolleagues.
Challengedonorsbeliefs.
Giveinformationyouarenotsureabout.
Disregarddonorsobservations.
Underestimatetheworryofdeferreddonors.
Whatnottodo
71
Weassumewhatdonorswant.
Theemotionalneedsofdonors.
Donorstakesomethingaway.
Askdonorswhatmatterstothem.
TheDonationExperience
72
324
ReliabilityIndex
Retentionrate
Frequencyrate
Newdonorintake
Donorattritionrate
Ageandgendergroupprofiles
Satisfactionmeasures
LoyaltyAssessment
73
Randomselectionofdonorpopulation
Scoreoutof10forthewholeexperience
Abouthowtheywerelookedafter
Abouthowtheywerevalued
Specificallyabouttheneedleinsertion
Specificallyaboutwaitingtimes
Specificallyaboutstaffattitude
DonationExperienceSurvey
74
Alldonorscommentsareopportunities
Activelylookforopportunities
Interactionwithdonorsmakesadifference
Assessandunderstandtheneeds
Modifyapproachinresponsetofeedback
PersonalContributionReinforcesPartnership
75
Shouldbemadetofeelextraspecial.
Maybefrightenedandoverwhelmed.
Mayhaveincorrectinformation.
Requirescarefulobservationallthrough.
Needsclearreassuringexplanation.
TheNewDonor
76
Thinkhowthedonormaybefeeling.
Upset,rejected,concernedandconfused.
Reassureandhandlewithempathy.
Giveadequateexplanation.
TheDeferredDonor
77
Selection
Professionalism
Donorclinicalcareskills
Donor/customerservicesskills
Dedicationandconviction
Newvisionandnewapproach
Staffing
78
325
X.RecordMaintenance
79
Eachactivityinthedonorarea,andtheoutcomeof
eachactivity,shouldbedocumented
Writewhatyoudo,dowhatyouwrite
Donorrecordsenableto:
Recordandanalyze successesandfailures
Monitorstaffcompliance
Assistinerroranalysis
Enableadocumenttrailforauditpurposes
Documentation
80
Donorrecordsarevitalinmaintainingthesafetyofthe
bloodsupply.
Minimumdonorrecordkeepingrequirements:
Recordofblooddonors.
o Blooddonorconsentfordonation
o Donor'snameandfather/husbandname
o Donation voluntaryorreplacement
o Dateofbirth(age),Sexandweight
o Address(office&residence)andtelephonenumber
o Historyofillness
DonorRecords
81
Recordofblooddonation.
o Dateofblooddonation
o Donationnumber(Identificationnumber)
o Physicalexaminationrecord pulse,temperature,andblood
pressure
o Hemoglobin
o ABOandRh(D)group
o ResultsofHBsAg,antiHCV,antiHIV1&2.VDRL/RPRand
malariatests
o Disposal:issuedfortransfusionordiscarded
DonorRecords
82
Confidentialityiscriticalinthemanagementof
blooddonors.
Alldonorinformationisprivilegedandmustbe
keptconfidential.
Recordsmustbekeptsecureatalltimes.
Onlydesignatedstaffshouldhaveaccesstorecords.
Confidentiality
83
XI.MonitoringandEvaluation
84
326
Itisnecessarytoperiodicallyevaluateeachpart
ofthedonationprograminordertoascertainthe
extentofitsinfluenceandsuccessasopposedto
whatitwasspent.
Developingindicatorswithwhichtomeasurethe
successorfailureoftheprograminasimple
sentence,"theprogramisconsideredsuccessful
iftheprovisionofsafebloodthroughoutthe
year.
MonitoringandEvaluation
85
Numberofregularandnewdonors.
Numberofpermanentlydeferreddonors.
Numberofdonorsexperiencedadversereactions.
Numberoforganizationsandcommunitiesinvolvedin
motivatingvoluntaryblooddonation.
Donorfeedbacksystem.
MonitoringandEvaluation
86
SetGoals
HireProfessionals
EducatethePublic
TreatDonorsWell&EnforceDonationBehavior
AskThemtoComeBack
ManageDonorsWithTheirBestInterestinMind
Finally whattodo?
87
Recommendation
Leaveittotheprofessionals
88
327
MinistryofHealth
Blood Component Preparation and
Storage
Institute Graduates
BloodComponentsDescription
2
WholeBlood
**FreshWholeBloodcontainsallbloodelements
plustheanticoagulantpreservativeinthecollecting
bag.
**After24hourstorage,itessentiallybecomesred
cellssuspendedinaproteinsolutionequivalentto
liquidplasma.
**Itisusedcommonlyasasourceforcomponent
production.
3
AnticoagulantPreservativeSolutions(mgin63mL)for450mL Collections
CPDCP2DCPDA1
Ratio(mL solutiontoblood)1.4:101.4:101.4:10
FDAapprovedshelflife(days)212135
Content
Sodiumcitrate166016601660
Citricacid206206206
Dextrose161032202010
Monobasicsodiumphosphate140140140
Adenine0017.3
%higher. 11 %to 10 andthecontent mL 70 collections,thevolumeis mL 500 With
4
Decreaseharmfuleffectofbloodtransfusion
Givingpatientsspecificcomponentneeded
Allowalongersurvivalforcomponents
Morethanonepatientwillusetheunit
5
BloodComponentsSeparation Goals
6
After centrifugation
WB separates into
plasma & platelets &
PRBCS
WholeBloodUnit
328
PackedRedBloodCells(PRBCs)
FreshFrozenPlasma(FFP)
PlateletConcentrates(PC)
Cryoprecipitate(CRYO)
LeukoreducedRedBloodCells
7
WhataretheComponentsofBlood? RedBloodCells
RedBloodCells(RBCs)areunitsofWholeBloodwithmostof
theplasmaremoved.
Thefinalhematocritmustbe80%.
AdditiveSolutionsupportsredcellsurvivalandfunctionupto
42day.
AdditiveSolutionallowsbloodcenterstouseorrecovera
maximumamountofplasma,yetstillpreparearedcell
component
withafinalhematocrit between55%and65%.
RBCscanbepreparedatanytimeduringtheirshelflife,butAS
mustbeaddedwithinthetimeframespecifiedbythe
manufacturer,generallywithinthefirst72hoursofstorage.
8
ContentofAdditiveSolutions(mg/100mL)

AS3AS5 AS1
(Adsol)(Nutricel)(Optisol)
Dextrose22001100900
Adenine273030
Monobasicsodiumphosphate02760
Mannitol 7500525
Sodiumchloride900410877
Sodiumcitrate05880
Citricacid0420
9 10
Howtomake(PRBCs)?
RBCshavehigherspecificgravitythanplasma,itmovesto
lowerportionofthebagbycentrifugation
WB(Lightspin) Twoproducts:
1) PRBCs
2) PlateletRichPlasma(PRP)
PRBCs
11
1 2 3
12
APRBCunitcontains~200ml
RBCand50mlPlasma
AllRBCtransfusionmustbe
ABO/RHcompatible,orin
emergency,transfusetypeO
PRBCs
RBCunitsmustbystoredat26C
PRBCareindicatedfor
Patientswithanemia
Surgery
Newbornexchangetransfusion
PRBC
329
Platelets
Plateletconcentrates(Platelets)arepreparedfromunits
ofwholebloodthathavenotbeenallowedtocool
below20C.
Plateletrichplasma(PRP)isseparatedwithin4hours
aftercompletionofthephlebotomyorwithinthetime
framespecifiedinthedirectionsfortheuseoftheblood
collecting,processing,andstoragesystemtypically8
hours
Thefinalcomponentshouldcontainresuspended
plateletsinanamountofplasmaadequatetomaintain
anacceptablepH;generally,40to70mL isused
13
HowtopreparePC?
PlateletRichPlasma(PRP)centrifugedusing(heavy
spin),thiswillproduce:
Freshfrozenplasma(FFP)
Plateletsconcentrate(PC)
PCarestoredatroomtemperatureonplatelet
agitator(preventplateletsclumping)
PChavea35daysexpirationdate
14
PlateletsConcentrate(PC)
Indications:
1. Topreventbleedingduetothrombocytopeniaor
dysfunction
2. Toapatientundergoinganoperation,iftheplatelet
countislessthan20,000/L
15
PlateletsConcentrate
16
1 2
3
FreshFrozenPlasma(FFP)
FreshFrozenPlasma(FFP)isplasmapreparedfrom
wholeblood,eitherfromtheprimarycentrifugation
ofwholebloodintoredcellsandplasmaorfroma
secondarycentrifugationofPRP.
Theplasmamustbefrozenwithin8hoursof
collection.
17
Plasma
Plasmacanbeseparatedatanytimeduringstorage,up
to5daysaftertheexpirationdateoftheWholeBlood.
Whenstoredfrozenat18Corcolder,thiscomponent
isknownasPlasmaandcanbeusedupto5yearsafter
thedateofcollection.
Ifnotfrozen,itiscalledLiquidPlasma,whichisstoredat
1to6Candtransfusedupto5daysaftertheexpiration
dateoftheWholeBloodfromwhichitwasprepared.
18
330
CryoprecipitatedAHF
Cryoprecipitated antihemophilic factor(AHF)isthecold
insolubleportionofplasmathatprecipitateswhenFFPis
thawedbetween1to6C.
Itcontains80IUFactorVIII (AHF),>150mgof
fibrinogen,andFactorXIII.
CRYOcontainsboththeprocoagulant activity(Factor
VIII)andthevonWillebrand factoroftheFactorVIII
vonWillebrand complex.
Onceseparated,CRYOisrefrozenwithin1hourof
preparationandstoredat18Corcolderforupto1
yearafterthedateofphlebotomy.
19
PlasmaCryoprecipitateReduced
Ifcryoprecipitatehasbeenremovedfromplasma,
thismustbestatedonthelabel.
Whenstoredat18Corcolder,thiscomponenthas
a12monthexpirationdatefromthedateof
collection.
Thiscomponentisusedprimarilyinthetreatment
ofthromboticthrombocytopenicpurpura.
20
ExpirationDatesforSelectedBloodComponents
1WholeBloodACD/CPD/CP2D21days
CPDA1 35days
2RedBloodCells(RBCs)ACD/CPD/CP2D 21days
CPDA1 35days
hours 24 Opensystem
Additivesolutions 42days
RBCsWashed24hours
3Platelets24hoursto5days,dependingoncollection
4Plateletspooledoropensystem4hours
5FFP12months(18C)
7years(65C),asapprovedbytheFDA
6FFPThawed24hours
7FFPThawed OpenSystem24hours
8PlasmaFrozenwithin24hours12months
9PlasmaCryoprecipitate12months
ReducedFrozen
10PlasmaCryoprecipitate24hoursto5days
ReducedFrozen
11CryoprecipitatedAHF12months
12CryoprecipitatedAHFThawed4hoursifopensystemorpooled,6hoursifsingle
unit
21
WhatareLeukoreduced RBCs?
PRBCsthathaveWBCsremovedbyspecialfiltersor
byamachine
AdvantageofLeukoreduced RBCs:
Maypreventfebrilenonhemolytic transfusionreactions
(FNHTR)
Reducesriskofcytomegalovirus(CMV)transmissionit
resideswithincytoplasmofWBCs
22
Preparationmethods:
InLinefiltration
Prestorage:Filterusedtoremove
leukocytesbeforestorageofRBCs,this
allowsupto42daysbefore expiry
Poststorage: filteration isdonewithin3
daysofstorage
Bedsidefiltration:Useaspecialfilter
duringtransfusionoftheunittothe
patient(rare).
23
24
331
Summary of Blood Components
25
BloodComponent
Storage
Indication
Temperature Time
1) PRBCs 26C
+SAGM
42days
Anemia
Newbornexchange
transfusion
2)PC R.T. 35 days
Bleeding
Operationifplt.Less
than2000/ul
3)FFP
18C
65C
1year
7 years
Clottingfactor
deficiencies
Severeburns
SummaryofBloodComponents
Blood
Component
Centrifugation
Storage
Indication
Temperature Time
4)Cryo
a)WBspecial heavyspin
3500rpmat4C
RBC+plasma11min
b)PlasmaStoreat18C
Thawat4C
Heavy spinat4C
30C 1year
Hemophilia
A Von
Willebrand
disease
26
332
MinistryofHealth
Quality Control of Blood
Component Preparation
Institute Graduates
QCofWholeBlood
Frequencyofcontrol:1%ofallunitswithminimum
of4unitspermonth
Storage: 2Cto6C,forCPDA1thestoragetimeis
35days,CPD&CD2D 22days.
Onexpiredate: measureHCT,pH,totalHb ,K
+
and
performsterilityassays
2
QCofWholeBlood
Volume:450ml10%ofbodyvolumeexcluding
anticoagulant
HCT:405%
pH>6.5
K<27mmol/L
Hb minimum45g/unit
Sterility:nogrowth
3
PerformthesameassayasforWholebloodonthe
expirydate
Storage:26C,for35daysifpreparedfromWB
collectedinCPDA1
QualityAssurance:
Volume:280ml50ml,frequencyofcontrol1%ofallunits
HCT:0.650.75
pH>6.5
K<78mmol/L
Hb:minimum45g/unit
Sterility:nogrowth
RedCellConcentrates
4
Preparedwithin6Hrsofbloodcollection
Mustevaluateatleast4plateletpreparationsmonthly
forplateletcount,pHandplasmavolume
Plateletsshouldbeselectedfromeachcentrifugeinuse
TheT atwhichpHismeasuredshouldbethesameas
stored
Labelthevolume,theactualvolumebymeasurement
mustbe10%ofthestatedvolume
Storage:2024C
Tshouldberecordedatleastevery4Hrsduringstorage.
PlateletConcentrates
5
Volume>40ml
pH:6.87.4
Plt count:atleast5.5x10
10
/baginatleast75%oftheunits
testedattheendofthestorage.Byapheresis:minimum3x
10
11
/bagplateletsinatleast75%unitstested
WBCcontamination:<2x10
3
/bag
RBCcontamination:<2x10
9
/bag
Macroscopicappearance:novisibleplateletsaggregates
Sterility:nogrowth
QCofPlateletConcentrates
6
333
QCOFFFP
Volume:220250ml
FactorVIIIc :>0.7IU/ml every2months
Noleakageafterpressureinplasmaextractor,before
freezingandafterthawing
oMacroscopic:noabnormalcolororvisibleclots
oResidualcell:
Redcell:<6.0x10
9
/l
Leukocyte:<0.1x10
9
/l
Platelets:<50x10
9
/l
7
Assayedonatleast4bags/month forfactorVIII
Storage:
12monthsatbelow18C
Mustbethawedat37Candusedwithin6Hrs
Cryoprecipitate
8
QCOfCryoprecipitate
Volume:1020ml
FactorVIII:>80IU/unit
Fibrinogen:>150mgperunit
Macroscopic:homogenous
Sterility:nogrowth
9
Transportation
Asystemmustbeusedtoensurethatallbloodandblood
componentsshippedbyorreceivedintoabloodbankor
bloodtransfusionservicehavebeenmaintainedwithinT
required.
AllliquidRBCcomponentskeptatTof110Cduring
transport
Allcomponentroutinelystoredat2024Cshouldbe
maintainTduringshipment
Allfrozencomponentsshouldbetransportinfrozenstate
at18Corcolder
PeriodicTcheckanddocumentedtoensurethe
transportationadequatetomeetthecriteria
10
**ATLEAST4UNITSTESTEDMONTHLY
Volume:280ml 50ml
HCT:0.650.75
pH>6.5
K<78mmol/L
Hb:minimum45g/unit
Sterility:nogrowth
UNITNO. HCT Volume PH K Hb Culture
RedBloodCellsQualityControlForm
11
UNITNO. WEIGHT/gm VOLUME/ml PH Platelets
count/ul
PLATELETS
COUNT/UNIT
** AT LEAST 4 EXPIRED (6
TH
DAY) UNITS TESTED MONTHLY
** ACCEPTABLE LIMITS:
* PLATELETS COUNT >.5.5 1010 platelets per unit
* PH > 6.2
PlateletsQualityControlForm
12
334
**ACCEPTABLELIMITS:
*FIBRINOGEN:>150mg/BAG
*FACTORVIII>80IU/BAG
UNIT NO. WEIGHT VOLUME
FIBRINOGEN
Mg/BAG
FACTORVIII
IU/BAG
CryoprecipitateQualityControlForm
13
PLATELETSCOUNT/UNITPLATELTSRICHPLASMA
PLATELETSYIELD PLATELETSRICHPLASMA= *100
PLATELETSCOUNT/UNITWHOLEBLOOD
PLATELETSCOUNT/UNITplateletsc.
PLATELETSYIELD PLATELETSCONCENTRATE= *100
PLATELETSCOUNT/UNITPRP
**ACCEPTABLEVALUE:
**PLATELETSYIELDinPLATELETSRICHPLASMA>75%
**PLATELETSYIELDinPLATELETSCONCENTRATE> 90%
UNIT
NO.
WHOLEBLOOD Platelets rich plasma PLATELETS CONCENTRATES
UNIT
NO.
PLT
COUNT
VOL/
ml
PLT/UNIT SPEED TIME PL/Ul Volume Plt/unit Yield
%
speed TIME PLT
/uL
VOLUME PLT
/ UNIT
CentrifugeCalibrationForm
14
335
MinistryofHealth
Leukoreduced and irradiated
blood components
Institute Graduates
Leukoreducedbloodcomponent
2
The name "white blood cell is derived from the fact
that after centrifugation of a blood sample, the white
cells are found in the buffy coat, a thin, typically white
layer of nucleated cells between the sedimented red
blood cells and the blood plasma. The scientific term
leukocyte directly reflects this description, derived
fromAncient Greek (white), and (cell).
Etymology
3

4
5
Leukocytecontentofwholebloodaveragestwo
billion(2x109)leukocytesper500mLofwhole
blood.
Duringbloodcomponentpreparation:
90%ofleukocytesfractionatewiththeredbloodcells
(RBCs).
8%isretainedwithinPlateletconcentrates.
2%arepresentintheplasmabeforefreezing.
6
336
Leukocytereductioncanbeachievedby
varioustechniques,including:
Centrifugation
Leukocytefiltration
Sedimentation
Washing
Freezethawing
Apheresis
7
Purpose Mechanism Pore Size Generation
No leukocyte filtration;
standard blood filter
Screen filter 170260 um First
Micro-aggregate filter;
leukocyte filtration, 90%
Screen filter 2049 um Second
Adsorption filter;
leukocyte filtration
99.9%
Adhesion filter Not applicable Third
LeukocyteReductionFilters
8
9
1. Nonhemolytic febriletransfusionreactions
2. Transmissionofleukocyteassociatedviruses
cytomegalovirus
3. Alloimmunization
4. Immunomodulatory effects
5. Cancerrecurrence
6. Postoperativeinfections
7. Transfusionstoragetimeforredbloodcells
8. Transfusionstoragetimeforplatelets
9. Transfusionrelatedacutelunginjury
10. Transfusionassociatedgraftversushostdisease
AdverseEffectsAssociatedwithDonor
Leukocytes
10
Definition: as a temperature increase of 1C after an
allogeneic blood transfusion.
Cause: alloantibodies in the recipients plasma
against antigens present on donor leukocytes and/or
platelets
Incidence:
0.5% in patients receiving a first transfusion
60% in Chronically transfused patients
1.FebrileNonhemolytic TransfusionReactions
11
2.Transmissionofleukocyteassociatedviruses
(e.g.cytomegalovirus)
TransfusionassociatedCMVinfectionisasignificant
causeofmorbidityandmortalityinimmuno
compromisedpatientsandespeciallyinorgan
transplantrecipients.
Aftereitherkidneyorlivertransplants,morethan
60%ofpatientsdevelopantibodiesagainstCMV.
12
337
3.PlateletRefractorinessand
Alloimmunization
Alloimmunizationcanreducetheclinical
effectivenessofplatelettransfusionsby50%.
Especiallyprevalentamongthose:
Patientsreceivingpooledrandomdonor
Plateletconcentrates
WhoarePregnant
13
4.Immunomodulationand
PostoperativeInfectiousComplications
ContaminatingleukocytesinRBCtransfusionsmight
beresponsiblefordownregulationof:
Naturalkiller(NK)cellactivity,
Tcellproliferation
Tlymphocyteantitumoractivity
CD4helpertoCD8suppressorratio
Lymphocyteblastogenesis
14
5.CancerRecurrence
Anassociationbetweenallogeneicbloodtransfusion
andcolorectalcancerrecurrenceaftersurgery.
Bloodtransfusionsincolorectalsurgerypatients
havebeenreportedtoincreasecancerrecurrenceby
37%alsohavebeenassociatedwithincreased
recurrenceofbreast,lung,kidney,prostate,
stomach,cervical,laryngeal,softtissue,andbone
malignancies.
15
6.Postoperativeinfections
Transfusionofbloodcomponentscontaining
bacteriamayleadtopotentiallyfatalsepsis.
Cause:inadequateskinpreparationbefore
venipuncture.
Commonpathogens:includeGramnegative
endotoxinproducingorganismssuchasYersinia
enterocolitica,pseudomonasandenterobacter
16
6.Postoperativeinfections
Optimalstoragetimebeforefiltrationtoallowfor
maximalleukocyteingestionofbacteriaappearedto
bebetween2and12hours.
Thebeneficialeffectofleukocytereductionmaylie
inremovalofleukocytescontainingingested
bacteria.
17
7.Transfusionstoragetimeforredbloodcells
DecreaseATP.
Glucoseconsumption.
IncreaselactateandK
+
production.
Thepresenceofleukocytesinbloodcomponents
reducesglucoseavailability.
Leukocytelysis leadstoreleaseofcytokinesthat
reduceRBCsurvival.
18
8.Transfusionstoragetimeforplatelets
DecreasesinpH
Increasesinglucoseconsumption
Lactateproduction
Lacticdehydrogenaserelease
Plateletsstoredwithleukocytesexpressdecreased
quantitiesofglycoproteinIb(GPIb)receptor,
resultinginableedingdisorders.
19
Storedbloodcontainsmicroaggregatesof
degeneratedleukocytes,plateletsandfibrin
Thesemicroaggregateshavebeenassociatedwith
pulmonaryinsufficiencyduetoagglutinationof
donorleukocytesbyrecipientantibodies
C/P:severedyspnea,noncardiogenicpulmonary
edema
9.Transfusionrelatedacutelunginjury
20
10.Transfusionassociatedgraftversushost
disease(GVHD)
GVHDisapotentiallylethalcondition.
Cause:donorTlymphocytes.
Mechanism:immunocompromisedrecipients,hostdefense
mechanismsfailtosuppressviabletransfuseddonorlymphocytes,
whichengraftwithintherecipientsmarrow,ultimatelyresultingin
death.
Occurrence:
WhenthedonorandrecipientshareanHLAhaplotype.
Useofdirecteddonorbloodfromfirstdegreerelatives.
Prophylaxis:gammairradiation.
21
AdverseEffectsofLeukocyteReduction
22
Fewsideeffectshavebeenreported
1. Hypotension:duetoreleaseofbradykinin like
vasoactivesubstanceesp.inpatientsreceivingACEI
prolongintravascularhalflifeofbradykinin :by
decreasingbradykinin degradation.
2. Complementactivationandformationofplatelets
aggregate
28%decreaseinpotencyofcellularcomponentsof
blood.
IrradiationofBloodComponent
23
Outline
Irradiation Source
Radiation Dose
Submission Contents
StandardOperatingProcedures(SOPs)
Records
Labeling
24
339
Irradiation Source
Cesium-137 sealed source irradiator
Cobalt-60 sealed source irradiator
Linear accelerator
X-ray irradiator
25
Radiation Dose
2500 cGy targeted to containers central portion
1500 cGy minimum dose at any other point of
the container
If product is irradiated more than once,
document total (additive) dose
An indicator should be used with each batch
that is irradiated
Followmanufacturersinstructionsforuse,including
temperaturecontrol
26
Submission Contents
Cover letter
Form FDA 356h
SOP for manufacturing irradiated blood
products
Typically, SOPs for equipment maintenance and
personnel training
27
Submission Contents (cont.)
Labels for each product with Form FDA 2567
Two months of irradiation records
Most recent dosimetry map
Contractor information, if applicable
Contractorwhoperformsirradiationmustregisterwith
FDA
28
SOPs
Description of the irradiator (e.g., radiation
source)
Description of the dose delivered to the center
of the container
Length of time required to deliver irradiation
Maximum irradiation dose limits
Description of procedures for re-irradiation, if
applicable
29
SOPs (cont.)
Indication of the maximum number of units to be
irradiated at one time
Description of procedures for monitoring to
determine actual dose delivered
Validation
Initially
AnnuallyforCe137
SemiannuallyforCo60
Aftermechanicalrepairs
UseThermoluminescent Dosimeter(TLD)chipsorother
directmethodsofmeasurement
30
340
SOPs (cont.)
Dating period for Red Blood Cell products
Notmorethan28daysfromthedateofirradiationbut
nomorethanthedatingperiodoftheoriginalproduct
Dating period for platelets remains unchanged
31
SOPs (cont.)
Maintenance of irradiator
Procedure for personnel training
Staff safety
If contract facility used for irradiation, your SOPs
should:
Describewhatstepsareperformedbyyouandbythe
contractor
Ensuremanufacturingstepsareperformedaccordingto
yourspecificationsandareincompliancewithall
applicableregulations
32
SOPs (cont.)
Quality control (QC) (review considerations)
Irradiator
DailyQC(e.g.,checkofturntablerotation)
Monthlycomparisonofirradiatortimerandbackup
timer,ifavailable,withcertifiedstopwatch
Irradiation indicators
Shippingandstoragetemperaturechecks
Expectedresultsofeachnewlot
Investigationoffailuresandcorrectiveactions
33
Records
Strength of source
Irradiation Records
QC
Equipment maintenance
34
Irradiation Records/QC
Operator ID
Site of irradiation
Date and time of irradiation
Duration of irradiation
35
Irradiation Records/QC (cont.)
Level/dose of irradiation
Documentation of QC for irradiator and
irradiation indicators
36
341
Labeling
Container Label (21 CFR 606.121)
Containerlabelmustincludepropernameofproduct,
andmodifier,ifapplicable(e.g.,RBCs,Irradiated)
ChangeexpirationdateforRBCsifappropriate
Circular of Information should include:
Indicationsforuseintreatingpatientsatriskof
transfusionassociatedGVHD
SideeffectsandhazardsofirradiatingRBCs
Highersupernatantpotassiumlevelsthannon
irradiatedRBCsduetocellmembranedamage
37
Labeling (cont.)
Circular of Information (cont.)
Removalofresidualsupernatantplasmapriorto
transfusionmayreducerisksassociatedwithelevated
plasmapotassium
License Number should not appear on the
container unless the product has been licensed
by the FDA
38
342
MinistryofHealth
Basic Immunology: Direct &
Indirect Antiglobulin Test
Institute Graduates
TheAntiglobulinTest
Antiglobulin serum(Coombs Serum)wasdiscoveredby
Coombsin1945.
Theantiglobulin testcanbeusedtodetectredcells
sensitizedwithIgG alloantibodies,IgG autoantibodiesor
complementcomponents.
Sensitizationofredcellscanoccurinvivoorvitro.
TheuseofAHGserumtodetectsensitizationofredcells
invitrocanbe:
Onestagetechnique,thedirectantiglobulin test(DAT).
Twostagetechnique,theindirectantiglobulin test(IAT).
2
Principle
Normalhumanredbloodcells,inpresenceof
antibodydirectedtowardstheantigentheypossess,
mayfailtoagglutinatewhencentrifugedand
becomesensitized.Thismaybeduetotheparticular
natureoftheantigenandantibodyinvolved.
SensitizationofRBCsmaybewithIgG or
complement.
Inorderforagglutinationtooccuranadditionalof
antiantibodyoranticomplements,whichreacts
withtheFcportionoftheIgG antibody,orwiththe
C3borC3dcomponentofcomplementalternatively.
3
Principle
Thiswillforma"bridge"betweentheantibodiesor
complementcoatingtheredcells,causingagglutination.
Thecoating(sensitization)ofredcellscanoccurinvivo
orinvitro followingincubationat37Cwithserum
containingantibody.
4
ProductionMethodsofAntiHumanglobulin
(AHGorCoombs)Reagent
Maybemadebyinjectingrabbits,goatsorsheep
withpurifiedhumanIgGorC3,thenharvestingthe
antibodiesproducedbytherabbit.
Monoclonaltechnologymaybeusedtomake
monoclonalantiglobulinreagent.
5
TypesofAHGreagent
PolyspecificAntihumanGlobulin:blendofAntiIgGand
AntiC3b,C3d
Monospecificreagents:AntiIgGaloneorAntiC3b,C3d
alone
Note: Reagentdoesnotcontainantibodiesto
IgM. InformationaboutIgMcoatingofcellscomesfrom
thepresenceofC3coatingthecellssinceIgMisastrong
complementactivator.
6
343
DirectAntiglobulinTest(DAT)
7
DAT
Thedirectantiglobulin test(DAT)detectssensitized
redcellswithIgG and/orcomplementcomponents
C3bandC3dinvivo.
InvivocoatingofredcellswithIgG and/or
complementmayoccurinanyimmunemechanism
isattackingthepatient'sownRBC's.
Thesemechanismcouldbe:
Autoimmunity
Alloimmunity
Oradruginducedimmunemediatedmechanism
Examplesofalloimmunehemolysis
Hemolytictransfusionreaction
Hemolyticdiseaseofthenewborn(alsoknownas
HDNorerythroblastosis fetalis)
RhesusDhemolyticdiseaseofthenewborn(also
knownasRhdisease)
ABOhemolyticdiseaseofthenewborn(theindirect
Coombstestmayonlybeweaklypositive)
AntiKell hemolyticdiseaseofthenewborn
Rhesusc,Ehemolyticdiseaseofthenewborn
Otherbloodgroupincompatibility(RhC,Rhe,Kidd,
Duffy,MN,Pandothers)
9
Examplesofautoimmunehemolysis
Warmantibodyautoimmunehemolyticanemia
Idiopathic
Systemiclupuserythematosus
Evans'syndrome(antiplateletantibodiesandhemolytic
antibodies)
Coldantibodyautoimmunehemolyticanemia
Idiopathiccoldhemagglutinin syndrome
Infectiousmononucleosis
Paroxysmalcoldhemoglobinuria (rare)
10
Druginduced immunemediatedhemolysis
Methyldopa(IgGmediatedtypeIIhypersensitivity)
Penicillin(highdose)
Quinidine(IgMmediatedactivationofclassical
complementpathwayandMembraneattack
complex)
11
BloodSample
WholeBloodSample Itshouldbeasfreshas
possiblenotmorethan24hoursold
Otherwise,thesampleshouldbetakeninEDTA.
12
344
ProcedureofDAT
1. Take23dropsofbloodtobetestedinacleanlabeledtube.
2. Washtheredcells34timesinalargevolumeofsalinetoremove
freeglobulinmolecules.Removeallsupernatantaftereachwash.
Completelydecantthefinalsupernatantwash.
3. Add2dropsofpolyspecific AHGserumin1dropofsensitized
washedredcellsorin1dropof35%suspensionofsensitized
cellsimmediately.
4. Mix,Centrifugeat1000rpmfor1minutesimmediately.
5. Gentlyshakethetubetodislodgethecellbuttonandseefor
agglutination,useopticalaidifneeded,Recordtheresult.
6. Add1dropofIgG coatedredcellstoanegativetest.Mix,
centrifugeat1000rpmfor1min.Immediatelylookfor
agglutination.Ifanegativeresult(noagglutination)isobtained
thetestresultisinvalidandwholetestshouldberepeated.If
agglutinationisobtained,theresultisvalid.
13
IndirectAntihumanGlobulinTest(IAT)
Indications
TheIATisdonetodeterminethepresenceof
sensitizationofredcellswithIgG and/or
complementinvitrointhefollowingconditions.
1. Compatibilitytesting.
2. Screeninganddetectionofunexpectedantibodiesin
serum.
3. DeterminationofredcellsphenotypeK,Lea,Fya Fyb,
Jka,Jkb andsubgroupofRhetc byusingknownsera.
14
Indirectantiglobulintest
15
Procedure:
1. Place23dropsofthetestseruminatube.Serumshouldbe
freshfordetectingcomplementcomponentsandcomplement
bindingantibodies,otherwise,freshABserumshouldbeadded
toit.
2. Add1dropof35%suspensionofwashedORh(D)positivered
cellstotheseruminthetube.
3. Mixandincubateat37Cfor3040minutes.
4. Centrifugeat1000rpmfor1minutes.
5. Examineforhemolysisand/oragglutination.Useopticalaidif
necessary.Agglutinationatthisstageindicatesthepresenceof
saline(complete)antibodies.
6. Ifnoagglutinationisseen,washcells34timesinlargevolumeof
saline.Decantsupernatantineachwashascompletelyas
possible.
16
7. Add2dropsofAHGserumtothecells.
8. Mixandcentrifugeat1000rpmfor1minutesimmediately.
9. Gentlyshakethetubetodislodgethebuttonandexamine
foragglutination,usingopticalaid.Recordtheresult.
10. Add1dropofIgG coatedredcellstoanytestthatis
negative.Mixandcentrifugeat1000rpmfor1minutes.
Lookforagglutination.Ifthereisnoagglutination,thetest
resultisinvalidandthewholetestisrepeated.If
agglutinationisobtainedtheresultisvalid.
11. AutocontrolshouldbekeptwithIAT.
Procedure:
17 18
345
BOVINEALBUMIN(22%)IAT
One Stage Method - Additive method
Procedure:
1. Twodropsofalbumin22.5%areaddedinstep(2)of
salineIAT
2. Mixandincubatefor2030minutesat37C
3. ProceedfurtherasinsalineIATprocedure.
19
SourcesofErrorinAHGtests
Falsenegativeresults:GeneralDAT&IAT
Failuretowashredbloodcellsadequately,sinceglobulinsnot
boundtoRBCswillneutralizetheAHGreagent.
ThewashingprocessandtheadditionofAHGreagentmustbe
undertakenasquicklyaspossibletominimizelossofbound
antibodiesbyelution.
Improperstorage,bacterialcontaminationand
contaminationwithhumanserumwillimpairtheAHG
reagentactivity.
NotaddingtheAHGreagent
Impropercentrifugation
Numberofcellspresentinthetest:
Toomanycellsgiveweakreactions
Toofewcellswillimpairthereadingoftheagglutination
20
AntigenAntibodyRatio
Theoptimumratiois80partsantibodyto1part
antigen. Therearespecifictermsforvariationsinthis
ratio.
Prozone antibodyexcess:Antibodiessaturatingall
antigensites;noantibodiesformingcrosslinkages
betweencells;noagglutination
Zoneofequivalence:antibodiesandantigenspresent
inoptimumratio,agglutinationformed
Zoneofantigenexcess(Postzone):toomanyantigens
anyagglutinationishiddenbymassesof
unagglutinated antigens
21 22
Falsenegativeresults
DAT
AllsamplesnegativeattheAHGphaseshouldbeincubatedat
roomtemperaturefor5minutestoachievemaximalsensitivity
neededforcomplementdetection.
IAT
Serumand/orRBCslosereactivityifimproperlystored.
Plasmausedinsteadofserumcanleadtofailuretodetect
antibodiesdependingonpresenceofactivecomplement(antiJka,
Jkb)
Temperatureandincubationtimeaffectattachmentofantibodyor
complementtocells.
Anoptimalproportionofserumtocellsshouldbeachieved:
usually23dropsserumtoonedropof5%cellsuspension.
23
Falsepositiveresults:
DATandIAT;
Inspecimenscontainingpotentcoldreactiveantibodiesagglutination
mayoccurbeforeaddingtheAHGreagent.
Dirtyglasswaremaycauseclumpingofcells.
Overcentrifugation
DAT
ApositiveDATfromaclottedsampleshouldberepeatedonanEDTA
sample
Samplescollectedfrominfusionlinesmayhavecomplementpresenton
thecells.
IAT
CellswithapositiveDATwillgiveapositiveresultinanyindirect
antiglobulin procedure.
24
346
CoombsCells
Toshowthattestcellswereproperlywashedandthatno
neutralizationorreagentdeteriorationhasoccurred,
antibodycoatedcellsareusedasapositiveindicator.
Inanegativeantiglobulin testtheantihumanglobulinshould
remainactiveandthiscanbedemonstratedbytheaddition
ofIgG sensitizedcells.
AgglutinationoftheIgG sensitizedcellsaftermixingand
centrifugingconfirmsthattheantihumanglobulinwas
addedtothetest,thatthetestcellswereproperlywashed
andallfreeglobulinmoleculeswereremovedandthatthe
antihumanglobulinwasactive.
FailureoftheIgG sensitizedcellstoagglutinateindicatesthat
theoriginalnegativeantiglobulin testresultisnotvalidand
testingmustberepeated.
25
PreparationofCoombscells
PreparingCoombscontrolcellsisveryeasy.Toabout10
dropsofwashedOPositiveredcellsadd56dropsof
antiDantisera.Incubateat37Cfor15minutes.Wash4
timesthenpreparea3to5%cellsuspension.
Toverifyreaction,addtwodropsofAHGintotesttube
andonedropofnewlypreparedCoombscells.
CentrifugeonHighspeedfor15seconds,Youshouldget
12+reaction.
26
347
MinistryofHealth
Blood Grouping Discrepancies:
ABO Discrepancies
Institute Graduates
Describethereactions,listtheclinicalsituationsinwhichtheymayoccur,
andexplainhowtoresolve eachofthefollowingcausesofABO
discrepancies:
*Decreasedimmunoglobulinlevels
*WeaksubgroupsofAwithantiA
1
*PassivelytransfusedantiA
1
*Unexpectedalloantibodyreactingatroomtemperature
*LossofAorBantigen
*AcquiredBantigen
*Rouleaux
*Coldagglutinins
Explain whatmustbedoneifanABOdiscrepancycannotberesolvedbefore
thepatient requiresatransfusion.
Explain whatmustbedonewhenadiscrepancyarisesinablooddonor.
ListcausesoftechnicalorclericalerrorsthatmaycauseABOdiscrepancies.
Listcausesformixedfieldagglutination.
Objectives AboDiscrepancies
2
Any deviation from the expected pattern of antigen on
the cell and the opposite antibody in the serum .
Definition
3
ABODiscrepanciesmustberesolved
In recipientsthediscrepanciesmustberesolved
beforeanybloodcomponentistransfused.Ifnot
resolvedbeforebloodisneeded,transfuseGroupO
(ONEGATIVEifthereisadiscrepancyintheRhtype
also).
In donorsthediscrepanciesmustberesolvedbefore
anybloodislabeledwithabloodtype.
4
Alwaysretestfirst.
Checkforclerical/technicalerrors
Weakestreactionisusuallytheoneindoubt.
Checkresultsofthescreeningcells.
Checkthepatientsage.
Checkthediagnosis
Checkthetransfusionhistory.
GeneralRulestoResolve
5
Clericalerrors(transcriptionerrors)
Technicalerrors
Problemswithserumtesting
Problemswithredcelltesting
Problemswithbothcellsandserum
KindsofDiscrepancies
6
348
Clericalerrorsarethemostcommon.
Recordtheresultsasyoureadeachtube
Ontherightworksheet.
Onepatientordonoratatime.
Recordontherightspotofworksheet(thisiswhy
labelingprocedureusescapitalAandBfortheforward
typeanda
1
CandbC forthereversetyping)
ClericalErrors(TranscriptionErrors)
7
Thereareanumberoftechnicalerrorsthatmayalsooccur:
1. Sample: mixup
2. Reagents:
o Failuretoaddserumorreagent.
RememberforbothABOandRhalwaysaddyourreagent
antiseraandserumbeforeaddingcells.
o Additionofwrongreagent
o Contaminatedreagentscouldresultineitherfalse
negativeorfalsepositiveresults
TechnicalErrors
8
3. Cellsuspension:Toomanycellsinyourcellsuspensioncan
leadtodecreasedornegativereactionssincetherearetoo
manycellsforthenumberofantibodiespresentinthe
reagents.
Rememberwewanttobeinthezoneofequivalenceforour
reactions.
4. Centrifugation: UnderorOver
5. Incubation:Warmingthetestcouldresultinafalse
negativereactionsinceABOantibodiesareIgMs thatreact
betterinthecold.
9
6. Interpretation:
o Failuretodetectweakresults canoccurifyouarenotwatchingthe
reactionswhileyouareshakingthemoutorifyoushaketoohard.
o Failuretodetecthemolysiscanbeadefiniteproblem.
o Rememberapositivereactioncanbehemolysisaswellas
agglutinationsincetheantigenantibodyreactioncanbind
complement. Whencomplementisbounditcanleadtohemolysis
thatisalsoanindicationofapositivereaction.
7. Dirtyglasswarecancausethecellstoartificiallyclump.
10
ProblemsWithSerumTesting
11 12
349
13
1.WeakorMissingAntibody(ies)
14
Causes:
1. Extremeage
2. Immunocompromized
1WeakorMissingAntibody(ies)
15
Anextremeexamplewouldbenoreactionfortheforwardand
reversetypings.
Thestepstofollowtoresolvethisdiscrepancyisto:
Checkbirthdatesincenewbornsandtheelderlyaremorelikelyto
demonstratethisdiscrepancy. Newbornantibodiesarenot
presentuntilatleast6months. DON'TATTEMPTTOSERUMCONFIRM
NEWBORNS. Asindividualsagesthey mayalsolosetheirabilityto
maintaintheirantibodylevels. Therefore,theveryelderlyhave
decreasedantibodylevels.
Checkdiagnosis sincepatientconditionssuchas: Immune
deficiencies, Chemotherapy, RadiationTherapy, and Bone
marrowtransplantationmayexplainthemissingantibodies.
1WeakorMissingAntibody(ies)
16
Addtwomoredropsofserumjustincaseyou
forgottoaddthemthefirsttimeandcentrifuge. If
negativethenincubateincold(418
o
C)1530
MINUTES
Includeautocontrol toruleoutinterferencefrom
naturalantiIwhenincubatingat(418
o
C).
(At4
o
CAntiAandAntiBenhancedsincetheyare
saline,coldactingantibodiesasseeninthisexample
foranOindividual.)
ResolutionofMissingAntibodies
17 18
Comparethiswitha4
o
CAutoAntiIenhancedwouldhavea
positiveautocontrol asseenintheexamplebelow:
19
GroupAorGroupBcanserveasitsownnegativecontrol.
4
o
CAntiBenhancedisshownbelow:
20
4
o
CAntiIenhancedontheotherhandwouldhaveapositive
autocontrol.
21
IfantiIenhancedalongwithantiAorantiB,canresetupand
incubateat18
o
C. Asseeninthisexampleof18
o
C:AntiBenhanced,
antiInonreactive
22
1SubgroupA
2PassivetransfusedAb
3Alloantibodies(coldreacting)
4Excessserumprotein(Rouleaux)
2.PresenceOfExcessAntibodies
23
A.Presence ofUnexpectedAntiAwhenthe
ImmediateSpinAntibodyScreeningisNegative
ThepresenceofAntiA
1
shouldbesuspectedwhentheantibodyisreactive
againsttheAcellsbutnotthescreeningcellsatimmediatespinasseenin
theexamplebelow
24
351
NaturallyantiA
1
occursinsubgroupsofAorarepassively
transfused fromGroupOplateletsandotherbloodproducts.
HowtoResolvetheIssueofUnexpectedAntiA:
1. CheckrecenttransfusionhistoryforgroupOproducts,
(especiallyplatelets)thatwouldexplainthepresenceofthis
antibody.
2. Testpatientcells withlectinA
1
. Subgroupswillbenegative
withthisreagentbutA
1
cellswillbepositive.
Lectin +A
1
CELL=4+
Lectin +AsubgroupsCELLS=0
25
3. Testpatientserum withthreeA
1
cellsandthreeA
2
cellsandifitis
anantiA
1
thefollowingreactionswilloccur:
AntiA
1
:SERUM+A
1
CELLS=+
SERUM+A
2
CELLS=0
AntiA
1
willreactonlywiththeA
1
cellsbutnotwiththeA
2
cells
4. InthecaseofpassiveAntiAfromGroupOPlateletsthe
reactionswouldbethefollowing:
SERUM+A
1
CELLS=+
SERUM+A
2
CELLS=+
Inthiscaseiftheantibodyisstrongenoughyoumayneedto
transfusegroupOblood.
26
YoumayhaveapositivereactionwiththereagentA1 orBcellthatisduetoaroom
temperatureantibodyreactingwithanantigenotherthanAorBonthecells
B.UnexpectedAOrBAntibodywhentheImmediateSpin
AntibodyScreeningisPositive
27
HowtoResolvetheIssueofUnexpectedAntiAthatisprobably
anotherantibodyduetotheresultsoftheAntibodyScreening:
(Alloimmunization)
Identifytheantibodybyperforminganidentificationpanel
atroomtemperature.
Prewarm away(usecaution)theeffectofthisantibodyby
doingthereversetypingwithprewarmed serumandreagent
cells.
TypereagentA
1
orBcellforthecorrespondingantigenonce
theantibodyisidentified.
Forexample,ifthepatienthadanantiN thatwasshowing
upatroomtemperatureaccordingtotheantibody
identificationprocess,youwouldthentypeforNonthe
reagentcellsusedforthereversetyping. IfantiNiscausing
yourproblem,thenthecellsshouldhaveNantigenpresent.
28
Rouleaux cangiveunexpectedagglutinationinallserumtests
Rouleaux FormationGivingUnexpected
AgglutinationinallSerumTests
29
Rouleaux mayalsogivefalsepositivecelltypingif
strongenoughandcellsareinsufficientlywashed. This
phenomenonisduetoalterationinserumprotein
concentrationsuchas:
Multiplemyeloma
Macroglobulinemia
Liverdisease(decreasedalbumin)
Alsoseenwithvolumeexpanders
30
352
Characteristicsofrouleaux isthatit:
Lookslikeagglutinationmacroscopically
Microscopicallyitappearsas"stacksofcoins"
Howwouldyouresolverouleaux problems?
Dosalinereplacementtechnique:
Recentrifugethetesttube.
Drawoffserumwithoutdisturbingcellbutton
Addtwodropsofsaline
Resuspend
Rouleax dispersesinsaline; TRUEAGGLUTINATION
REMAINS
31
ProblemswithCellTyping
32
Mixedfieldagglutinationisseenaslargeorsmallagglutinates
withmanyunagglutinatedcells.Usuallymixedfield
agglutinationmeansaMIXEDCELLPOPULATION Thecausesof
mixedfieldagglutinationcanbe:
1.MixedFieldAgglutination
33
MixedFieldAgglutination
1. Massivetransfusion ofanotherbloodgroup("O"redblood
cell)
2. Bonemarrowtransplantpatients
3. WeaksubgroupsofA
3
.
4. Chimerism duetointrauterineexchangeoferythrocyte
precursorsbetweentwinsor2fertilizedeggsfuseintoone
individual.
Checkthepatient'stransfusionrecordsandclinicalhistory. Ifit
appearstobeaweaksubgroupperformedthetestsdiscussed
under UnexpectedAntiA.
34
Maybedueto:
1)VeryweaksubgroupofAorB,
2)Lossoftransferase inacuteleukemia,
3)MassivetransfusionofGROUPO,or
4)Bonemarrowtransplant
2.WeakorMissingAntigen
35
Obtainrecenttransfusionhistory andanyclinicalhistory
ofbonemarrowtransplant
Readforwardgroupingmicroscopically
UseantiA,Bandincubateat422
o
Catleast15minutes
Usemonoclonalantisera thatisknowntoreactwith
antigenslikeA
x
andB
x
Performspecializedtestsiftheabovestepsdonot
resolvetheproblem:
Specializedtestswouldincludeabsorption/elution
techniquesandsalivastudies.
Howwouldyouresolveaweak,ormissing,
antigen?
36
353
AcquiredBantigensareseeninproblemswiththecolonorinfectionswith
Gramnegativerods
Bacterialenzymesmodifythe"A"antigentoa"B"antigenandthepatient
forwardtypesasanABbutreversesasanA.
3AcquiredBAntigen
37
AcquiredB
Bacteria(E.coli)haveadeacetylatingenzymethat
effectstheAsugar.
GroupA
individual
Nacetylgalactosamine
AcquiredB
Phenotype
Galactosaminenow
resemblesD
galactose(foundin
GroupB)
38
Setupanautocontrol. Thepatient'sownantiBwillnot
agglutinatetheirownABcells.
CheckclinicalhistorytoevidenceofcolonproblemsorGram
negativerods.
CheckmonoclonalantiBproductinserts sincesomewillnot
reactwithBacquiredantisera
Acidify somereagentsantiB topH6andretest. Modified
(acquired)Bantigenswillnotreactintheacidifiedantiserum,
normalBantigenswillstillreact.
HowwouldyouresolveapossibleacquiredB
antigen?
39
MostmonoclonalantiAandantiBwillshowproblems
withpolyagglutinable cellsifitisaproblemwiththe
cellmembranethatleadstotheagglutination.
Themostlikelycausesof dueto:
1Wharton'sJelly,foundincordblood,andstrong
positivedirectantiglobulin testduetoacold
agglutinin.
2StrongpositiveDAT,itwouldappeartobeanAB
intheforwardtypeandanOonreverse.
4Polyagglutinablecells
40
41
Wharton'sjelly
Coatsnewborncordcellsandthechild'stypemayappearAB. Youdonot
doareverseonnewbornbloodsincetheyhavenotmadeanyantiAor
antiByet.
IfthebabytypesasanAB recheckbywashingcellsseveraltimesandre
testingsinceyouneedtomakesureyouhaveremovedtheWharton's
JellyandthebabyistrulyanAB. BetteryetAlwayswashcordbloodat
least4TO5X'sbeforedeterminingthetypeofthebaby.
StrongpositiveDAT
Maybeseenincoldautoimmunehemolyticanemia
Ifduetocoldagglutinin,washseveraltimesinwarmsalineandretest
Cellswashed3Xat37
o
Cwouldprobablylooklikethis:
42
354
43
ProblemswithBothCellsandSerum
44
45
AstrongcoldautoagglutininismostoftenduetostrongautoantiI.
1.Toresolvecelltypingdifficulties:
Washcells34Xwithwarm(37
o
C)saline
Retestwarmwashedcells
2.Toresolveserumtypingdifficulties:
Performserumtestingat37
o
C (Usecautionthatweakisoagglutinins
(antiAandantiB)arenotmissedusingthistechnique)
Autoabsorb coldagglutininsontopatientcellsat4
o
C.
StrongColdAutoAgglutinins
46
47
355
MinistryofHealth
Guidelines Of Pretransfusion
Compatability Procedures and Neonatal
Transfusion Policy
Institute Graduates
TheBloodTransfusionServiceperformstestsfor
serologiccompatibilitybetweenpatientanddonor
bloodpriortotransfusion,exceptincaseofurgent
bloodneed.
Tominimizetheriskofhemolytictransfusion
reactionandmaximizeposttransfusionredcell
survival.
StatementofPurpose
2
TheMajorityofABO incompatibletransfusionare
duetodocumentation/Identificationerrors.
Transfusionrequestsmustbeprescribedbya
medicalofficer.
Therequestformandsamplecontainthefollowing
minimumidentification:
(a)Surname
(b)Firstname(s)
(c)Dateofbirth
(d)Hospitalnumber/accidentandemergencynumber.
Transfusionrequest/Samples
3
Informationconcerningthesexofthepatientandobstetric
andrecenttransfusionhistoryshouldbeobtained.
Requestsshouldincludethedateandtimerequired,the
numberorvolumeandtypeofcomponentsrequired,the
reasonforrequestandanyotherspecificrequirements
relatingtothepatientorrequesti.e.irradiated,filtered
blood.
Samplesreceivedfromtraumaorfromunconsciousaccident
oremergencypatientmustcontainatleastoneidentifierlike
traumaoremergencynumberandsexofthepatientand
mustbesignedbymedicalofficer.
Ifoneidentifiernotavailableandinlifethreateningsituation
sogivehim(O)Negativeblood.
TransfusionRequest/Samples
SampleRequirements
ClottedorEDTAsamplemaybeusedforpretransfusion
testing.
Wholebloodsampledeteriorateduetoredcelllysis,
lossofcomplement,decreasepotencyofredcell
antibodiesandbacterialcontamination.
Insituationsinwhichpatientsarebeingrepeatedly
transfuseditisnotnecessarytorequiredailyantibody
screeningasitisvalidfor72h.andmakeonly
immediatespincrossmatching
Patientswithnohistoryoftransfusionorpregnancyin
thelast3monthsantibodyscreeningcanbevalidfor
oneweek
5
18 25C 4C 30C
EDTAwholeblood Upto48h. Upto7days N/A
Serum N/A Upto7days Upto6months
StorageofSamples
6
356
Receptionistmustsuretherequestdataandsamplefullfilledthe
requirementsmentioned.
Therequestformandsampletubeshallcarryidenticalpatient
identificationinformation.,incaseofdiscrepancyordoubt,the
officerinchargeofthebloodbankshallbenoted.Unlabelled
samplesshallbediscarded.
Searchforthehistoryofthepatientincomputersystemifavailable
orintype&screenfilewhichcontainstheresultsofbloodgroup
andantibodyscreeningofallpatientsreceivingbloodinthelast
72h
Ifthereisanydatafortherecipient(patient)intherecordsof
bloodbankmustwritebloodgroupandresultofantibody
screeningandlasttimereceivedblood.
Receptionistmustwritethetimeofreceivingtherequestandsign.
ReceivingBloodRequest
7
PretransfusionCompatibility
TestsforPatients>4MonthsOld
8
1. CellandserumtypingusingIDGelcard(seeSop#CBB035)
andifnotavailable,usetubemethod
2. Controlsmustbeincludedineachbeginningofshift,using
newreagentsornewlotnumberofIDGelcard.
3. TheABO&Rh groupingmustbeverifiedagainstprevious
resultsfromtype&screen fileorfromcomputerrecordsif
available
4. Anydiscrepanciesmustberesolvedpriortransfusionofred
cellsandinthe
5. Incaseofemergentrequestchoose(O)RHnegativeunits.
6. InRhnegativepatientnotmakeweakDtestandincaseof
partialDconsider
1.ABO&Rhgrouping
9
Antibodyscreeningmorereliableandsensitivethan
crossmatchingagainstdonorredcellssoshould
performedinallpretransfusiontesting.
Antibodyscreeningisvalidfor72hinnegativeresult.
soifnegativecangivepatientsbloodwithout
repeatingantibodyscreeningonlyrepeatABO&Rh
groupingthenimmediatespincrossmatching
2.Antibodyscreening
10
Whenirregularantibodyisdetectedintheantibody
screeningidentificationmustbedonetodetermine
itsspecificityandclinicalsignificant.
Iftheantibodyscreeningispositivemustrepeat
identificationforeachrequestofbloodtransfusion
3.AntibodyIdentification
11
IncaseoftransfusionofwholebloodmustbeABO
RhD identical.
RedcellcomponentsofthesameABOandRhD
groupasthepatientmustbeselectedwhenever
possible.
4.SelectionofBlood
12
357
Donewhenantibodyscreeningisnegativefromsample
takenwithin72h.whichisthetimelimitforthevalidity
ofthetest.
A shortincubationtimeof25min.beforecentrifugation
isrecommended.
Doneonlywhenthebloodinneedsodonotbegin
selectionandimmediatespincrossmatch untilbloodin
needandmadewithin10minutes.
Itisrecommendedallproceduresofcompatibilitydone
byonepersonbutincaseofstandbyrequestevery
techniciansignforworkdonebyhim.
5.ImmediateSpinCrossmatch
13
Donewhenantibodyscreeningresultispositiveand
mustberepeatedforeveryrequestaftermaking
identificationbutifrequestisurgentcanbemade
withoutidentificationusingtrialsunits.
Theremustbeacompatibilitylabelwhichshouldbe
securelyattachedtothebloodbagandinclude
patientname,hospitalnumber,bloodgroupandthe
datebloodrequired/crossmatched.
6.IATcrossmatch
14
Beforetheunitisplacedinbloodissuerefrigeratorit
shouldbeinspectedfor:
(a)Integrityofthepackbycheckingforleaks
(b)Evidenceofhemolysisinplasma(c)evidenceof
discoloration
(d)Presenceoflargeclots
Ifthereisanyevidenceoftheabovetheunitshould
benotused
Visualinspectionofredcellunit
15
PretransfusionCompatibilityTests
forNeonates(0 4MonthOld)
16
1. CelltypingandDCTusingID gelcardforneonate
andifnotavailableusetubemethod
2. Verifytheresultagainstpreviousresultfromtype&
screenfileorfromcomputerrecord
1.ABO&RhGrouping&DCT
17
MakeAntibodyscreeningusingmotherserumwhich
isthefirstchoiceandifnotavailableuseneonatal
eluate orneonateserum.
Antibodyscreeningisvaliduntilnewbornis4
monthsold
2.Antibodyscreening
18
358
IftheDCTisnegativeandbloodgroupofthemotheris
knownsogivehimPRBCSaccordingtothefollowing:
IfDCTisnegativeandbloodgroupofmotherisunknownso
givehim(O)andRhofneonate.
IfDCTisnegativeandbloodgroupofmotherunknownand
PRBCSbloodgoup (O)notavailablesomustmakereverse
groupinguntilAHGphaseforneonateserumtodetectAnti
Aor/andAntiBwhichmaybetransferredtohimfrom
motherandchoosebloodgroupcompatiblewiththat.
IfDCTispositiveyoumustselect(O)Rhnegativeunits
3.SelectionofBlood
19
Antibodyscreeningresultisvaliduntilnewbornis4
monthsold.
Ifantibodyscreeningisnegativenoneedfordoing
crossmatching andgiveABO&Rh compatibleblood
only.
IfantibodyscreeningispositiveAntibody
Identificationmustbedoneandselectbloodunit
negativeforantigenwhichcanreactwithantibody
specifiedbyantibodyidentificationfollowedbyAHG
crossmatching
4.Crossmatching
20
Bloodshouldbenotmorethan5daysoldfor
exchangetransfusion.
PackedredcellsshouldbereconstitutedwithAB
plasmaatthetimeofissueforexchangetransfusion.
Ifthemothersantibodyisreactiveagainstahigh
frequencyantigenandnocompatiblebloodis
availableMotherssiblingscanbetestedfor
compatibleblood.Aunitofbloodcanbecollected
frommotheriftheobstetricianagreesthatissafe.
MothersredcellsshouldbeconstitutedinAB
plasma
5.SpecialNotes
21
AABBTechnicalManual,14
th
Edition;2008.
AABBStandard23rdEdition;2005
References
22
359
MinistryofHealth
Antibody Identification
Institute Graduates
TheBasics..
Asyourecall,
AntibodyScreensuse2or3ScreeningCellstodetect
ifantibodiesarepresentintheserum
Ifantibodiesaredetected,theymustbeidentified
present
Notpresent
2
WhydoweNeedtoIdentify?
Antibodyidentificationisneededfortransfusion
purposesandisanimportantcomponentof
compatibilitytesting
Itwillidentifyanyunexpectedantibodiesinthe
patientsserum
Ifapersonwithanantibodyisexposedtodonor
cellswiththecorrespondingantigen,seriousside
effectscanoccur
3
KeyConcepts
Inbloodbanking,wetestknownswithunknowns
Whendetectingand/oridentifyingantibodies,wetest
patientserum(unknown)withreagentRBCs(known)
Unknown Known
Patientserum +ReagentRBCs
PatientRBCs +Reagentantisera
4
ReagentRBCs
ScreeningCellsandPanelCellsarethesamewith
minordifferences:
Screeningcells
Antibodydetection
Setsof2or3vials
Panelcells
Antibodyidentification
Atleast10vialsperset
5
AntibodyPanelvs.Screen
Anantibodypanelisjustanextendedversionofan
antibodyscreen
Thescreenonlyuses23cells:
6
360
AntibodyPanel
Anantibodypanelusuallyincludesatleast10panel
cells:
7
Panel
GroupOredbloodcells
8
Panel
Eachofthepanelcellshasbeenantigentyped
(shownonantigram)
+referstothepresenceoftheantigen
0referstotheabsenceoftheantigen
Example: PanelCell#10has9antigens present:c,e,f,M,s,Le
b
,k,Fy
a
,andJk
a
9
Panel
AnautocontrolshouldalsoberunwithALLpanels
Autocontrol
PatientRBCs+
Patientserum
10
Panel
Thesamephasesusedinanantibodyscreenare
usedinapanel
IS
37
AHG
11
AntibodyIDTesting
Atubeislabeledforeachofthepanelcellsplusone
tubeforAC:
AC
1 2 3 4 5 6 7 8 9 10 11
1dropofeachpanelcell
+
2dropsofthepatientsserum

12
361
ISPhase
Performimmediatespin(IS)andgrade
agglutination;inspectforhemolysis
Recordtheresultsintheappropriatespaceas
shown:
2
+
0
0
Last
tube
13
(LISS)37CPhase
2dropsofLISSareadded,mixedandincubatedfor
1015minutes
Centrifugeandcheckforagglutination
Recordresults
14
15 16
(LISS)37CPhase
2
+
0
0
2
+
0
0
2
+
0
0
2
+
0
0
0
0
17
IATPhase(orAHG)
IndirectAntiglobulinTest(IAT) weretesting
whetherornotpossibleantibodiesinpatients
serumwillreactwithRBCsinvitro
TodothisweusetheAntiHumanGlobulinreagent
(AHG)
Polyspecific
AntiIgG
Anticomplement
18
362
AHGPhase
Washcells3timeswithsaline(manualor
automated)
Add2dropsofAHGandgentlymix
Centrifuge
Read
Recordreactions
19
AHGPhase
2
+
0
0
2
+
0
0
2
+
0
0
2
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
20
Anddontforget.
.add check cells to
any negative AHG !
21
IS LISS
37
AHG CC
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
Allcellsare
at negative
,soadd AHG
CheckCells
22
Youhaveagglutinationnowwhat?
2
+
0
0
2
+
0
0
2
+
0
0
2
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
??
CC
23
InterpretingAntibodyPanels
Thereareafewbasicstepstofollowwheninterpreting
panels
1. Rulingoutmeanscrossingoutantigensthatdidnot
react
2. Circletheantigensthatarenotcrossedout
3. Considerantibodysusualreactivity
4. Lookforamatchingpattern
24
363
Anantibodywillonlyreact withcells
thathave thecorrespondingantigen;
antibodieswillnotreact withcells
thatdonothave theantigen
AlwaysRemember:
25
1.RulingOut
2
+
0
0
2
+
0
0
2
+
0
0
2
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
CrossoutantigensthatshowNOREACTIONinanyphase;doNOT crossout
heterozygousantigensthatshowdosage.
26
2.Circleantigensnotcrossedout
2
+
0
0
2
+
0
0
2
+
0
0
2
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
27
3.Considerantibodysusualreactivity
2
+
0
0
2
+
0
0
2
+
0
0
2
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
Le
a
isnormallyaColdReactingantibody(IgM),soitmakessensethatwe
seethereactionintheISphaseoftesting;TheEantigenwillusuallyreact
atwarmertemperatures
28
4.Lookforamatchingpattern
2
+
0
0
2
+
0
0
2
+
0
0
2
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
Yes, there is a matching pattern!

E doesnt matchandits a
warmer rxAb
29
Interpretation
anti
Le
a
30
364
Guidelines
Again,itsimportanttolookat:
Autocontrol
Negative alloantibody
Positive autoantibodyorDTR(i.e.,alloantibodies)
Phases
IS cold(IgM)
37 cold(somehavehigherthermalrange)orwarmreacting
AHG warm (IgG)significant!!
Reactionstrength
1consistentstrength oneantibody
Differentstrengths multipleantibodiesordosage
31
Aboutreactionstrengths
Strengthofreactionmaybeduetodosage
Ifpanelcellsarehomozygous,astrongreactionmaybe
seen
Ifpanelcellsareheterozygous,reactionmaybeweakor
evennonreactive
Panelcellsthatareheterozygousshouldnotbe
crossedoutbecauseantibodymaybetooweakto
react(seefirstexample)
32
Guidelines(continued)
Matchingthepattern
Singleantibodies usuallyshowsapatternthatmatches
oneoftheantigens(seepreviouspanelexample)
Multipleantibodies aremoredifficulttomatchbecause
theyoftenshowmixedreactionstrengths
33
Ruleofthree
Theruleofthree mustbemettoconfirm the
presenceoftheantibody
Apvalue0.05mustbeobserved
Thisgivesa95%confidenceinterval
Howisitdemonstrated?
PatientserumMUSTbe:
Positivewith3cellswiththeantigen
Negativewith3cellswithouttheantigen
34
Ourpreviousexamplefulfillstheruleof
three
2
+
0
0
2
+
0
0
2
+
0
0
2
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
3Negative
cells
3Positive
cells
Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin
Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin
35
Whatiftheruleofthreeisnotfulfilled?
Iftherearenotenoughcellsinthepaneltofulfillthe
rule,thenadditionalcellsfromanotherpanelcould
beused
Mostlabscarrydifferentlotnumbersofpanelcells
36
365
Phenotyping
Inadditiontotheruleofthree,antigentyping the
patientredcellscanalsoconfirmanantibody
Howisthisdone?
OnlyperformthisifthepatienthasNOTbeenrecently
transfused(donorcellscouldreact)
Ifreagentantisera(ofthesuspectedantibody)isadded
tothepatientRBCs,anegative reactionshould
resultWhy?
37
RememberLandsteinersRule
IndividualsDONOT makealloantibodiesagainst
antigenstheyhave
38
Multipleantibodies
Multipleantibodiesmaybemoreofachallengethan
asingleantibody
Why?
Reactionstrengthscanvary
Matchingthepatternisdifficult
39
Sowhatisatechtodo?
Severalprocedurescanbeperformedtoidentify
multipleantibodies
SelectedCells
Neutralization
Chemicaltreatment
Proteolyticenzymes
Sulfhydrylreagents
ZZAP
40
SelectedCells
Selectedcellsarechosenfromotherpanelor
screeningcellstoconfirmoreliminatetheantibody
Thecellsareselectedfromotherpanelsbecause
oftheircharacteristics
Thenumberofselectedcellsneededdependson
howmayantibodiesareidentified
41
SelectedCells
Everycellshouldbepositiveforeachofthe
antibodiesandnegativefortheremainingantibodies
Forexample:
Letssayyouranapanelandidentified3different
antibodies:antiS,antiJk
a
,andantiP
1
Selectedcellscouldhelp
42
366
SelectedCells
Selected
cells
S Jk
a
P
1
IS LISS
37
AHG
#1 + 0 0 0 0 2+
#5 0 + 0 0 0 3+
#8 0 0 + 0 0 0
Theseresultsshowthatinsteadof3antibodies,thereareactually
2:antiSandantiJk
a
43
Neutralization
Someantibodiesmaybeneutralizedasawayof
confirmation
Commercialsubstancesbindtotheantibodiesin
thepatientserum,causingthemtoshowno
reactionwhentestedwiththecorresponding
antigen(inpanel)
44
Neutralization
Manufacturersdirectionsshouldbefollowedanda
dilutionalcontrolshouldalwaysbeused
Thecontrolcontainssalineandserum(nosubstance)
andshouldremainpositive
Acontrolshowsthatalossofreactivityisduetothe
neutralizationandnot tothedilutionoftheantibody
strengthwhenthesubstanceisadded
45
Neutralization
Commonsubstances
P
1
substance (sometimesderivedfromhydatid cystfluid)
Le
a
andLe
b
substance (solubleantigenfoundinplasmaandsaliva)
I substance canbefoundinbreastmilk
Sd
a
substance derivedfromhumanorguineapigurine
**Youshouldbeawarethatmanyofthesesubstancesneutralize
COLDantibodies;Coldantibodiescansometimesmaskmore
clinicallysignificantantibodies(IgG),animportantreasontouse
neutralizationtechniques
46
Enzymes(proteolytic)
Canbeusedtoenhanceordestroycertainblood
groupantigens
Severalenzymesexist:
Ficin(figs)
Bromelin(pineapple)
Papain(papaya)
Inaddition,enzymeproceduresmaybe
Onestep
Twostep
47
Enzymes
Enzymesremovethesialicacid fromtheRBC
membrane,thusdestroyingitandallowingother
antigenstobeenhanced
Antigensdestroyed:M,N,S,s,Duffy
Antigensenhanced:Rh,Kidd,Lewis,I,andP
48
367
Enzymetechniques
Onestage
Enzymeisaddeddirectlytotheserum/cellmixture
Twostage
Panelcellsarepretreated withenzyme,incubatedand
washed
Patientserumisaddedtopanelcellsandtested
49
Enzymetechniques
Ifthereisnoagglutinationaftertreatment,thenitis
assumedtheenzymesdestroyedtheantigen
50
Enzyme
treament
AntiK
Perfectmatchfor antiFya
Duffyantigensdestroyed
Kellantigensnotaffected
Enzymetreatment
51
SulfhydrylReagents
CleavethedisulfidebondsofIgMmoleculesand
helpdifferentiatebetweenIgMandIgGantibodies
GoodtousewhenyouhavebothIgGandIgM
antibodies(warm/cold)
Dithiothreitol(DTT) isathiolandwilldenatureKell
antigens
2mercaptoethanol(2ME)
52
ZZAP
Acombination ofproteolyticenzymesandDTT
DenaturesKell,M,N,S,Duffy andotherless
frequentbloodgroupantigens
DoesnotdenaturetheKxantigen
Goodforadsorptiontechniques
freesautoantibodyoffpatientscell,sothatautoantibodycanthen
beadsorbedontoanotherRBC
53
Autoantibodies.
Warm&ColdReacting
54
368
Autoantibodies
Autoantibodiescanbecold orwarm reacting
ApositiveautocontrolorDATmayindicatethatan
autoantibodyispresent
Sometimestheautocontrolmaybepositive,butthe
antibodyscreeningmaybenegative,meaning
somethingiscoatingtheRBC
55
GettingapositiveDAT
WehavefocusedalotontheIATusedinantibody
screeningandID,butwhatabouttheDAT?
Thedirectantiglobulintest(DAT) testsforthein
vivo coatingofRBCswithantibody(inthebody)
AHGisaddedtowashedpatientredcellsto
determinethis
56
WhatcantheDATtellus?
Althoughnotalwaysperformedinroutine
pretransfusiontesting,apositive DATcanoffer
valuableinformation
Ifthepatienthasbeentransfused,thepatientmayhave
analloantibodycoatingthetransfusedcells
IfthepatienthasNOTbeentransfused,thepatientmay
haveanautoantibody coatingtheirowncells
57
Identifyingautoantibodies
Autoantibodiescansometimesmaskclinically
significantalloantibodies,soitsimportantto
differentiatebetweenauto andalloantibodies
58
Coldautoantibodies
Reactatroomtemperaturewithmost(ifnotall)of
thepanelcellsandgiveapositiveautocontrol
TheDATisusuallypositivewithantiC3AHG(detects
complement)
CouldbeduetoMycoplasmapneumoniae,
infectiousmono,orcoldagglutinindisease
59
Coldautoantibodies
Minicoldpanelscanbeusedtohelpidentifycold
autoantibodies
SinceantiIisacommonautoantibody,cordblood
cells(noIantigen)areusuallyincluded
GroupO
individualwith
coldautoantiI
GroupAindividual
withcoldautoanti
IH
AntiIHis reacting weakly withthecordcells (someH
antigenpresent)
60
369
Avoidingreactivity
Coldautoantibodiescanbeanuisanceattimes.
Hereareafewwaystoavoidareaction:
UseantiIgGAHGinsteadofpolyspecific.Mostcold
antibodiesreactwithpolyspecificAHGandantiCAHG
becausetheyfixcomplement
SkippingtheISphaseavoidstheattachmentofcold
autoantibodiestotheredcells
Use22%BSAinsteadofLISS
61
Othertechniques
Iftheantibodiesremain,thenprewarmed
techniques canbeperformed:
Redcells,serum,andsalineareincubatedat37 before
beingcombined
Autoadsorption isanothertechniqueinwhichthe
autoantibodyisremovedfromthepatientsserum
usingtheirownredcells
Theserumcanbeusedtoidentifyanyunderlying
alloantibodies
62
Warmautoantibodies
More commonthatcoldautoantibodies
PositiveDATduetoIgGantibodiescoatingthered
cell
Again,themajority ofpanelorscreeningcellswillbe
positive
TheRhsystem(eantigen)seemstobethemain
targetalthoughothersoccur
63
Warmautoantibodies
Causewarmautoimmunehemolyticanemia
(WAIHA)H&H
Howdoyougetawarmautoantibody?
Idiopathic
Knowndisorder(SLE,RA,leukemias,UC,pregnancy,
infectiousdiseases,etc)
Medications
Severaltechniquesareusedwhenwarm
autoantibodiesaresuspected
64
Elution(wheneverDATispositive)
Elutiontechniquesfreeantibodiesfromthe
sensitizedredcellssothattheantibodiescanbe
identified
Y
Y
Y
Y
SensitizedRBC
PositiveDAT
Y
Freesantibody AntibodyID
65
Elution
Theeluate isatermusedfortheremovedantibodies
Testingtheeluateisusefulininvestigationsofpositive
DATs
HDN
Transfusionreactions
Autoimmunedisease
TheredcellscanalsobeusedafterelutionforRBC
phenotypingifneeded
Whentestedwithpanelcells,theeluateusuallyremains
reactivewithallcellsifawarmautoantibodyispresent
66
370
ElutionMethods
Acidelutions(glycineacid)
Mostcommon
LowerspH,causingantibodytodissociate
Organicsolvents(ether,chloroform)
DissolvebilipidlayerofRBC
Heat(conformationalchange)
FreezeThaw(lysescells)
ABO
antibodies
67
Adsorption
Adsorptionprocedurescanbeusedtoinvestigate
underlyingalloantibodies
ZZAP orchloroquinediphosphatecanbeusedto
dissociateIgGantibodiesfromtheRBC(maytake
severalrepeats)
AfterthepatientRBCsareincubated,theadsorbed
serumistestedwithpanelcellstoIDthe
alloantibody(ifpresent)
68
Adsorption
Twotypes:
1. Autoadsorption
Norecenttransfusion
AutoantibodiesareremovedusingpatientRBCs,so
alloantibodiescanbeidentified
2. Allogenic(Differential)adsorption
Ifrecentlytransfused
Usesothercellswiththepatientsserum
69
Washx3 after
incubation
Centrifugeafter
incubating; andtransfer
serumto2
nd
tubeof
treatedcells; incubateand
centrifugeagain
2tubes
Removeserum
andtestfor
alloantibody
70
Morereagents.
Manyofelutiontestscandamagetheantigenson
theRBC
Choroquinediphosphate (CDP)andglycineacid
EDTAreagentscandissociateIgGfromtheRBC
withoutdamagingtheantigens
VeryusefuliftheRBCneedstobeantigentyped
71
Chloroquinediphosphate
Quinilonederivativeoftenusedasanantimalarial
MaynotremoveautoantibodycompletelyfromDAT
positivecells
Partialremovalmaybeenoughtoantigentypethe
cellsortobeusedforautoadsorptionofwarm
autoantibodies
72
371
MinistryofHealth
Transfusion Transmitted
Diseases (TTD)
Institute Graduates
Theinfectiousmicrobesthattransmittedbybloodtransfusion:
1 HepatitisBandC
2 HIV
3 HTLV
4 Cytomegalovirus(CMV)
5 EpsteinBarrvirus
6 HumanParvovirus(B19)
7 HumanHerpesvirus
8 Bacterialcontamination
9 Syphilis
10 Malaria
11 WestNileVirus(WNV)
TheInfectiousDiseasesTransmittedbyBlood
Transfusion:
2
1 ViralWindowPeriodistheperiodbetweentheonsetofviral
infectionandtheappearanceofdetectableantibodiestothe
virus.
2 TheGeneticVerticaltransmissionofviruses.
3 Donorimmunestatus(Asymptomaticimmunocompetent
patients).
4 Laboratory andpersonal error.
5 Bacterial contamination.
FactorsThatPlayaRoleinEstablishmentof
BloodTransfusionInfection
3
Whatarebloodbornepathogens?
MicroOrganism:
HepatitisB(HBV)
HepatitisC(HCV)
HumanImmunodeficiencyVirus(HIV)
Substancesthatarecarriedbythebloodorotherbody
fluids andcauseillnessorinjurytothebody.
VirusandbacteriaarepathogensandmanyareBlood
borne.
BloodBornePathogents
4
Include:
Hepatitis:
A, B, C, D, E
Execution
Viruses or bacteria
that are carried in blood
and cause disease in
People.
Malaria
TypesofBloodBornePathogens
Human
Immunodeficiency
Virus (HIV)
Brucellosis
Syphilis
5
Hepatitis
Aninflammationoftheliverusuallycausedbydrugs,toxins,
autoimmunedisease,orinfectiousagents.
Potentiallylifethreateningbloodbornepathogen.
Potentialforcarrierstopassdiseasetoothers.
Effectscanbebothacuteandchronic.
Hepatitis
A
Hepatitis
E
Hepatitis
C
Hepatitis
B
Hepatitis
D
BloodBornePathogens
6
372
ThemostcommonlyconcernedofHepatitisare:
BloodBornePathogens
7
Transmittedviacontaminatedfoodorwater
whichcontainsfecalmattercontainingthe
virus.ThereisavaccinetopreventHAV.
TwoTypesofHAV:
1. Infectious(transmittedpersontopersonbythe
fecaloralroute)or
2. Serum(transmittedbytransfusionofblood
products).
HepatitisA(HAV)
8
Transmittedbyinjectionstransportingavirus
bearingserum,mostoftenduringbloodtransfusions
andbycontaminatedneedlesandsyringes.
TransmittedprimarilythroughBloodtoBlood
contact.
Verydurable,anditcansurviveindriedbloodforup
tosevendays.
Thisvirusistheprimaryconcernforhousekeepers,
custodians,laundrypersonnelandotheremployees
whomaycomeincontactwithbloodorpotentially
infectiousmaterialsinanonfirstaidormedicalcare
situation.
HepatitisB(HBV)
9
Transmittedthroughparenteral,sexualexposure
Meanincubationtime90days
50%ofinfectionsaresymptomatic
1/500infectionsarelethal
610%ofinfectionsbecomechronic
VaccinationmakesdonorantiHBs+,HBsAg,anti
HBc
Riskoftransmission1/66,000
HepatitisB
10
HepatitisBVirusGeographicDistribution
11
Mildflulikesymptoms
Fatigue
YellowEyesandSkin
Possiblestomachpain
Lossofappetite
FeverandVomiting
Nausea
Jaundice
DarkenedUrine
SymptomsofHBV
12
373
HBVSeroconversioninEarlyInfection
13
MarkersofHBVInfection
14
Transmittedinbloodorbodyfluids.Novaccination
existsforHCV.
Chronicliverdiseasedevelopsinabout70%of
personswhobecomeinfectedwithHCVandnearly
all(85%100%)personswithacuteHCVinfection
becomepersistentlyinfected.
Antibodyappearsinserum54 192dayspost
infection
HepatitisC(HCV)
15
HepatitisCVirus GeographicDistribution
16
Injection drug use
Receipt of donated blood, blood products, and organs.
Needlestick injuries in healthcare settings (3%).
Birth to an HCVinfected mother.
Sex with an HCVinfected person.
Sharing personal items contaminated with infectious
blood (razors or toothbrushes).
HepatitisCVirus ModeofTransmission
17
Indevelopingcountries,theprimarysourcesofHCVinfectionareunsterilized
injectionequipmentandinfusionofinadequatelyscreenedbloodandblood
products.
HepatitisCTransmissioninDeveloped
Countries
18
374
HCV RNA
Anti-HCV EIAs
1
st
gen 150 d
2
nd
gen 80 d
3
rd
gen 70 d
0 10 20 30 40 50 60 70 80 90 100
Days following infection
Ramp-up phase
Plateau phase viremia: 10
5
-10
8
gEq/mL
Pre-ramp-
up blip
viremia
- HCV Ag EIA
- HCV MP-NAT
- HCV ID-NAT
ALT
Viral set-point:
10
2
-10
7
gEq/mL
Glynn et al, Transfusion 2007
Viraemia andSeroconversion DuringEarlyHCV
Infection
19
Oneofthenewertypesanditistransmitted
primarilythroughinjecteddruguseand
sexualcontact.
Prevention:
Educationtoreduceriskbehaviorsforthosewith
chronicHBVinfection.
HepatitisD(HDV)
20
Defective single stranded RNA virus.
Only in patients with HBV infection.
Requires HBsAg in order to synthesize an
envelope protein.
Screening donors for HBV infection eliminates
the risk for HDV.
HepatitisDVirus
21 22
HepatitisGVirus
AlsocalledGBVCisaFlavivirusdistantlyrelatedto
HCV.
RecentreportsdonotimplicateHGV/GBVCasa
causeofhepatitisoranyotherdisease
manifestation.
23
HumanImmunodeficiencyVirus(HIV)
HIVisthevirusassociatedwithAIDS
Thereisnospecifictreatmentforit
Thereisnocure
Thereisnopreventativevaccine
HIVattacksthebodyimmunessystem,weakeningit
sothatitcannotfightotherdeadlydisease.
HIVisveryfragileandwillnotsurviveverylong
outsideofthehumanbody.
24
375
SymptomsofHIVinfectioncanvary,butofteninclude:
Weakness
Fever
Sorethroat
Nausea
Headaches
Diarrhea
Whitecoatingonthetongue
Weightloss
Swollenlymphglands
SymptomsofHIV
25
TransmissionofHIV
Sexualintercourse(0.11 1.7%).
bloodorbloodproducts(90%).
NeedlestickinjuryofHealthcareworkers(0.3%
transmission).
Intravenousdrugusers(0.67%).
Frommothertochild(25 35%):
Transplacentally
Duringbirth
Breastfeeding
26
IncubationPeriodofHIV
TimefromexposuretoHIVuntilonsetofacute
clinicalillnessis1 4weeks.
Afterprimaryinfectionthereisaperiodranging
fromafewmonthstomorethan10yearswithno
ormildsymptomsbeforetheappearanceofsevere
immunodeficiency.
27
Adults&ChildrenEstimatedtobeLivingwith
HIVin2009
28
HIV RNA (plasma)
HIV Antibody
11
0 10 20 30 40 50 60 70 80 90 100
HIV p24 Ag
16 22
Ramp-up viremia
DT = 21.5 hrs
1
st
gen
2
nd
gen
3
rd
gen
p24 Ag EIA -
HIV MP-NAT -
HIV ID-NAT -
Peak viremia: 10
6
-10
8
gEq/mL
blip viremia
Viral set-point:
10
2 -
10
5
gEq/mL
Closing the WP through improved screening tests
HIVViraemiaMarkers
29
HumanTCellLymphotropic Viruses(HTLVI&
II)
ModeofTransmission:
Sexualtransmission.
Intravenousdrugabuse.
Bloodtransfusion.
Breastfeeding.
30
376
HumanTCellLymphotropic Viruses(HTLVI&
II)
SymptomsandsignsofATL:
IsFatalassociatedwithacuteinfiltrationofskinand
visceraltissuewithmonoclonalproliferationofCD4
bearingTlymphocytes &thefollowingclinical
manifestations:
Skinlesionsduetoinfiltratingleukaemic cells.
Interstitialpneumonia.
Hepatosplenomegaly.
Bonelesions.
31
LaboratoryDiagnosis
ELISAforscreeningandWesternblotfor
confirmation.
ThereisextensivecrossreactivitybetweenHTLVI
andHTLVII,makingitdifficulttodistinguish
betweenthesetwoviruses.
PCRmethodscanalsobeusedtodetectHTLVIand
HTLVIIinperipheralbloodandspinalfluid.
32
Seropositivity rate:5090%amongblooddonors.
1%ofcellularcomponentstransmittheviruses.
Hepatitisisrare&generallymildintheabsenceof
severeimmunosuppression(e.g.highriskneonates
andtransplantrecipients).
Removalofleukocytesfromdonatedblood=reduce
ifnotpreventposttransfusiontransmission.
CMVandEBV
33
Benignand/ortransientnatureofmostparvovirus
disease.
Effectivetreatment:IVImmunoglobulin.
Extremerarityoftransmissionbyblood
components.
Parvovirus
34
Aninfectiousdiseasecausedbythebacteriaof
thegenusBrucella.
Thesebacteriaareprimarilypassedamong
animals,andtheycausediseaseinmany
differentvertebrates.
Commonlytransmittedtosusceptibleanimalsby
directcontactwithinfectedanimalsorwithan
environmentthathasbeencontaminatedwith
dischargesfrominfectedanimals.
Brucellosis
35
ASexuallyTransmittedDisease(STD)causedbya
bacteria
Itcanalsopassthroughbrokenskinonotherpartsof
thebody
SignsofSyphilisinclude:
Chancres"("shanker"),orsores.
Skinrash.
Mildfever.
Feelingverytired.
Headache.
Sorethroat.
Hairloss.
Syphilis
36
377
Causes:
Riskofdiseasetransmissionbyblood
transfusionisexceedinglyrare.
CausativeagentsTreponemapallidum
diagnosedbyexaminationofmicrobeby
darkfieldorbyimmunofluorescent
microscopy.
Prevention:
PreventedbyVDRLorRPRtestingforblood
donors.
AllpositivecasesconfirmedbyTPHA
testing
Syphilis Malaria
Causes:
Mostfrequentlyplasmodiumfalciparum.
Riskrateis<1:1,000,000
Signsandsymptoms:
Ofmalariainfection
Management:
Antimalaria medication
Prevention:
Exclusionanddeferralofhighriskdonorsduringdonorselection.
Testingthickbloodfilmformalariaforallblooddonors.Orusemore
sensitivetestsfordetectionofmalariaifavailable.
38
Malaria
39
Causes:
InfectiousagentisBabesia microti Riskfactor<1:
1,000,000
Prevention:
Deferdonorsfromendemicareasorthosewhohave
previousinfection.
Babesiosis
40
Transmittedbyarthropodexposure(incidental)
Birdsareprimaryhosts
50nmspherical,lipidenvelopedflavivirus
SinglestrandedpositivesenseRNAgenome(11,000
nts)
Encephalitis,meningitis
Asymmetricflaccidparalysis(poliomyelitislike)
WestNileVirus
41
WestNileVirus
2002:4156casesreportedinU.S.(284fatalities)
510%mortality
Initialscreeningbyquestionnaire
VoluntaryNATtesting:through2004,1017units
withdrawnduetopresumptiveWNVinfection
PeakexposureinAugustSeptember
Viremia6.5to56.4days
42
378
ChagasDisease
Causes:
Riskfactoris1:42,000
InfectiousagentisTrypanosoma cruzi.
Prevention:
Deferdonorsfromendemicareasorthosewhohave
previousinfection.
43
PrionDiseases:Transmissiblespongiform
encephalopathies (TSEs)
Fatalneurodegenerativediseasesofmanand
mammals.
Longincubationperiodwithlittleornohostimmune
response.
Associatedwiththeconversionofnormalhostprion
proteintoadiseaseassociatedisoform(PrP
Sc
).
Characteristicneuropathologywithspongiform
changesinthegreymatterofthebrain,usually
concurrentwithdepositionofPrP
Sc
.
44
Firstreportedcasein2004.RecipientclinicalonsetofvCJD
6.5yrs afterbloodtransfusion.DonordevelopedvCJD
approx 40monthsafterdonation(nonleucodepleted red
cells)
Secondcaselaterin2004(Peden etal:Lancet;364:527).
Recipientdiedofaorticaneurism5yrs posttransfusion.No
signsofvCJD butabnormalprionaccumulationinspleenand
onelymphnode.BloodDonordevelopedvCJD approx 18
monthsafterdonation(nonleucodepleted redcells)
Thirdcase2006(Wroe etal;Lancet;368:2061).Recipient
clinicalonsetofvCJD 7.8yrs afterbloodtransfusion.Donor
developedvCJD 21monthspostdonation(non
leucodepleted redcells)
TransmissionofvCJDbyBloodTransfusion
45
TransmissionofvCJDbyBloodTransfusion
Fourthcase2007(HPApressrelease).DevelopedvCJD 9
yrs aftertransfusion.Blooddonorhadalreadybeen
implicatedinprevioustransmissioncase
Hemovigelance ReportreviewinUK:Nocasesyetfrom
transfusionofplasmaorleucodepleted blood.
AlsonoevidencethatdonorswhodevelopsCJD have
transmittedvCJD inblood
Bloodappearstobehighlyinfective.Someindication
thatleucodepletion mayreducerisk.
Asymptomaticpatientwithprionaccumulationin
spleensuggestscarrierstatusispossible
46
MeasurestoProtecttheBloodSupplyFrom
TransmissionofvCJD
1. Leucodepletion ofbloodfortransfusion.
2. Blooddonationsnotacceptedfromdonorswhose
bloodwastransfusedtopatientswholaterdeveloped
vCJD.
3. Blooddonationsnotacceptedfromrecipientsofblood
transfusionsintheUKsinceJanuary1980.
4. Filtrationofredcellstoremoveorreducepriontitre.
5. Deferdonorswithfamilyhistoryof(CJD),exposureto
riskfactors,orresidenceinendemicareaswith(CJD),
formorethan6months(UK).
47
PotentialTransmission
Bloodborne Pathogensmayalsotransmittedby:
Contactbetweenbrokenordamagedskinandinfected
bodyfluids.
ContactbetweenMucousMembranesandinfectedbody
fluids.
Example:Eyes,Nose,andMouth
Anytimethereisbloodtobloodcontactwithinfected
bloodorbodyfluids,thereisaslightpotentialfor
transmission.
Accidentalpuncturefromcontaminatedneedles,broken
glass,orothersharpsispotentiallyhowtransmission
couldoccurforcustodialemployees.
48
379
Work Practice
Controls
Personal Protective
Equipment
Personal
Hygiene
Universal Precaution
ComplianceControlMethods
49
P
r
e
c
a
u
t
i
o
n
Treatallbloodandbodilyasiftheyarecontaminated.
Allbodyfluidsmustbeconsideredaspotentiallyinfectious
materials.
Propercleanupanddecontamination
AlwayswearappropriatePPE
ReplacePPEthatistornorpunctured
RemovePPEbeforeleavingtheworkarea
ChangePPEbetweenpatientsandwashhandseachtimeafter
removalofglove.
Standard
Precaution
50
PPEControls
PPEmustbeusedtopreventpotentiallyinfectious
materialsfromcomingincontactwithworkclothes,
streetclothes,undergarments,skinormucous
membranes.
Employeesmustweargloveswhenthereispotential
contactwithblood,potentiallyinfectiousmaterials,
mucousmembranesorbrokenskin.
Removeglovespromptlyafteruse,andbeforetouching
noncontaminateditemsandenvironmentalsurfaces.
51
SafeWorkPracticeControls
RemovecontaminatedPPEor
clothingassoonaspossible
Cleananddisinfectcontaminated
equipmentandworksurfaces
Thoroughlywashupimmediately
afterexposure
Properlydisposeofcontaminateditems,including
contaminatedPPE
52
PersonalHygiene
Donottouchanythingthatiscontaminated,suchas
sharpsorbodyfluids.
Takecaretominimizesplashingofallinfectious
materials.
Eating,drinking,smoking,applyingcosmeticsorlip
balm,andhandlingcontactlensesareprohibitedin
areaswherethereisapotentialforoccupational
exposure.
53
PersonalHygieneCont.
UseCDCguidelinesforhandhygiene:
Ifhandsarenotvisiblysoiled,usealcoholgel.
Whenhandsarevisiblysoiled,washhandswith
soapandwater.
Alwayswashyourhandsbeforeeatingandafter
usingtherestroom.
54
380
Summary
Alwaysknowwhatyouareworkingwith.
AlwayswearappropriatePersonalProtective
Equipment(PPE)whenhandlinganytypeofbodily
fluid.
Alwayswashyourhandsafterhandlinganytypeof
bodilyfluid,evenwhenwearinggloves.
Donothandlesharpsorbrokenglasswithyour
handsandwithoutprotection.
Properlydisposeofpathogenwaste,includingPPE.
Alwaysreportallsuspectedexposures.
55
381
MinistryofHealth
Bacterial Contamination in Blood
Products: Risks, Prevention and Detection
Institute Graduates
BacterialContaminationinBloodProducts
WhatistheProblem?
WhataretheRisks?
WhatOrganismsareAssociatedwithBacterial
Contamination?
WhataretheSourcesofContamination?
WhatOptionsexisttoDetectBacterial
Contamination?
WhatCorrectiveActions(Preventionand
Management)arePlanned?
2
Whatistheproblem?
3
Firstrecognizedinfectiousriskofbloodtransfusion.
Riskgreatlyreducedinthe1960sbytheuseof
closed,sterilesystemsforthecollectionandstorage
ofblood.
Recentdramaticimprovementsinsafetyfromviral
screeningandtestinghavereducedtherisksfrom
HepatitisandHIV.
Bacterialsepsisisnowthemostcommoninfectious
diseaseeventfollowingtransfusion.
4
Bacterialcontaminationoccursprimarilyinroom
temperaturestoredproducts(platelets)butcan
occurinredbloodcellsandplasmaalso.
Thebloodbankingcommunityistakingstepsto
improvepreventionanddetectionofbacterial
contamination.
TheAmericanAssociationofBloodBanks(AABB),as
wellastheCollegeofAmericanPathologists(CAP)
haveestablishedcompliancecriteriafortransfusion
services.
5
TheAmericanAssociationofBloodBankshasissued
twonewstandards(March,2003):
5.1.5.1Thebloodbankortransfusionserviceshall
havemethodstolimitanddetectbacterial
contaminationinallplateletcomponents.
5.6.2Thevenipuncturesiteshallbepreparedsoasto
minimizetheriskofbacterialcontamination.Green
soapshallnotbeused
6
382
CollegeofAmericanPathologistsAccreditation
Checklist(December,2002):
TRM.44955Phase1Doesthelaboratoryhavea
systemtodetectthepresenceofbacteriainplatelet
components?
7
Whataretherisks?
8
Althoughuncommon,butthistypeofspecificreactioncan
havearapidonsetandhighmortalityinrecipients.
Thepresenceofbacteriaintransfusedbloodmayleadeither
tofebrilereactionsintherecipient(duetopyrogens)or
seriousmanifestationsofsepticorendotoxicshock.
Commonlycausedbyendotoxinproducedbybacteria
capableofgrowingincoldtemperaturessuchas
Pseudomonasspecies,E.coli,Yersiniaenterocolitica.
BacterialContaminationReaction
9
Usuallyappearrapidlyduringtransfusionorwithin
about30minutesaftertransfusionwithdryness,
flushingofskin.
Fever,Hypotension,Chills,Musclepain,vomiting,
Abdominalcramps,Bloodydiarrhoea,
Hemoglobinuria,Tachycardia,Shock,Renalfailure,
DIC.
ClinicalManifestation
10
BacterialContamination
BacterialContaminationoccursatamuchhigher
frequencythananyotherinfections(Incidence:
0.3%)andisassociatedwithsubstantialmortality.
Rateofbacterialinfection/contamination:
RBCs1in30,000
RandomPlatelets1in2,000
Thehigherratewithplateletsisbecausetheyare
storedatroomtemperatureandtheunitsare
generallypooledbetween6and10donorunits.
11
BacterialContamination
1. Bacterialtestingofapheresisproductsinitiatedin
2004
2. Assessmentofriskbaseduponreporting
Pretesting:
Septicreactions1:40,000
Fatalities1:240,000
Posttesting:
Septicreactions1:75,000
Fatalities:1:500,000
12
1 AmericanRedCross Ederetal.(Transfusion2007)
20septicreactions(3fatal)
1,004,000testedproducts~1:59,000
2 Canada RamrezArcosetal.(Transfusion2007)
82,004testedproducts~1:41,000
3 Holland deKorte etal.(Transfusion2006)
2septicreactions
113,092testedproducts~1:56,500
4 Germany Schmidtetal.(Vox Sanguinis 2007)
52,243productstested~1:26,000
ResidualRiskofBacterialSepsisAfterBacterial
CultureofAphaeresisPlatelets
13
WhatOrganismsareAssociatedwithBacterial
Contamination?
14
15
Exogenous
Staphylococcusepidermidis
Diphteroidsspp
Micrococcusspp
Pseudomonasspp
Bacilluscereus
Propionibacteriumacnes
Flavobacteriumspp
Nor mal sk i n
OrganismsInvolved
16
Endogenous
Osteomyelitis
Staphylococcus
S.cholerasuis
Staphylococcusspp
Teeth Streptococcusviridans
Serratialiquefaciens
Yersiniaenterocolitica
Intestines Salmonellaspp
Campylobacterspp
OrganismsInvolved
S. epidermidis, 30.2%
S. aureus, 10.5%
E. coli, 9.3%
B. cerus, 9.3%
S. cholerae-suis, 8.1%
B-hem. Strep, 5.8%
E. aerogenes, 2.3%
10 others, 1.3% each
n =86
Compilation of data from Clin Micro Rev
1994; 7:290-302; Transfusion 2001;41:1493-
99; www.shot.demon.co.uk/toc
BacterialSpeciesinPlts ImplicatedinClinical
Sepsis
17
S. epidermidis 9.6%
S. aureus 17.3%
E. coli 5.7%
Bacillus 5.7%
Salmonella 7.7%
Enterobacter 5.7%
Streptococcus 7.7%
Klebsiella 17.3%
Serratia 15.4%
P. mirabilis 2.2%
n =52
BacterialSpeciesinPlts ImplicatedinSeptic
FatalitiesReportedtotheFDA(19761998)
18
384
S.epidermidis islesscommonlyobservedinseptic
fatalitiesandmorecommonlyobservedinseptic
reactions.
Klebsiella iscommonlyobservedinsepticfatalities.
Gramnegativeorganismsareimplicatedinmore
fatalities(60%) thangrampositiveorganisms(40%);
grampositivescauseamajorityofsepticreactions
(56%).
DifferencesBetweentheSpeciesImplicatedin
SepticMorbidity&MortalityinPlatelet
Components
19
Approximately30%areassociatedwithnormalskin
flora.
Approximately56%aregrampositive.
Allareaerobicorfacultativeanaerobes;
Arare(singlecase)exception: Clostridiumperfringens
fatalityfromapooledplateletunitTransMed1998;8:19
22.
OrganismsImplicatedinSepsisFromPlatelets
20
BacterialContaminationofBloodProducts
Whatarethesourcesofcontamination?
21
SourcesofBacterialContamination
SkinSurfaceContamination.
PhlebotomyCore.
DonorBacteremia.
ContainersandDisposables.
Environment.
22
Infectionofstoredbloodisextremelyrare.
Skincontaminantsarenotinfrequentlypresentin
freshlydonatedbloodbuttheseorganisms
(predominantlystaphylococci)donotsurvive
storageat4Calthoughtheywillgrowprofuselyin
plateletconcentrates storedat22C.
23
Healthydonorwhoarebacteremicatthetimeof
donation.ThemajorityareduetoYersinia
enterocolitica,whichgrowswellinredcell
componentsduetoitsdependenceoncitrateand
Iron.
Gramnegative,endotoxin producingcontaminants
foundindirt,soilandfaecesmayrarelygrowinthe
storageconditionofblood.
24
385
BacterialContaminationofBloodProducts
WhatOptionsexisttoDetectBacterial
Contamination?
25
AABBAssociationBulletin#0307May16,
2003
MethodstoDetectContamination:
Culturemethodsoptimal.Twoapprovedproducts
cited.Otherculturemethodscanbevalidated.No
labelclaimsallowed
Duetoinsensitivity,staininganddipstickmethods
shouldbeusedascloseintimetoissueaspossible
Validationofallmethodsisrequired
Swirlprocedureusefulforinspectionbutdoesnot
byitselfmeetAABBStandard5.1.5.1
26
Visualexaminationfordiscoloration,clumpingorabnormal
morphology
Microscopy
Gramstain
Acridine orange
MeasuringBiochemicalchanges
LoweredpH
ReducedGlucose
Bacterialculture
Detectionthroughoxygenconsumption
DetectionthroughCO
2
production
BacterialDetectionOptionsinPlatelet
Products
27
3devicesareclearedforqualitycontrolmonitoringofplateletcollection
processofleukoreduced platelets:
BioMeriuex BacT/ALERT
PalleBDS
hemoSystems Scansystem
Useofnonvalidatedtests(glucoseandpHbydipstick,swirling).
Nonstandardizedmethodologyevenwithculturebaseddevices.
BacterialDetectionTests
28
Products
VisualExamination
Inspectproductpriortotransfusionfordiscoloration
orabnormalclumping.
Performswirlproceduretodetectmorphologic
changesinplatelets.
Normalshapedplateletswillalignwithfluidflowand
shimmerwhenswirled.
Contaminatedplatelets,amongothers,losediscoid
shapeanddonotshimmerwhenswirled Nota
specificmarkerforcontamination.
29 30
386
Low pH
Metabolic disturbance
No alignment with flow
Alignment with flow
SENSITIVITY: 75%
SPECIFICITY: 95%
Leach MF et al. Vox Sang 1998;74(suppl 1):1180.
Swirling
31
Products
MicroscopicMethods
GramStainorAcridine Orangepreferredmethods
Limitations:
MustbeperformedbytheTransfusionServicepriorto
productissuefortransfusion
Lacksensitivitywithlowbacterialload
32
MeasuringBiochemicalChanges
Measurechangesinglucoseconsumptionagainsta
control.Variancesof>2S.D.mayindicatebacterial
contamination.
Dipsticktesting.
Limitations:
Boththismethodandstainingmethodsaresubjective,
requirehighlevelsofcontamination,andmustbe
performedpriortoissuebytheTransfusionService.
Products
33
The majority of proliferating bacteria will
metabolize glucose and produce acid, lowering
the pH. Measurement of glucose and pH has been
evaluated and, indeed, utilized in the USA for the
detection of bacteria in platelet concentrates
(Burstein et al,. 1997; Choo et al., 2004; Cocco et
al., 2004; Hahn etal., 2004; Werch et al., 2002). An
advantage of this system is that it is extremely
rapid and conclusive , PH of all platelet Units
must be 6.2.
MetabolicChanges(pH)
34
Must be performed immediately before issue because of its relative
insensitivity and the need for high bacterial counts.
ChemicalTests Dipsticks
35 36
ValidationofBacterialDetectionMethods
pH&glucosetestsareanalyticallyinsensitive
(Yomatovian R,Brecher ME.Transfusion2005;45:6478)
Validationrequired:
Variableplasticbags.
Variableanticoagulants.
Variablehandling.
Gramstaininsensitiveunless106CFU/mL.
FacilitiesmaynothaveFDAapprovedequipment.
ValidateforQCuse
Sensitivity
TypesofUse
Growthtime
387
BioMeriuexBacT/AlertSystem
Detectsbacterialgrowthinculturebottlesby
measuringCO2production.
Automatedreadercontinuouslymonitorssamples.
Samplingintervalof>24hourspostphlebotomy
Culturingintervalof>24hourspostsampling
(aerobicandanaerobiccultures).
Culturesincubatefor57days;mayidentifypositive
culturesposttransfusion.
FDAApprovedforQ.C.purposesonlyon
Leukoreduced AphaeresisPlatelets.
37
BioMeriuexBacT/ALERT
Colorimetric
technology/SensorCulture
bottles
CO
2
releasecausessensor
bottletoturnyellow
Instrumentmeasures&
detectscolorchange,
analyzesdatatodetermine
positivity,alertswhen
positiveculture
38
PallBiomedicalBDSSystem
DetectsbacterialcontaminationbymeasuringO
2
consumption.
AutomatedreadermeasuresO
2
levelsinheadspace
ofculturepouch.
Samplingintervalof>2448hours.
Cultureperformedfor>2430hours.
FDAApprovedforQ.C.onleukoreducedplatelet
concentratesandleukoreducedapheresisplatelets.
39
PalleBDS
Sampleset/OxygenAnalyzer
Sterileweldplateletcomponentto
set
Fillpouchwith~3mLofproduct
Disconnectsamplepouchfromset
andincubateat35Cfor2430hrs
MeasuretheO
2
contentintheair
abovetheplasmasamplewith
insertionofanalyzerprobeinto
pouch
LEDdisplaywillreadPASSorFAIL
40
LimitationsofBloodCultureMethods
Earlysampling/testingmaynotdetectsmall#
bacteriaperbag.Approvedmethodsrequire2430
hourwaitbeforesampling
TwoFDAApprovedmethodsrequirebacteriato
growupaftersamplingtodetectablelevels,so
culturemustbedonewellbeforeplanned
transfusion(BloodCenter)
Thetwotimeintervals(collectiontosamplingand
samplingtorelease/transfusion)dominatethe
logisticconsiderations
41
Bothoptionsrequireleukoreducedplatelets.
BacT/Alertrequirescontinuedcultureafterproduct
release.
Releaseandrecall(BacT/ALERT)orholdtoendof
culturetorelease(PALLBDS).
Needtobalancetheriskofplateletshortagesversus
theriskofplateletcontamination.
Complexandexpensive.
42
388
ThetwoavailabledevicesareFDAApprovedforQ.C,
andnotapprovedasprereleasetests.
Probablenegativeimpactonoutdates.
Possibleextensionofplateletstoragetosevendays
orpooling/storingwholebloodderivedplatelets.
Falsepositives,indeterminants,falsenegatives,
Followupofsuspectedproducts.
Limitedeffectiveness:delayedbacterialgrowth
43 44
1. Rapid ~ 25 minutes (3 min hands on)
3. Sensitivity ~ 10
3
CFU/ mL
Singleuse disposable test
Verax rapid Platelet PGD
Test
2. Positives typically < 10 minutes
4. Specificity > 99.7%
NewDeviceforTransfusionServices
45
Verax PGD BacT/ALERT Pall eBDS
Technology
Conserved Bacterial
Ag Immunoassay
Culture
CO
2
Measure
Aerobic Culture
O
2
measure
Sample Volume 500 uL 420 mLs 35 mLs.
Time to Result
10 30 min
(positives typically
within 10 minutes)
24 96 hours 24 30 hours
Detect Aerobic and
Anaerobic bacteria?
Yes Yes, but time varies Misses Anaerobes
Clinical Specificity 99.7% 99.299.8% ~ 99%
Source: Abbott, Biomerieux and Pall Medical web site.
MethodsComparison
46
WhatCorrectiveActions(Prevention&
Management)arePlanned?
47
Prevention
Strictadherencetopolicies&proceduresregarding
bloodcomponentcollection,storage,handling,and
preparationisessentialtoreducetherisk.
VisualInspectionofcomponentsbeforereleasefrom
thetransfusionserviceincludeanydiscolouration,
visibleclots,orhemolysis.
Ensurethebloodcomponentsareinfusedwithin
standardtimelimits(4hours).
48
389
Bloodpacksshouldneverbeopenedforsampling,if
anyopenmethodofpreparationhasbeenused,the
unitshouldbetransfusedwithin24hours.
Bloodshouldalwaysbekeptinaccuratelycontrolled
refrigerators(withalarms),maintainedstrictlyat2
6C,thebloodshouldneverberemovedandtaken
tothewardorOTuntilitisrequired.
Prevention
49
PrecautionstobeObservedinPreparing
Components
Incollectionofblood
Properselectionofdonor
Clean&asepticvenepuncture sitetominimize
bacterialcontamination
Cleanvenepuncture withminimumtissuetrauma
andfreeflowofblood
Theflowofbloodshouldbeuninterruptedand
continuous.Ifanyunittakesmorethan10minutes
todraw,itisnotsuitableforpreparationofblood
components.
50
PrecautionstobeObservedinPreparing
Components(contd)
Acorrectamountofbloodproportionatetoanti
coagulantshouldbecollectedinprimarybagthat
hassatellitebagsattachedwithintegraltubing.
Monitorthecollectionofbloodwithautomatic
mixerwhichisusedforcollectingthedesired
amountofbloodandmixingthebloodwith
anticoagulant
Ifplateletsaretobeharvestedthebloodbagshould
bekeptatroomtemperature2024Cuntilplatelets
areseparated.Plateletsshouldbeseparatedwithin
6hoursfromthetimeofcollectionofblood.
51
AABBAssociationBulletin#0307May16,
2003
MethodstoLimitContamination:
Carefulphlebotomy Nogreensoapprep.
Iodinebasedscrubrecommended.
Considerphlebotomydiversion samplefirst
technologies.
Considerincreaseduseofaphaeresisplatelets.
52
Skindisinfectionmethods
Someagentsmayreducethenumberofsurface
bacteriamorethanothers.
Methodofapplicationandapplicatormayhave
someimpactontheextentofreductionofsurface
bacteria.
Minimumscrubof30secondsrequiredtobe
effective.
53
CFU per
plate
PVPI Isopropyl
Alcohol +
Tincture
of
Iodine
Chlor-
hexidine
Gluconate
Green
soap +
Isopropyl
alcohol
0 34-40% 60% 0%
1-10 35-43% 34% 25% 17%
11-100 10-14% 2% 12% 47%
>100 0-13% 1% 3% 36%

Goldman et al, Transfusion 1997;37:309-12
63%
ImpactofSkinDisinfectiononSurfaceBacteria
54
390
AvoidingSkinContamination
Diversionoftheinitialbloodflow.
Improvementinprephlebotomyskincleansing.
55
DiversionofInitialBloodFlow
Diversionofinitialbloodflowintosamplingtubes
Reducestheloadofskinassociatedbacteria
enteringbloodcontainer
Phlebotomycoredirectedintosamplingpouch
insteadofbloodcontainer
56
Total bacterial
prevalence
S. epidermidisprevalence
Standard
Collection
0.35%
(0.27-0.44)
0.14%
Collection with diversion 0.21%
(0.12-0.35)
0.03%
P-value <0.05 <0.02
deKorteetal.VoxSang2002;83:1316
Collectedbloodnormallyordivertedthefirst10mLofwhole
bloodintoasatellitebag
Performedbacterialtestingbyautomatedbloodculture
(BacT/Alert)inalaminarflowhood
ClinicalDataSupportingDiversionofInitial
BloodFlow
57
Bloodshouldbeinspectedbeforetransfusionfor
possiblebacterialcontamination,hemolysis,visible
clots,brownorredplasma.
Segmentclosesttounitishemolyzed.Mayindicate
bacterialcontamination.
InspectionofDonorBlood
58
Ifaunit'sappearancelooksquestionabledothe
following:
Quarantineunituntildispositionisdecided.
Gentlymix,allowtosettleandobserveappearance.
Ifbacterialcontaminationissuspectedtheunit
shouldbeculturedandagramstainperformed.
Positivebloodculturesusuallyindicativeof:
Inadequatedonorarmpreparation.
Improperpoolingtechnique.
Healthofdonor bacteremiaindonor.
Ifonecomponentiscontaminated,other
componentspreparedfromthesamedonorunit
maybecontaminated.
DonorBloodInspectionandDisposition
59 60
391
Bacterialcontaminationofblood
componentscontinuestoposea
threattotransfusionrecipients
Progresstopreventadverse
reactiontobloodtransfusionsdue
tobacterialcontamination
continuestobeseen
Microbiologistsnowplayamajor
roleinthisprogress
Summary
61
392
MinistryofHealth
Screening & Confirmatory tests for TTDs
Institute Graduates
NAT CONFIRMATORAYTEST SCREENINGTEST TTDS
NAT WESTERNBLOT ELIZA
Chemiluminescent
HIV
NAT RIBATEST ELIZA
Chemiluminescent
HCV
NAT NEUTRILIZATION ELIZA
Chemiluminescent
HBV
WESTERNBLOT ELIZA
Chemiluminescent
HTLV
TPHA ELIZA,RPR, TPHA
Chemiluminescent
SYPHILIS
THICKFILM ELIZA,THICKFILM MALARIA
Screening&ConfirmatoryTestsforTTDs
2
3
ELIZATechniques Definitions
Antibodies(alsoknownas
immunoglobulins
abbreviatedIg)aregamma
globulinproteinsthatare
foundinbloodandare
usedbytheimmune
systemtoidentifyand
neutralizeforeignobjects,
suchasbacteriaand
viruses.
4
Antigens
A substance that when introduced
into the body stimulates the
production of an antibody
Immunoassay
A laboratory technique that makes use
of the binding between an antigen
and its homologous antibody in order
to identify and quantify the specific
antigen or antibody in a sample
Definitions(cont)
5
EnzymeLinkedImmunoSorbentAssay
UsedtodetectthepresenceofAg,AbfromtheSerum
Bymeasuringtheenzymaticactivitywhichinteracts
withsubstratethatchemicallychangedtoproduce
color.
Thedensityofcoloriseitherdirectlyorindirectly
proportionaltotargetaccordingtothetypeofELISA.
ThisEnzymemaybelinkedtoAgorAb.
Performedasasolidphaseassayinwhichthetargetto
bedetectedisboundtosolidsurface(microtitiration
well)
6
393
The techniques are divided into:
1 CompetitiveELISA
2 SandwichELISA(alsocalleddirectELISA)
3 IndirectELISA
TypesOfELISATechniques
7 8
The labelled antigen
competes for
primary antibody
binding sites with the
sample antigen
(unlabeled). The
more antigen in the
sample, the less
labelled antigen is
retained in the well
and the weaker the
signal).
CompetitiveELISA
IndirectELISAprotocol(forscreening
monoclonalantibodies
9
SandwichELISAProtocol
10
11
ChemiluminescentImmunoassays
Theprocessofchemiluminescenceoccurswhen
energyintheformoflightisreleasedfrommatter
duringachemicalreaction.
Lightemissionrangesfromquickburstorflashto
lightwhichremainsforalongertime.
Differenttypesofinstrumentsarerequiredbasedon
emission.
12
394
Canbeusedforheterogeneousorhomogeneous
assays.
Heterogeneousassaysusecompetitiveand
sandwichassay.
Competitiveassaysusedtomeasuresmaller
analytes.
Sandwichassaysareusedtomeasurelarger
analytes.
Manyapplications.
Canmeasureantigenorantibody.
Addchemiluminescentlytaggedanalyte.
Measurelightwhichisemittedwhichisdirectlyrelatedto
concentrationalthoughcompetitivebindingassaysare
available.
14
Notrequiredlongincubationtime.
Noadditionofstoppingreagent(colorimetric
methodisrequired).
Moreeconomicalcomparedtoconventional
colorimetricmethod(Ex:ELISA)
Moresensitivecomparetothecolorimetricmethod.
AdvantageofChemiluminescent
Immunoassays
15
TheRPRtestisanontreponemalcardaggluitination
testforserologicscreeningforsyphilis.
TheknownRPRantigenconsistsofcardiolipinand
cholesterolboundwithcharcoalparticles.
Charcoalmakesthereactionvisible.
Ifthedonorhassyphilis,theantilipidantibodies
(reagin)inhisserumwillcrossreactwiththeknown
RPRlipidantigensgivingavisibleclumpingofthe
charcoalparticles.
RapidPlasmaReagin(RPR)Test
16
Serum.
Plasma(centrifugetoremovefibrinbeforetesting).
Samplesnotsuitablefortesting:
Haemolysed.
Lipemic.
Highconc.ofBillirubin.
TypesofSamples
17
Confirmatorytestingisprimarilyconcernedwiththe
statusofthedonorandthesubsequentactiontobe
taken.
Donationsthatarerepeatreactivemaybe
confirmedasbeingofnegative,inconclusiveor
positivestatus.
Interpretation&UseofConfirmatoryResults
18
395
Anegativeconclusiononconfirmatorytesting
indicatesthatthedonorisnotinfectedwiththe
specificinfection.
However,adonorshowingrepeatreactiveresultson
screeningandnegativeresultsonconfirmatory
testingshouldbecounseledandtemporarily
deferreduntilscreennonreactiveonfollowup.
Thedonorcanthenbeacceptedforfuture
donations.
NegativeConclusion
19
Aninconclusiveoutcomeisusuallyduetonon
specificreactivitynotrelatedtothepresenceofthe
infectiousagent.
Thedonorshouldbecounseled,deferredforblood
donationandfollowedupforfurtherinvestigations.
InconclusiveOutcome
20
Apositiveconclusionconfirmsthatthedonoris
infectedandshouldbedeferredfromfutureblood
donation.
Thedonorshouldbecounseledandreferredfor
appropriatemedicalcare.
PositiveConclusion
21 22
AWesternBlotlooksforthe
presenceofantibodiesinthe
patientsbloodtocertain
componentsoftheHIVvirus.
Whenthoseantibodiesare
present,abandofcolor
appearsonthetestresult.
Tobeconsideredapositive
test,acertainnumberof
differentantibodies,and
consequentlycolorbands,
needtoappear.
Atestisconsidered
indeterminatewhensome,
butnotall,colorbands
appear.
WesternBlot
23
IndeterminateResult:some,butnotall,bandsare
present.
Causes:recentinfection,advancedHIV,certainstrainsof
HIV,crossreactiontootherantibodies,HIVvaccineOr
Laberror.
Resetin>6weeks.Riskcounselingifindicated.
WesternBlot
24
396
WesternBlot
25
Expensive.
Technicallymoredifficult.
Visualinterpretation.
Lackstandardisation:
performance
interpretation
indeterminatereactions
DisadvantageofWesternBlot
26
ContainsthestructuralandnonstructuralHCV
antigens.
IndividualHCVantigensaredisplayedona
nitrocellulosestrip.
AntibodiesagainstspecificHCVantigenscanbe
identified.
RecombinantImmunoblotAssay(RIBA)
27
ApositiveRIBAassayrequiresatleasttworeactivebands.
Testswithonlyonereactivebandareconsidered
indeterminate.
ThesensitivityofRIBAtestsarenothigherthansensitivityof
EIAtests
IfNATtestgivesthenegativeresult,RIBAtestscanbeusedto
distinguishfalsepositiveEIAresultsfrompriorexposureto
HCV.
ResultsofRIBAs
28
DisadvantageofRIBAAssay
DetectonlyIgG antibodies.
RIBAsaretechnicallymoredemandingthanELISA.
Manyintermediateresults.
Highcost.
29
NeutralizationTest
Inthefirststage:SampleallowedtoreactwithspecificAb
boundtoasolidphase,whereuponeitherthecomplexsolid
phaseAb/AgorthecomplexsolidphaseAb/interfering
substanceisformed.
Inthesecondstage:AspecificunlabeledantiHBsAbis
allowedtoreactwithoneofthetwoduplicateseries,whilea
serumnonreactiveforantiHBsAbisallowedtoreactwith
thesecondduplicateseries.
30
397
NeutralizationTestcont..
Inthethirdstage:ThelabeledantiHBsAbisaddedtoboth
duplicate.Ifthepositivereactioninthescreeningtestwas
causedbyaninterferingsubstance,theunlabeledspecificAb
willnotinhibitthereactionofthelabeledAbwiththesolid
phasecomplex.Conversely,ifthepositivereactioninthe
screeningtestwascausedbythepresenceofHBsAg ,the
unlabeledspecificAbwillinhibitthebindingbetweenthe
labeledAbandthesolidphasecomplex.
Areductionofthevalueoftheneutralizedsamplewith
respecttothevalueofthenonneutralizedsamplewill
confirmthepresenceofHBsAg.
31
TPHATestForSyphilis
the SyphilisTPHAtest isaclassic,indirecthemagglutination testusedfor
thedetectionandtitrationofantibodiesagainstthecausativeagent
of syphilis,Treponema pallidum.
Inthetestredbloodcells(erythrocytes)aresensitizedwithantigens
from T.pallidum.Theerythrocyteswillthenaggregatetogethertoform
distinctivepatternsonthesurfaceofamicroplate wellswhenexposedto
syphiliticserum.
32
ThickBloodFilm
NumerousringformofPlasmodiumcanbeseenasindicated
bythearrows.
Notesizeofneutrophils (forcomparison
Theonlydefinitivediagnosisthatcanbemadefromthisfilm
isthatMalariaispresent.
Thickfilmconsideredgoldstandardfor
detectionofparasitesduetobeingabletouse
largervolume(10lofblood).
33
Preparingthickfilms
5.
Touchthedropof
bloodtotheslide
frombelow.
4.
Slidemustalwaysbe
graspedbyitsedges.
2.
Punctureattheside
oftheballofthe
finger.
3.
Gentlysqueeze
towardthe
puncturesite.
1.
Thesecondorthird
fingerisusually
selectedandcleaned.
6.
Spreadthefirstdrop
tomakea1cm
circle.
34
Adropofbloodisspread
overasmallarea.Whendry,
theslideisstainedwith
FieldsorGiemsa stains.The
redcellslyseleavingbehind
theparasites.
Usedtodetect
parasites,evenif
parasitaemia islow
Lessusefulfor
speciation
Back
Thickbloodfilm
35
Microscopy TheGoldStandard
Benchmarkdiagnosticstandardforover100years.
Inexperthands:highlysensitive,specific.
Resultsprovideawealthofclinicallyimportantdata.
Stainedslideservesaspermanentrecord.
36
398
MicroscopyLimitations
Microscopyskillsmaybelackinginareasnotroutinelydoing
malariaevaluations
Smearpreparation,staining
Interpretation
Mixedinfections canbedifficulttodiagnose.
Lowparasitemia canbedifficulttodiagnose.
Handsontimeisveryhigh.
37
399
MinistryofHealth
NAT & Blood Safety
Institute Graduates
Transfusionofbloodandbloodcomponentsisan
importantissueofanyhealthcaresystem.
Itisnecessarytoensurethatblood&blood
componentsadministeredare100%safe.
Transfusiontransmittedinfectionsarebyfar,the
morecomplexandvexingproblemfacedbyBTS.
2
Introduction
Morethan92millionblooddonationsarecollected
globallyperyear
1
Annually,unsafebloodtransfusionsareestimated
havebeenresponsibleforupto:
16millionnewHBVinfections
5millionnewHCVinfections
160,000casesofHIVinfections
GlobalBloodSafetyandAvailability:FactsandFiguresfromthe2007
BloodSafetySurvey,WHOFactSheet#279,Nov.2009.
3
TransfusionTransmittedInfectionsAreAReal
Concern
ScreeningforTTIstoexcludeblooddonationsat
riskoftransmittinginfectionfromdonorsto
recipientsisacriticalpartoftheprocessofensuring
thattransfusionsareassafeaspossible WHO,
2010.
4
TheLancet
ImprovingBloodSafetyWorldwide
Developingcountriesaremorelikelytouseblood
thatiscontaminatedthanindustrializednations:
o Higherdiseaseprevalence
o Useofpaid,family/replacementdonors
o Concealingmedicalhistoryand/orriskybehaviorin
questionnaire
o Inadequateserologybasedscreening
AsianJTransfusionSci.;v4(2),July2010
TTIRisks&ImplicationsinDeveloping
Countries
5 6
BloodSupplySafety
TheGoal
Preventingtransfusiontransmissionofbloodborne
pathogens.
OverallImpact
Asinglewholeblooddonationcanbetransfusedto
4recipients.
Maybeaddedtopoolsofmorethan1,000unitsto
manufacturebloodderivatives.
400
NucleicAcidAmplificationTesting(NAT):
NATisamoleculartechnologythatfocusedonthe
detectionofviralDNAorRNAofintendedviruses.
NATutilizeseitherPolymeraseChainReaction
(PCR)orTranscriptionMediatedAmplification
(TMA)methodspermittingtheamplificationof
viralsequencesinvitro.
Highlysensitiveandspecifictechnique.
Fullyautomatedtechnique,eitherbasedon
individualtestingorpoolingsystem.
7
WhatisNAT?
8
AdvantagesofNAT
Highlysensitive&specific.
Targetsspecificviralnucleicacidsequences.
DirectdetectionoflowlevelofviralRNAorDNA.
ShortenstheWindowPeriodfrominfectionto
detection.
Helpspreventtransfusiontransmitteddisease.
Providesadditionallayerofsafetytotheblood
supply.
Improvesconfidenceinbloodsupply.
Antibody negativewindow
NAT negative
window
INFECTION Detection
minipool
Seroconversion
Detection
Single
donation
9
WhydowedoNAT?
10
WindowperiodWP
ThemostimportantfactorforTTIsresidualrisk.
TheWPisdefinedasthetimefrominfectivitytotest
reactivity.
Thechanceoftransmissionisafunctionofboth
incidenceandlengthofWP.
Bloodtransfusionauthoritiesandbloodbankswere
concernedabouttheabilitytoclosethegapof
windowperiodbyadditionalstepstoensure
qualityandsafetyofbloodandbloodproducts
All volunteer donors
HBsAg test
AI DS high-risk exclusions
Anti-HI Vtest
ALT/ HBcAb tests
Anti-HCVtest
Improved
HCVtests
1965 1970 1975 1980 1985 1990 1995 2000
Year of Transfusion
%
R
e
c
ip
ie
n
t
s

I
n
f
e
c
t
e
d

B
y

B
lo
o
d
B
o
r
n
e
D
is
e
a
s
e
s
25
20
15
10
5
0
NAT
Implementation
11
StepstoSafety
Viral
RNA/DNA
Detection
ViralAntigen
Detection
Antibody
Testing
Surrogate
Marker
SerumALT
Tcellcount
AntiHIV
AntiHBc
AntiHCV
AntiHTLV
HIVp24Ag
HBsAg
HCVAg
NAT
HIV1/2
HCV
HBV
NATistheonlydirecttestfortheinfectiousagent.
Shorterwindowperiodtodetection
12
ProgressinPathogenDetection
TransfusionTransmittedPathogens
401
Source: Busch et al. Transfusion.2005;45(2):254-264. Kleinman and Busch. Transfusion.
2006;36:S23-S29
13
WindowPeriod
Residualrisk=
Incidencerate windowperiodduration
Incidencerate=
Seroconversions /Person/Years
15
SourcesofResidualRisk
Windowperioddonations.
Viralvariantsnotdetectedbytraditionalserological
tests.
Immunosilent donors.
Laboratorytestingerrors.
16
PrerequisitestoperformingNAT
Serologicalscreeninginplaceandeffective.
Technicaldevelopmentatmolecularlevel(trained
staff).
Indepthtrainingofstaff.
Sufficientbloodsupplytoaffordquarantining.
Donatedoraffordableequipment.
Supply,transport&storageofreagentscoldchain.
Methodsadaptedtosmallorlargenumbers,
affordable.
1. Individual sample screening
- Duplex HCV/HIV-1 RNA . Expensive.
- Triplex HCV/HIV/HBV . Sensitive.
. Specific.
2. Minipool screening (pools of 96, 48, 24, 16, 10, 8, 6)
- Duplex HCV/HIV, Triplex HCV/HIV/HBV
. Less expensive
. Less sensitive
. Less specific?
All commercial duplex or triplex requireidentification of positives with three
discriminatory singlevirus assays except Roche.
17
TestingmodesforNAT
Single Unit
NAT
Mini Pooled
NAT
Serology
testing
1:3,000,000 1:1,900,000 1:1,300,000 HIV
1:2,300,000 1:1,600,000 1:230.000 HCV
1:410,000
Data from FDA December 2001 Workshop:
Presented by M. Busch, M.D. (Current risk
estimates in bold)
1:210,000 1:180,000 HBV
18
CalculatedRisk/UnitTransfused
402
Country Author Serology Mode NAT Yield
Brazil Levi HIV MP6-12 -
Mexico Chiquete
HBV IDT -
HCV IDT -
Mexico Garcia-Montalvo HBV IDT <1:865
Lebanon El-Zaatari HBV IDT 1:501
Lebanon Ramia HBV IDT 1:358
Mongolia Tsatsralt-Od HBV IDT 1:81
China Ren HBV MP8 1:1,430
Malaysia Lam HBV IDT 1:3,616
India Makroo
HBV IDT 1:2,037
HIV IDT 1:6,112
HCV IDT -
India Chaudhuri HBV IDT <1:617
Iran Behzad-Behbahani HBV IDT 1:125
Kuwait Al Radwan HBV - 1:24,275
19
PublishedYieldCases DemonstratedNATYieldin
DevelopingCountries(Ekiaby etal.2010)
TranscriptionMediated
Amplification
PolymeraseChain
Reaction
Idea
Theamplificationis
performedbymakingmany
RNAtranscriptionsat1fixed
temperature
(41.5C Isothermal
Reaction)
Theamplificationis
performedbychainof
reactionsat3different
temperatures
(94Cthen50 60Cthen
72C thermoCycling)
Amplified
product
RNA
(Labileinenvironment
Minimalcontamination
issues.)
(stableinenvironment
Veryproneto
contamination)
Amplified
Productfor1
Cycle
100 1000Amplicons pilcons
20
TMAVsPCR
TMA PCR
After1Hourof
Amplification
Approximately:abillionfoldof
RNAcopies
Approximately:1millionof
copies
Detection
Chemiluminescentdetection
(2timesofmagnitudemore
sensitivethanFluorescence)
Fluorescencedetection
RESULT
Therearebillions sitesof
detectionlabeledwith
Chemiluminescence Molecule.
(moredetectionsensitivity)
Therearemillionsiteof
detectionlabeledwith
FluorescenceMolecule.
21
TMAVsPCR
Cobas S201/MPX /ULTRIOPlus
LimitofDetection
95%
11IU/mL 3.1IU/mL HCV(AllGenotypes)
3.8IU/mL 2.1IU/mL HBV(AllGenotypes)
49IU/mL
27.6IU/mL
HIVM
152Copies/mL
HIVO
NotAvailable
HIVN
2.2Copies/mL NotAvailable
HIV2
22
TMAVsPCR
23
LimitationsofcurrentHIVNAT
TheUltrioAssayDetectsonlyHIV1RNA
HIVNRNAarenotdetectedbytheTaqscreenMPX
(Roche)
HIVNATdoesnotdetectHIVDNA
CurrentantiHIVassayscoverallHIVtypes
HepatitisBvirus(HBV)isamajorhumanpathogen
thatcausesacuteandchronichepatitisinhumans.
Mostprimaryinfectionsinadultsareselflimited,
thevirusisclearedfrombloodandliver,and
individualsdevelopalastingimmunity.
24
HepatitisBvirus(HBV)
403
Lessthan5percentofinfectedadultsdevelop
persistentinfectionsthatcanbeasymptomatic(i.e.a
carrierstate).
Twentypercentofchronicallyinfectedindividuals
candevelopcirrhosis.
Chronicallyinfectedsubjectshave100timeshigher
riskofdevelopinghepatocellularcarcinomathan
noncarriers.
25
ChronicHepatitisB
PresenceofHBVDNAintheliver(serum)ofindividualswith
undetectableHBsAg withcurrentlyavailableassays
SeropositiveOBI(antiHBc+;antiHBs)
Seronegative (antiHBc andantiHBsnegative)
Tobedistinguishedfrom:
Primaryinfectionwindowperiod
SproteinescapemutantsundetectedbyHBsAg assays
Genomicassaycontamination
26
DefinitionofOBI
(Taorminaworkshop03/2008)
27
MechanismsforOBIoccurrence
LackofHBsAg production:primaryOBI
Incompletecontrolofhostimmunesystem
InHBVgenotypeA2,B,C,D
EscapemutantswhenantiHBspresent
AccumulationofrandommutationsinantiHBc
Deficiencyorinhibitionofviralreplication/gene
expression
IngenotypeA1andEfordeficiency
InallgenotypesforinhibitionofHBsAg expression
Vaccinebreakthroughacuteinfections
28
TestingforbloodHBVsafety
1. HBsAg
o widevariationinsensitivity
o generallyexcellentspecificity
o Inexpensive
2. AntiHBc
o lowspecificity,recentlyimproved
o needconfirmationwithalternativetest(s)
o Inexpensive
3. NAT
o variablesensitivity550IU/mlor35350copies
o excellentspecificity
o expensive,delicate,contamination,timeconsuming.
29
EfficacyofantiHBcvsNAT
TypeofHBV
Infection
HBsAg AntiHBc NAT*
WindowPeriod No No Yes
PrimaryOBI No No Yes
2dWP No Yes Yes
Chronic No Yes Yes
AntiHBc+OBI No Yes Yes
AntiHBsonlyOBI No Yes Yes
ChronicNAT Yes Yes No
30
404
31
Results
HBsAg+orantiHBc+/ antiHBs=Windowperiod
Repeatinitialresults=confirmpreliminarydiagnosis
HBVDNAundetected
Fluctuation
Contamination
AntiHBsundetected=potentialfluctuation
Allnegative=contamination
Conclusions
BloodsafetycanbeimprovedbyNAT.
NATcannotbetheonlybloodscreeningtest.
Multiplexingincreasescostefficiency.
Poolingdecreasescostbutalsosensitivity.
NATdetectsWindowPeriodforHIV,HCV,HBV.
NATalsodetectsHBVOBI.
OBIsaremultifacet&difficulttodiagnose.
OBIscanbeinfectiousevenwhenantiHBspresent.
32
405
Ministry of Health
Hematology
406

Hematology

Introduction to Hematology
- Slide 8 Stability of anti-coagulated blood
Accuracy of results for EDTA bloods reduces dramatically after 4 hours in the fridge

407
MinistryofHealth
Institute Graduates
IntroductiontoHematology
Contentoutline
Main Function of hematology
Development of blood cells
Anticoagulants used in hematology
Stability of anticoagulated blood
Blood Film :
Value
Preparation
Staining
2
3
Nutrients absorbed fromthe digestive tract, e:g. monosaccharides
(especially glucose), amino acids, fatty acids, glycerol, and
vitamins; are transported to
Waste products of metabolismare transported fromthe tissues to
site of excretion, e.g carbon dioxide produced fromcellular
activity is carried to the lungs for excretion, and the waste
products of protein metabolism(urea, creatinine, uric add) are
transported to the kidneys for excretion.
Hormones are carried fromendocrine glands to the organs where
they are needed.
Continued
4
Developmentofbloodcells
5
EDTA (ethylene diamine tetra-acetic acid), also. called
sequestrene, and tri-sodiumcitrate. These chemicals prevent
blood fromclotting by removing calcium.
EDTA anticoagulated blood can be used for most tests, e.g.
haemoglobin, PCV,WBC count platelet count reticulocyte
count; and reporting blood cell morphology. It is not suitable
for coagulation tests. The ICSH, (International Committee for
Standardization in Haematology) recommends the use of
dipotassiumEDTA at a concentration of 1.5 O.25' mg\ml of
blood.
.
Anticoagulants Used in Hematology
6
408
Trisodium citrate 32 g\l is used to anticoagulate
blood for:
Measuring the ESR, with 1.6 - ml of venous blood
(or previously collected EDTA blood) being mixed
with 0.4 ml of sodium citrate anticoagulant.
Coagulation tests, with 9 ml of venous blood being
mixed with 1 ml of sodium citrate anticoagulant.
Continued
7
Stability of anti-coagulated blood
8
EDTA anticoagulated blood
When EDTA anticoagulatedblood cannot be tested within
1-2 hours it must be refrigerated at 4-8 C to prevent cellular
changes affecting test results.
Manual or automated blood cell counts, reticulocyte
counts, and PCV change little in EDTA blood at 4-8
0
C
when stored for up to 24 hours .
Hemoglobin concentration is stable for 2-3 days at 4-8 C
providing there is no hemolysis
Important
Blood which has been refrigerated must be allowed to warmto roomtemperatureand
bewell mixed beforebeing tested.
In EDTA anticoagulated blood, morphological blood cell changes occur soon after
blood is collected when is stored at roomtemperature(18-25
0
C) and within 3 hours
when stored at 4-8 C
It is thereforerecommended that blood films bemadeand methanol-fixed as soon as
possibleafter blood is collected and never madeafter overnight storage.
Continued
9
Someof theblood cell changes which occur in EDTA blood include:
. Neutrophil degeneration with neutrophils becoming moreirregular in shape,
nudear lobes separating, and vacuoles .appearing in thecytoplasm. Thereis also loss of
granules.
Segmentation (budding) of thenucleus of lymphocytes and monocytes and vacuoles
appearing in thecytoplasm.
Erythrocytes becoming crenated and spherocytic.
Platelets disintegrating
Continued
10
Citrate anti coagulated blood:
Even when citrated blood is stored at 4-8 C there is a decrease in the
ESR due to changes in erythrocyte shape affecting rouleaux. The
ESR should be measured within 4 hours. of collecting the blood.
Coagulation tests should be carried out as soon as possible after
blood is collected into citrate anticoagulant.
Continued
11 12
409
Important:
Reliablebloodfilmreportingisonlypossiblewhenlaboratory
staffaretrainedadequatelyintherecognitionofbloodcellsand
followstandardizedproceduresforpreparingandstainingblood
films,reportingmorphologicalchangesandperforminga
differentialwhitecellcount.
Continued
13
Romanowskystains
ThesestainscontaineosinYwhichisanacidicanionicdye
andazureBandotherthiazinedyes(derivedfromthe
oxidation,orpolychroming,ofmethyleneblue)whichare
basiccationicdyes.Whendilutedinbufferedwater,
ionizationoccurs.Eosinstainsthebasiccomponentsof
bloodcells,e.g.haemoglobinstainspinkred,andthe
granulesofeosinophilsstainorangered.AzureBand
othermethylenebluederiveddyes,staintheacidic
componentsofcells.
Bloodfilmsstaining
14
Nucleicacidsandnucleoprotein,stainvariousshadesofmauve
purpleandviolet
Thegranulesofbasophilsstaindarkblueviolet,andcytoplasmof
monocytesandlymphocytesstainsblueorbloodgrey.The
stainingreactionsofRomanoskystainedaredependentwhichis
whythestainsaredilutedinbufferedwaterofspecificpH
Continued
15
Note:
ThereissomevariationbetweenbatchesofRomanoskystains
duetothedifferentthiazinesandirontiestheycontain.Ahighly
purifiedRomanoskystainbeendevelopedwhichcontainsonly
azureBandeosinY.IrecommendedbyICSHforstandardized
Romanostainingbutitisveryexpensiveandnotneededforrout
work.
.
Continued
16
Manyofthedifficultiesinreportingbloodfilms,particularlyredcell
morphology,areduetovariablestaining.Itisimportanttherefore
forlaboratoriesuseareproduciblestandardizedstainingtechnique.
Leishman stainingtechniquecont.
Continued
17
Redcells.Pinkred cont.
NucleusofcellsPurpleviolet
Cytoplasm
Neutrophils,eosinophils Palepink
LargeLymphocytes Clearblue
Smalllymphocytes.... Darkerdearblue
Monocytes Greyblue
Stainingresults
Continued
18
410
Granules
Eosinophils.....................Orangered
Neutrophils.Mauvepurple
ToxicgranulesDarkViolet
Basophils..Darkblueviolet
Platelets:.Purple
Continued
19
Inclusions:
Malariapigment(inmonocytes)'Brownblack
HowellJollybody,Purpleviolet
Auerbody(inmyeloblast):Purplered
Wright'sstainissimilartoLeishman stain*
*Adifferentpolychroming techniqueisusedintheproductionOfWright'sstainandnuclear
stainingisusuallypalerthan
Continued
20
411
MinistryofHealth
Graduates
RedCells
RedCells
(erythrocytes)formthemaincellular
cormponent ofblood,i.e.about450/0oftotal
bloodvolumeinanadult,givingblooditsred
coulor.Eachlitre ofbloodcontainsabout5x
10
12
redcells,theexactnumbervaryingwith
age,gender,andstateofhealth
Productionofredcells Continued
Redcellsareproducedinthebonemarrow
Tissuehypoxia(lackofoxygen)leadstothe
releaseofthehormoneerythropoietin
whichstimulatesprogenitorcellsBFUEand
CFUE,todevelopintopronormoblasts
(proerythroblasts).
Developmentinbonemoitiw (about5days)
Continued
BFUE
CFUEPronormoblast Early
Normoblast
Late
normoblast
Reticulocyte
Bloodcirculation
Normalbloodalsocontains
Upto2%reticulocytes
Erythrocyte
Note:Normalerythropoiesis isdependentontherebeingadequate
amountsoferythropoietin,proteinandparticularlyitemvitaminB12
andfolicacidwhichareessentialforhemoglobinsynthesis.
Reticulocyte count
Valueoftest :Reticulocytes areimmature,redcells
normallypresentinsmallnumbersintheblood(up
to2%).Reticulocyte numbersincreasewhen.there
isanincreaseinerythropoietinactivity.
Areticulocyte countassessesbonemarrowactivity,
e.g.whetherthereisaneffectiveerythropoietic
responsewhenthereisareductioninthenumberof
redcellsduetohaemolysis orhaemorrhage.
Areticulocyte countisalsoofvalueinmonitoring
theerythropoietic responseofananaemic patient
followingtreatment
412
Hemoglobinsynthesis
ThemainfunctionofredcellsistocarryO
2
tothetissues
andtoreturncarbondioxide(C0
2
)fromthetissuestothe
lungs.Inordertoachievethisgaseousexchangethey
containthespecializedproteinhaemoglobin.Eachredcell
containsapproximately640millionhaemoglobin
molecules.Eachmoleculeofnormaladulthaemoglobin
(Hb)A(thedominanthaemoglobin inbloodaftertheage
of36months)consistsoffourpolypeptidechains,a.2~2'
eachwithitsownhaem group.Themolecularweightof
Hb Ais68000.Normaladultbloodalsocontainssmall
quantitiesoftwootherhaemoglobins:Hb FandHb
A
2
Thesealsocontainachains,butwithyandI)chains,
respectively,insteadofB
Continued
Haemoglobin synthesisinthedevelopingredcell.Themitochondria
arethemainsitesofprotoporphyrin synthesis,iron(Fe)issupplied
fromcirculatingtransferrin;globin chainsaresynthesizedon
ribosomes.bAl.A,l)..aminolaevulinic add;Coa,coenzymeA.
NormalHemoglobinTypes PCVandredcellindices
Valueoftest:Thepackedcellvolume(PCV)also
calledhaematOOit,isusedtocalculatethemean
cellhaemoglobin concentration(MCHCand
meancellvolume(MCV).Theseredcellindices
areusedintheinvestigationofanaemia.TheTV
isalsousedtoscreenforanaemia whf not
Possiblle tomeasurehaemoglobin,andto.
diagnosepolycythaemia vera andtomonitorits
treatmentissuitableforscreeningfarge dinic
Populations,e.g.antenatalclinics.
Continued
Thepackedcellvolumeisthat
proportionofwholeblood
occupiedbyredcells,expressed'as
a'ratio,(Iitrellitre).whenusingan
electroniccellanalyter which
computesthevaluefromtheMCV
andredcellCount
(PCV=MCVxRBC).
Measuringredcellsindices
Redcellindicesmostfrequently'usedinthe
invesgation ofanaemia are:
Meancellhaemoglobin concentration(MCHC)
Meancellvolume(MCV)
Meancellhaemoglobin (MCH)
413
MCHC
TheMCHCgivestheconcentrationof
haemoglobin gld inlitre ofpackedredcells.It
iscalculatedfromthehaemoglobin (Hb)and
PCVasfollows:
Hbg/l=MCHCg/l
PCV(l/I)
MCV
Themeanredcellvolume(MCV)
providesinformationonredcellsize.Itismeasured
infemtolitres (fl)andisdeterminedfromthePCV
andelectronicallyobtainedRBCcount.Itcanbe
calculatedasfollows:
PCV(l\l)=MCVfl
RBC10
12
/l
.
MCV
TheMCHgivestheamountofhemoglobinin
picograms (pg)inanaverageredcell.Itis
calculatedfromthehemoglobinand
electronicallyobtainedRBCcount:
Hb ing\l
RBCx10
12
/1=MCHpg
Apicogram (pg)is10
12
ofagram.
Disorderofredcells
Themaindisordersofredcellsare:
Anaemia
Haemoglobinopathies (thalassaemias,abnormal
haemoglobins).
Disordersduetoredcellenzymedefects,eg G6PD
deficiency
Disordersduetoredcellmembranedefects,eg
hereditaryspherocytosis
Polycythaemia
414
MinistryofHealth
Institute Graduates
Anemia
2
Anemia
Worldwideanaemia isthecommonestredcelldisorder.It
occurswhentheconcentrationofhaemoglobin fallsbelow
whatisnormalforapersonsage,gender,andenvironment,
resultingintheoxygencarryingcapacityofthebloodbeing
reduced.
Haemoglobin valuesarelowerinwomenthanmenprobably
duetomenstruallossandtheinfluenceofhormoneson
erythropoiesis.Hemoglobinlevelsfallinnormalpregnancy
duetoanincreaseinplasmavolume.
3
Morphologicallyanemiacanbeclassified
by:
Redcellsizewiththeterms:
Normocyticreferringtonormalsizeredcells
(approximately8umindiameter)
Microcyticreferringtosmallerthannormalredcells
Macrocyticreferringtolargerthannormalredcells.
4
Haemoglobinizeltion ofredcellswiththe
terms:
Normochromic,describingnormalstainingofred
cellsasseenwhenhaemoglobinization isadequate.
Thecellscontainasmallareaofcentralpallor(no
morethanonethirdofthecell'sdiameter)dueto
thebiconcavityofredcells.
Hypochromic,describingpalestainingofredcells,as
seenwhenhaemoglobinization isinadequate.
Hypochromic cellsshowanincreasedareaofcentral
pallor.
5
Microcytichypochromicanemiacausedby:
Irondeficiency(commonestcause)
Thalassaemia syndromes
Someanaemias ofchronicdisease
Sideroblastic anaemia*
6
MacrocyticAnemiaCausedby:
Megaloblastic changes:
Folatedeficiency
VitaminB
12
deficiency
415
7
Normocyticchangescausedby:
Liverdisease
Alcoholism
Haemolyticanaemia(associatedwithraised
reticulocytecount.)
8
NormochromicAnemia
Inanormocytic normochromic anemiatheredappear
normocytic andnormochromic inastrainedbloodfilmand
theMCHC;MCVandMCHarenormal.
Anormocytic normochromic anaemia may,foundin:
Acutebloodloss
Anaemia ofchronicdisease
Aplastic anaemia
9
Haemolyticanaemias
Haemolyticanaemiasarecharacterizedbya
fallinghemoglobin,jaundice,darkurine,
increasingreticulocytosis(whenthereis
effectiveerythropoiesis)andusually
splenomegaly.
10
Bloodfilmfindings
11
Redbloodcell(RBC)inclusions
Maybeseenintheperipheralbloodfilminvarious
conditions
The reticulocyteRNAandHeinzbodiesareonly
demonstratedbysupravital staining(e.g.withnew
methyleneblue
Heinzbodiesareoxidizeddenaturedhaemoglobin.
Siderotic granules(Pappenheimer bodies)containiron.
Theyarepurpleonconventionalstainingbutbluewith
Perls'stain
TheHowellJollybodyisaDNAremnant.
BasophilicstipplingisdenaturedRNA.
12
416
13
HEREDITARYREDCELLMEMBRANE
DISORDERS
Hereditaryspherocytosis:
Whichusuallycausesonlyamildchronic
haemolytic anaemia withsplenomegaly.
Obstructivejaundiceduetogallstonesisa
commoncomplication.Redcellshavea
reducedlifespan.Anaemia canbecomemore
severewhenthereisalsofolate deficiency,
anaemia ofchronicdiseaseandaplastic crisis
inducedbyparvovirusB
19
infection.
14
Continued
Thebloodfilmshowsspherocytes (whichmaybescanty)
withpolychromasia.
Theosmoticfragilitytestshowsincreasedfragilityofthe
cells,particularlyfollowing24hincubation.
Thedirectantiglobulin test(DAT)isnegativewhichhelps
todifferentiatethisconditionfromimmunecausesof
spherocytosisinwhichtheDATispositive.
15
Hereditaryelliptocytosis
Maycausemildanaemia Inhomozygotes.Itis
foundinWestandNorthAfrica.Ahemolytic
crisismayoccurduringsevereinfections,eg
malariaTheblood,typicallyshowselliptocytes
(elongatedredcells)andalsoredcell
fragments
16
Hereditaryovalocytosis
A commondisorderinSoutheastAsia,
Indonesia,PhilippinesandMelanesia.Itis
usuallyanasymptomaticcondition.Theblood
filmcontainslargenumbersovalocytes
17
(a)Thebonemarrowaspirationneedle andasmear
madefromabonemarrowaspirate.(b)Thebone
marrowtrephine(biopsy)needle andnormaltrephine
section.
417
MinistryofHealth
Institute Graduates
AbnormalHemoglobins
Abnormal Haemoglobins
HbS which has a wide distribution in tropical Africa,
parts of India, the Caribbean Mediterranean region,
Arabian peninsula, arid elsewhere in people of
African descent
HbC which is found only in West Africa and else
where in people of West African descent
HbE which is found in Southeast Asia and the Indian
subcontinent.
2
Alkalinecelluloseacetateelectrophoresis
Used to separate and idetify the different
hemoglobins by there migration within an electric
field . Hemoglobin variants separate at different
rates due to differences in their surface electrical
charge as determined by their amino acid structure
3
Severaltechniquesareavailabletoseparate
haemoglobin variantsbyelectrophoresis.For
routinework,electrophoresisinanalkalinebuffer
atpH8.48.6usingacelluloseacetatemembrane
isadequate.
ThisgivesgoodseparationofHbA,HbF,Hbs,andHbC
Cont.
Continued
4
Continued
5
OnalkalineelectrophoresisHbDandHbShave
thesamemobilityandHbC,HbEandHbOalso
comigrate.Inspecialistlaboratoriesagarose
gelelectrophoresisatanacidpH(6.0)canbe
usedtoseparatethesehaemoglobinsandalso
toprovideaclearseparationofHbFfromHbS
andHbC
Anelectrophoresischamber(tank),model
6
PlateS.S(a)HelenaBioSciencesZipZoneelectrop,.chambersuitableforalkaline
celluloseacetatehaemelectrophoresis
418
Continued
7
(b)Applicatorsystem
usedwiththe
electrophoresis
chamber.Courtesyof
HelenaBioSciences
G6PDDeficiency
8
G6PDdeficiencyisthecommonestcauseofthe
inheritedredcellenzymedisorders.Deficiency
oftheenzymeglucose6phosphate
dehydrogenase(G6PD)inredcellsreducesthe
abilityofthecellstowithstandlysis from
oxidantdamage,particularlyduringinfections
andfollowingexposuretooxidantdrugsor
chemicals.
419
MinistryofHealth
Institute Graduates
TheWhiteCells
Granulocytes,monocots
2
Whitebloodcells Normalbloodcounts
4
Adults Bloodcount Children Bloodcount
Totalleucocytes
Neutrophi1s
Eosinophils
Monocytes
Ba50phils
Lymphocytes
4.0011.0xH)9/L*
2.57.5x109/L*.
040.4x109/L
0.20.8x109/L
0.010.1x109/L
1.53.5x109/L
Totalleucocyies
Neonates
1year
47years
812years
10.025.0X10
9
/L
6.018.0x10
9
/L
6.015.0x10
9
/L
4.513.5x10
9
/L
Abnormalwhitebloodcells.
5
Continued
6
(a)Neutrophilleucocytosis:
Toxicchangesshownbythepresenceofredpurplegranulesin
thebandformneutrophils.
Dahle bodycanbeseeninthecytoplasmoftheneutrophil.
420
(c)Megaloblastic anaemia:
hypersegmented oversizedneutrophilin"peripheralblood.
(d)MayHegglin anomaly
theneutrophilscontainbasophilicinclusions25indiameter
thereisanassociatedmildthrombocytopeniawithgiant
platelets.
Continued
7
(e)PelgerHuet anomaly:
Coarseclumpingofthechromatinin configuration.
(f)ChediakHigashisyndrome:
Bizarregiantgranulesinthecytoplasmofamonocyte
(g)Alder'sanomaly:
Coarsevioletgranulesinthecytoplasmofaneutrophil.
Continued
8
421
MinistryofHealth
Institute Graduates
Lymphocytesandtheirbenigndisorders
Lymphocytesandtheirbenigndisorders
Lymphocytes :
In postnatal life, the bone marrow and thymus are the
primary lymphoid organs in which lymphocytes develop. The
secondary lymphoid organs in which specific immune
responses are generated are the lymph nodes, spleen and
lymphoid tissues of the alimentary and respiratory tracts.
2
Lymphocytes:
(a)smalllymphocyte;
(b)activatedlymphocyte;
(c)largegranularlymphocyte;
(d)plasmacell.
3
Lymphocytosis
Lymphocytosisoftenoccursininfantsandyoung
childreninresponsetoinfectionsthatproduce
neutrophilreactioninadults.
4
Causesoflymphocytosis
Infections
Acute: infectious mononucleosis, pertussis, mumps,
acute infectious lymphocytosis, infectious hepatitis'
cytomegalovirus, HIV, herpes simplex or zoster
Chronic: tuberculosis, toxoplasmosis, brucellosis. syphilis
Chronic lymphoid leukaemias
Acute lymphoblastic leukemia
NonHodgkin's lymphoma (some)
Thyrotoxicosis
5
TheSpleenfunctionsofthespleen
The spleen is the largest filter of the blood it and
several of its functions are derived from
Control of red cell integrity
The spleen has an essential role in the control of red
cells.
6
422
Continued
Extramedullary haemopoiesis
The spleen like the liver undergoes a transient
period of haematopoiesis at around 37 months of
fetal life but is not a site of erythropoiesis in the
adult. However,haemopoiesis may be restablished in
both organs as extramedually haemopoiesis
7
Indisorderssuchasmyelofibrosis orinchronicsevere
haemolytic andmegaloblastic anaemias.Extramedullary
haematopoiesis mayresulteitherfromreactivationof
dormantstemcellswithinthespleenormovingofstemcells
fromthebonemarrowtothespleen
Continued
8
Continued
The lymphoid tissue in the spleen is in a unique position to
respond to antigens filtered from the blood and entering
the white pulp. Macrophages and dendritic cells in the
marginal zone initiate an immune response and then
present antigen to B and T cells to start adaptive immune
responses
9
423
MinistryofHealth
Institute Graduates
Leukemia
Leukemia
The leukaemias are a group of disorders
characterized by the accumulation of malignant
white cells in the bone marrow and blood.
2
Classificationofleukemia
Acute
Acute myeloid leukaemia (AML): MoM
7
Acute lymphoblastic leukaemia (ALL): L
1
L
3
Chronic
Chronic myeloid leukaemias (CML)
Chronic lymphoid leukaemias (CLL)
3
Continued
4
AML Cytogenetic
Abnormality
M
o
undifferentiated
M
I
.withoutmaturation
M
2
withgranulocyticmaturationt(8,21)
M
3
acutepromyelocytic t(15%17)
M
4
granulocyticandmonocytic maturation inv(16)
M
5
monoblastic (M
5a
)ormonocytic (M
5b
)
M
7
megakaryoblastic.
Continued
5
ALL:
L
1
:blastcellssmall,uniformhighnuclearto
cytoplasmicratio.
L
1
:blastcellslarger,heterogeneous,lower
nucleartocytoplasmicratio.
L
3
:vacuolatedblasts,basophiliccytoplasm
(usuallyBALL)
DifferentiationofALLfromAML
6
424
Acutemyeloidleukemia
7
Continued
8
TheFrenchAmericanBritish(FAB)classificationofacutemyeloidleukaemia.
(a) M
1
blastcellsshowfewgranulesbutmayshowAuerrods,asinthiscase
(b) M2cellsshowmultiplecytoplasmicgranules
(e) M3blastcellscontainprominentgranulesormultipleAuerrods
(d) M
4
blastshavesomemonocytoiddifferentiation
(e) M5amonoblasticleukaemiainwhich80%ofblastsaremonoblasts
(f) M5bmonocyticbut<80%ofblastsaremonoblasts.
Continued
9
(g) M
6
showingpreponderanceoferythroblasts
(h)M
7
7megakaryoblasticleukemiashowing
cytoplasmicblebsonblasts.
10
Favourable Umfavourable
Cytogenetics
Bone marrow response to remission
Induction
AgeOnset
(t15; 17)
t(S;21)
inv (16)
NPM mutation
5% blasts after first
course
<60 years
Primary
Deletion. of
chromosome5 or 7
FLt-3 mutation
1lq23
1(6;9)
abn(3q)
Compiex.rearrmgeme
nts
>20% blasts after first
course
>60 years
Secondary
Prognosis in acute myeloid leukemia (AML)
11
MinistryofHealth
Institute Graduates
Chronicleukemia
Haematological Tests
ChartMainlaboratoryfeaturesofacuteand
chronicleukemia's
2
3
Acute
leukaemias
Acute
lymphoblastic
Kaemia(ALL)
Acute
myeloblastic
kaemia(AML)
Chronic
Leukaemias
Chronic
lymphocytic
leukaemia(CLL)
Chronic myeloid
leukaemia
(CML)
*Chronic stage
*Accelerated/acute
stage
2
Erythrocytesedimentationrate
(ESR)(Westergren technique)
Value of test:
The erythrocyte sedimentation rate (ESR) is a
nonspecific test. It is raised in a wide range of
infectious, inflammatory, degenerative and
malignant conditions assciated with changes in
plasma proteins, particularly increases in fibrinogen
immunoglobulins, and Creactive protein.
4
TheESRisalsoaffectedbymanyotherfactorsincluding
anaemia,pregnancy,haemoglobinopathies,
hemoconcentrationandtreatmentwithanti
inflammatorydrugs.
Continued Equipment
6
DisposableplasticWestergren pipettesare
used.Itisgraduatedfrom0200mm.The
diametershouldnotbelessthan2.55mm
Timercapableoftimingaccurately1hour
Reagent
TriSodiumcitrate,32g/l
(3.2%wIv)anticoagulant
426
Specimen
Eithervenousbloodcollecteddirectlyintosodiumcitrateandtested
within2hours
7
Eithervenousbloodcollecteddirectlyintosodium
citrateandtestedwithin2hours,or
EDTAanticoagulatedblooddilutedinsodiumcitrate
canbeused.
IfEDTAbloodisusedandkeptrefrigeratedat48C
citratedilutionofthebloodandtestingcanbe
delayedforupto6hours
Specimen
8
Qualitycontrol
9
ThemostpracticalwayofcontrollingESRtestsisto
followthetestmethodexactly.
Problems(erroneousresults)occurwhen:
Usingthewrongvolumeofbloodtoanticoagulant
Bloodnotsufficientlymixedwithanticoagulant
Clotsintheblood.Eventhesmallestfibrindotinthe
samplewillinvalidatethetestresult
Airbubblesatthetopofthecolumn.
Continued
10
Testingbloodsamplesatthehottesttimeoftheday,or
leavingtestsindirectsunlight.Temperaturesover25.C
increasesedimentation.
Pipettenotpositionedvertically.Evenslightvariationsfrom
theuprightincreasesedimentation.
NotcheckingwhethertheESRstandislevelonthebench.
PlacinganESRstandonthesamebenchasacentrifuge
wherevibrationwillinterferewithsedimentation.
MeasuringtheESRwhenapatientisdehydrated.
InterpretationofESRtestresults
Reference range :
Men .. Up to 10 mm/hour.
Women . Up to 15 mm/hour.
Elderly Up to 20 mm/hour.
11
CausesofasignificantlyraisedESR
Anemia due to any cause .
Acute and chronic inflammatory conditions and
infections including.
HIV disease.
Tuberculosis.
Acute viral hepatitis.
Arthritis.
Bacterial endocarditis.
Pelvic inflammatory disease.
Ruptured ectopic pregnancy
Systemic lupus erythromatosis.
12
427
MinistryofHealth
Institute Graduates
Investigationofbleedingdisorders
Causesofbleedingdisorders
2
Abnormalbleedingmaybecausedby:
Damagetovascularendothelium
Reductioninplateletnumbers
Defectiveplateletfunction
Disordersofbloodcoagulation
Laboratoryinvestigation:
Hemoglobin test
Blood film report
Platetet count
Bleeding time test
3
APTTtestmethod
Test the control plasma and patient's plasma in
duplicate
Reference APTT range :
Normal plasma clots in 3645 seconds
4
Continued
Causes of a prolonged APTT include:
DIC (involving several clotting factors) .
Deficiency of clotting factors
On heparin anticoagulant
5
SourcesoferrorwhenperformingAPTTtests
(alsoandTTtests)
Difficulty in obtaining a venous sample resulting in
hemolysis or small clots. in the sample.
Delay in testing the plasma and leaving
sample at room temperature.
Using tubes or pipettes which are not clean and dry or are
contaminated with detergent. when ever possible use
disposable tubes.
Not timing accurately the different stages of the test (a stop
watch must be used).
6
Continued
Using unsatisfactory reagents (will be detect when
testing control plasma).
Not pipetting correct volumes of plasma and
reagents
Not correctly filling tubes to specified line
Clotting in collection tube
Inadequate mixing
7
Reconstitutingreagentsandcontrolplasmacontaminated
deionizedwater.Thisisacommoncauseofincorrect
coagulationtests(usuallyshortertimesareobtained).
Wheneverpossiblefreshdistilledwatershouldbeused.When
itispossibletoobtaingoodqualitywater,puredistilledwater
fromapharmacyormanufactureofthecoagulationreagents
beingused.
Continued
8
Prothrombintime(PT)test
The PT is a screening test for the extrinsic clotting system,
i.e. factor VII. It will also detect deficiencies factors,
prothrombin, V, X and fibrinogen. It is mainly used to
monitor patients receiving warfarin anticoagulation.
9
Continued
10
INR=
ISI
i.eprothrombinratiotothepoweroftheISICont.
ReferencePTrange
Normal plasma samples (patients not on anticoagulant) in
1116 seconds. Each laboratory should establish its own
normal reference range.
11
429
MinistryofHealth
Institute Graduates
Platelets,bloodcoagulation
Continued
The normal hemostatic response to vascular damage depends
on closely linked interaction between the blood vessel wall,
circulating platelets and blood coagulation factors
2
Continued
3
Theinvolvementofblood
vessels,plateletsandblood
coagulationinhaemostasis.
ADP.adenosinediphosphate
Thrombocytopenia
Infections, e.g. typhoid and other septicaemias .
Deficiency of folate or vitamin B
12
Aplastic anaemia .
Drugs (e.g. cytotoxic, quinine , aspirin), chemicals
(e.g. benzene), some herbal remedies.
Leukaemias, lymphoma, myeloma, myelofibroses,
carcinoma.
Hereditary thrombocytopenia (rare condition).
4
Continued
Increased destruction or consumption of platelets.
Infections, e.g. acute malaria, dengue, trypanosomiasis,
visceral leishmaniasis.
Disseminated intravascular coagulation (DIC)
Hypersplenism.
Immune destruction of platelets
5
Thrombocytosis
Causes of an increase in platelet numbers include:
Chronic myeloproliferative diseases. E.g. essential
thrombocythaemia, polycythaemia vera, chronic
myeloid leukaemia, myelofibrosis.
Carcinoma (disseminated).
Chronic inflammatory disease, e.g. tuberculosis
Haemorrhage.
Sickle cell disease associated with a non functioning
spleen or after splenectomy.
Iron deficiency anaemia, associated with active bleeding.
6
430
Institute Graduates
Thrombintime(TT)test
Thrombintime(TT)test
The TT test is sensitive to a deficiency of fibrinogen
or inhibition of thrombin. It measures the formation
of a fibrin clot by the action of thrombin on
fibrinogen.
2
ReferenceTTrange
Normal plasma samples clot within 1215 seconds
3
Fibrinolytic system
Fibrinolysis is the enzymatic process used by the
body to remove a fibrin thrombin to restore normal
blood flow once damaged endothelium is
repaired.
4
Duringtheclottingprocess,tissue,plasminogenactivator(tPA)
releasedfromthebloodvesselwall.andtheplasmaproenzyme
plasminogenbindtotheformingfibrinthrombus.When
activatedplasminogenisconvertedtoplasminwhichdegrades
thefibrinnetwork,.Causingtheclottodissolve,Duringthis
processfibrindegradationproducts(FDPs)i.e fragmentscalledD
Dimersareproduced
Continued
5
Continued
6
Laboratorytestsareavailabletodetectandsemiquantify,
FDPs(DDimers)inplasma.Mostaresimpletoperform
slidetestsusinglatexparticlescoatedwithantiDDimer.
FDPtitres inexcessof500ng/mlcanbefoundinDICalsoin
thrombosis,phelibitis,andembolism.
431
MinistryofHealth
Institute Graduates
Automatedbloodcellcountingusinganelectronic
bloodcellanalyzer
Principleofimpedanceanalyzers
Blood cells are diluted in a buffered electrolyte solution.
A measured volume of the sample passes through an
aperture tube (e.g. 100 !tm in diameter) between two
electrodes. Interruption of the current by the non
conducting blood cells alters the electrical charge and a
pulseis produced.
Continued
3
Theamplitudeofeachpulseisproportionedtothevolumeof
thecellwhichcausedit.Athresholdcircuitensuresonly
thosepulsesthatexceedthepresetthresholdlevelare
counted,Thecellcountisdeterminedfromthe.totalnumber
ofpulsesobtainedfromameasuredvolumeofblood.
Continued
4
Analysisofthepulseheightsenablesmeancellvolume
(MCV)tobemeasuredandthehaematocrit tobe
calculatedfromtheMCVvalueandredcellcount
Continued
The haematocrit is determined from voltage pulse data and
the MCV calculated from the haematocrit value. The
haemoglobin concentration is used with the red cell count,
MCV and haematocrit, to calculate the MCH and MCHC
5
SemiAutomatedInstruments
Which require blood samples to be externally diluted (with
separate dilutions for counting RBCs and WBCs)
6
432
FullyAutomatedInstruments
Withinternaldilutionandsimultaneouscountingofwhite
cells,redcells,andplatelets.Themoreadvancedanalyzers
Inadditiontodetermininghaemoglobin,WBC,RBC,
platelets,haematocrit,MCV,MCHC,andMCH,alsoprovide
redcelldistributionwidth(RDW),plateletdistributionwidth
(PDWl)andawhitecelldifferential
7
Continued
In addition to determining haemoglobin, WBC, RBC,
platelets, haematocrit, MCV, MCHC, and MCH, also provide
red cell distribution width (RDW), platelet distribution width
(PDW) and a white cell differential
8
Useofanelectronicbloodanalyzer
An electronic blood cell analyzer is appropriate to use when:
The work load is sufficiently high.
The capital cost and running costs of the analyzer are
affordable.
9
433
Ministry of Health
Specimen
Collection
434

Specimen Collection

Specimen Collection
- Slide 8 Blood
Universal precautions (old term) has been relabeled as Standard Precautions. This should be
for ALL patients with anticipated blood, body fluid, or pathogen exposure.

435
MinistryofHealth
Graduates
SpecimensCollection
Introduction
As part of the decision process for diagnosing,
confirming, treating or monitoring diseases, physicians
rely heavily on the results of the clinical laboratory tests.
So, physicians use laboratory tests to diagnose disease,
to monitor its progress or its response to treatment, and
to screen for disease in seemingly healthy individuals.
Many physicians consider disease as the only possible
cause of abnormal test results. Yet, many factors besides
disease affect the composition of body fluids; these
factors may be either preanalytical or analytical.
2
Introduction
Whenever possible, preanalytical variability should be
controlled so that correct interpretation of test results is
facilitated.
The variability of test results due to biological factors is
often greater than the variability due to analytical
factors.
Control of biological variability begins with proper
preparation of an individual before proper specimen
collection.
3
Introduction
When factors are not controllable (e.g. genetic and other
longterm influences) their possible effect on test values
should be recognized and considered in the evaluation of
laboratory data.
The diagnostic testing process can be divided into three
phases:
1. Preparation of the patient and collection of a specimen (pre
analytical phase).
2. Analytical measurement by the laboratory (analytical phase).
3. Reporting of the testing results to the physician (postanalytical
phase).
The preanalytical phase is being concerned in this lecture.
4
SpecimenLabelling
Before collecting a specimen of any type, specimen
containers must be labeled with the patients name,
hospital or identification number, location in the
hospital, and date and time of collection.
All specimens from patients with dangerous infections
should be labeled with a yellow dangerous specimen
sticker. A similar label should be attached to the request
form. Of most concern to the laboratory staff are
hepatitis and HIV viruses, but all specimens should
always be handled as potentially hazardous.
5
TypesofSpecimens
There are different types of samples on which laboratory
investigations will be done according to certain diagnostic
purposes for respective diseases. These types include:
1. Blood.
2. Urine.
3. Other body fluids:
4. Stools.
5. Solid tissue (tissue biopsy).
6. Swabs.
7. Calculi.
8. Investigations done on the individual himself.
6
436
TypesofSpecimens
Other body fluids:
2. Peritoneal (Ascitic) fluid.
3. Pleural fluid.
4. Pericardial fluid.
5. Synovial fluid.
6. Seminal fluid.
7. Amniotic fluid.
8. Saliva and sputum.
9. Wound exudates, pus and others.
7
1.Blood
Before collecting any blood sample, a phlebotomist should put on
disposable gloves.
If a patient is known to have an infectious disease, universal precautions
should be observed.
These precautions include the wearing of a facemask, glasses or goggles,
and gown in addition to gloves.
8
Bloodspecimentubesforspecificlaboratory
tests
9
1) Plain tube Containing no anticoagulant.
2) Lithiumheparin tube Containing lithiumheparin as anticoagulant.
3) Fluoride/oxalatetube
Containing sodiumfluorideand potassiumoxalateas
anticoagulants.
4) EDTA tube Containing EDTA as anticoagulant.
5) 1 : 9 citratetube Containing 1 : 9 sodiumcitrateas anticoagulant.
6) 1 : 4 citrate tube Containing 1 : 4 Sodiumcitrateas anticoagulant.
7) Metal-freetubes Containing no anticoagulant and aredemineralized.
8) Heparinizedsyringe Containing heparin as anticoagulant.
9) Blood culturebottle
Containing liquoid (sodium polyanethol sulphonate
and nutrient broth) as anticoagulant and bacterial
growth medium.
Bloodspecimentubesforspecificlaboratory
tests
10
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BloodFormsforInvestigations
Serum:
If blood is collected into a plain tube and allowed to clot, after
centrifugation (within one hour of clotting) a serum specimen is
obtained. It is the recommended specimen for many biochemical,
serological, immunological and hormonal analyses.
Plasma:
The blood is collected into a tube containing an anticoagulant and
when centrifuged, the supernatant is called plasma. It is preferred
when the analyte in question is unstable and speed is necessary.
Lithium heparin tube: For most biochemical tests.
1 : 9 citrate tube: For PT, PTT and INR, and other coagulation studies.
Fluoride/oxalate tube: For blood glucose and lactate.
11
BloodFormsforInvestigations
Whole blood:
It is obtained by collecting blood into a tube containing anticoagulant as
EDTA for CBC, glycosylated haemoglobin (HbA1C) and glucose6
phosphate dehydrogenase (G6PDH), as 1:4 sodium citrate for ESR, as
heparin for arterial blood gases (ABGs), and as liquid for blood culture.
Red blood cells:
This type of samples is obtained by collecting blood into a tube containing
EDTA. Then after centrifugation, the supernatant is removed, a suitable
amount of normal saline (NaCl 0.9%) is added with gentle mixing and
after second centrifugation the supernatant is removed. This process will
be repeated until the supernatant becomes clear and colourless where it
is removed, and the deposit now contains only red blood cells (RBCs)
which are recommended for G6PDH assay and haemoglobin
electrophoresis.
12
437
BloodSampling
Blood for analysis may be obtained from:
Veins by venipuncture: Venous blood.
Capillaries by skin puncture: Capillary blood.
Arteries by arterial puncture: Arterial blood.
13
A.Venipuncture
Before performing a venipuncture, phlebotomist should:
Verify that the patient is fasting if this is necessary to
ensure medically useful results. The patient should be
comfortably seated for about 20 min. before the specimen
is withdrawn. This standardization minimizes differences in
concentrations of blood due to variations in the blood
volume.
Estimate the volume of blood to be collected and select
the appropriate number and types of tubes for the plasma
or serum tests requested. An appropriate needle must also
be selected. The most commonly used sizes are gauges 19
to 22 for adults (21 23 for children). The larger the gauge,
the smaller the bore.
14
A.Venipuncture
Venipuncture is accomplished by using a vacutainer set or a
syringe.
The median cubital vein in the anticubital fossa is the preferred site
for collecting venous blood in adults because the vein is both large
and close to the surface of the skin. Veins on the back of the hand
or at the ankle may be used, although these are less desirable and
should be avoided in diabetics and other individuals with poor
circulation. In severely ill patients and those requiring many
intravenous injections, an alternative blood drawing site should be
chosen to preserve the good veins for the patients treatment.
After cleaning the skin by alcohol swab (by benzalkonium chloride
in specimens for alcohol determinations), a tourniquet is applied
about 10 cm above the intended puncture site.
15
A.Venipucture
A tourniquet should not be left in position for more than 1 minute and
the patient should not be allowed to pump his or her fist while the
tourniquet is in place.
When blood collection is complete, the tourniquet should be released
and the needle is withdrawn. Then, the patient should hold a dry gauze
pad over the puncture site, with the arm raised to lessen the likehood of
leakage of blood. The pad can subsequently be held in place by a
bandage.
The phlebotomist must separate the needle from the syringe or
vacutainer tube and discard it in a container (sharps container) designed
specifically for this purpose.
Vigorous suction on a syringe during collection, or forceful transfer from
the syringe to the receiving tube may cause haemolysis of blood.
Haemolysis is usually less when blood is drawn through a smallbore
needle because turbulence of the blood is less than when a largerbore
needle is used.
Do not collect blood from the arm with a drip attached. Use opposite
arm.
16
B.SkinPuncture
Skin puncture is the method of choice in paediatric age
group although it is done when only small volume of
blood is required for a blood test in adults (e.g. glucose
estimation by glucometer). It is often preferred in
geriatric patients because of thinness of skin and the
loss of elasticity.
The puncture site is:
The centre of the palmer side of the tip of the third or fourth
finger of the nonwriting hands in adults or grown children.
Earlobe in adults or grown children.
The lateral or medial plantar surface of the foot in infants
younger than 1 year of age.
The plantar surface of the big toe in older children.
17
B.SkinPuncture
18
Acceptable sites for skin puncture to
collect blood from an infants foot
438
B.SkinPuncture
After selection of the puncture site, it is warmed by warm and moist
towel, cleaned by alcohol swab and then the puncture is made with
sterile lancet (manually or automatically). The depth of incision should be
less than 2.5 mm to avoid contact with bone.
The first drop of blood is wiped off and subsequent drops are transferred
to the appropriate tube by gentle contact. Blood may be collected into
capillary tubes by capillary attraction. Filling should be done rapidly to
prevent clotting, and the introduction of air bubbles should be avoided.
Massage of the finger to stimulate blood flow should be avoided because
it causes the outflow of debris and of tissue fluid which does not have the
same composition as plasma. The finger should be held in such a way that
gravity assists the collection of blood on the fingertip.
The site of puncture must not be oedematous or a previous puncture site.
Skin puncture is timeconsuming and there is a greater risk of infection
than from venipuncture.
19
C.ArterialPuncture
Arterial blood is used to measure arterial blood gases (ABGs).
Arterial punctures require considerable skill and are usually
performed only by physicians or specially trained technicians
or nurses.
The preferred sites of arterial puncture are (in order) the
radial artery at the wrist, the brachial artery in the elbow and
the femoral artery in the groin.
Because leakage of blood from the femoral artery tends to be
greater, especially in elderly, sites in the arm are most often
used.
The artery to be punctured is identified by its pulsation and
thick wall. The skin surrounding the puncture site should be
cleaned. No tourniquet is required for arterial puncture.
20
C.ArterialPuncture
A needle with a gauge size 18 20 should be used for one of
the larger arteries but a smaller needle with a gauge size 23
25 should be used to collect the blood from the smaller
arteries.
The needle and syringe should be flushed out with heparin
solution both to ensure adequate anticoagulation and to
eliminate trapping of air in the needle and in the dead space
of the nozzle.
Evacuated blood tubes should not be used for the collection
of specimens for blood gas analysis because the residual air
in the tube may cause false results.
Once an arterial puncture has been performed, firm pressure
should be applied over the puncture site for at least 5
minutes to minimize bleeding.
21
C.ArterialPuncture
After collecting the specimen for blood gas analysis, the
nozzle of the syringe should be sealed and the syringe should
be placed in ice water for immediate transport to the clinical
laboratory.
Analysis should be performed within 15 minutes because
chilling will not completely inhibit the metabolic activity of
white blood cells.
The best method for blood gas specimen collection in
neonates is the indwelling umbilical artery catheter.
In older children or adults in whom it is impossible to
perform an arterial puncture, a capillary puncture may be
performed to obtain arterialized capillary blood. Such a
specimen yields acceptable values for pH and pCO
2
but not
for pO
2
.
22
AbnormalColourofSerumorPlasma
Normally, serum or plasma is clear and its color is
yellowish like the color of cooking oil. According to
the abnormal color of the sample, there are 3 types
of samples:
1. Haemolyzed sample.
2. Lipaemic sample.
3. Icteric sample.
23
1) Haemolyzed sample:
Serum or plasma is reddish in color that results from
hemolysis of red blood cells. This can affect some
laboratory investigations as follows:
Direct effect: Some constituents in red blood cells or in their
membranes go out into the plasma or serum affecting the
concentration of its constituents e.g. potassium, lactate
dehydogenase, acid phosphatase, etc.
Indirect effect: The color affects the investigations done by
colorimetric method e.g. glucose and bilirubin, and
interferes with the kinetic determinations of certain
constituents e.g. creatinine, CPK and alkaline phosphatase.
24
2) Lipaemic sample:
Serum is whitish in color and may be milky due to its high
content of lipids especially triglycerides. The color of this
sample interferes with most (if not all) the methods
employed for biochemical tests except those for lipid
profile. This sample should be divided into 2 parts:
The first part is used directly for lipid profile assay.
To the second part, equal volume of ethyl acetate will be
added and well mixed. Then, it is separated by
centrifugation into 2 layers: the upper layer containing the
ethyl acetate with fat, and the lower clear layer for all
investigations except lipid profile.
25
3) Icteric sample:
Serum or plasma is greenish yellow in color that
interfere with the colorimetric methods for many
investigations e.g. glucose and cholesterol, and with
the kinetic determination of creatinine that should be
measured by deproteinization method.
26
TimingofBloodSampling
All investigations for metabolites with dietary sources
will be assayed after fasting for 8 12 hours except for
lipid profile; the fasting should be 12 14 hours.
Since the most satisfactory specimen is a post
absorptive one, the best time for sampling is after
fasting overnight. This helps the laboratory as regards
planning the days work.
The time at which a specimen is obtained is important
for those blood constituents that undergo circadian
(diurnal) variation (e.g. cortisol, growth hormone, iron)
and those used to monitor drug therapy (e.g. digoxin,
lithium or prothrombin time) because the interval after
drug administration affects the drug concentration.
27
TimingofBloodSampling
Filaria migrates into peripheral blood at night. So,
midnight is the best time for diagnosis of filariasis.
Metabolic challenge studies (such as xylose
absorption test and glucose tolerance test) require
specimen collection at specified timed intervals.
It is preferred to collect blood sample for the assay
of female sex hormones according to their relation
to menstrual cycle, e.g. at the luteal phase for
progesterone, at the follicular phase for oestrogen
and FSH, and in the midcycle for LH.
28
TransportandStorage
Transport of blood samples from collection site to the
laboratory is an important component of sample
processing. Specimen should reach the laboratory
without undue delay.
All laboratory specimens must be transported in a safe
and convenient manner to prevent biohazard exposure
or contamination of the sample.
Agitation of blood specimens should be avoided to
minimize haemolysis.
Specimens should be protected from direct exposure to
light, which causes breakdown of certain metabolites
e.g. bilirubin which should be analyzed as soon as
possible.
29
TransportandStorage
For analysis of unstable constituents, such as ammonia,
plasma rennin activity and acid phosphatase, specimens
must be kept at 4C immediately after collection and
they should be transported on ice.
Some serum specimens as that of liver enzymes, can be
stored at 2 8C for 24 48 hours, and others, as glucose
and lipids, can be frozen at 20C for months or even
years.
The effect of freezethaw cycles on constituent stability
is an important consideration. Once the frozen sample is
thawed, it should be analyzed as soon as possible.
30
ImportantAdvisesforPatientPreparationand
SampleProcessing
Perrectum examination, prostatic massage, urinary
catheterization and chronic constipation should be avoided
for about one week before collecting the sample for acid
phosphatase assay.
Muscular exercise or straining and intramuscular injection
should be avoided before collecting the sample for total CPK,
total LDH, and aldolase assays.
Individuals for lipid profile estimation should be on lipidfree
diet for 72 hours before specimen collection.
Individuals for glucose tolerance test (GTT) should be on the
ordinary carbohydrate diet for 72 hours before specimen
collection.
Mouth pipetting of any sample must be avoided.
31
SamplingErrors
There is a number of potential errors that may contribute to the
success or failure of the laboratory to provide the correct answers to
the clinicians questions. Some of these problems may arise when the
clinician first obtains specimens from the patient.
1. Blood sampling technique:
o Difficulty in obtaining a blood specimen may lead to hemolysis with
consequent release of red cell constituents.
2. Prolonged stasis during venepuncture:
o Plasma water diffuses into the interstitial space and the serum or
plasma sample will be concentrated. Protein and proteinbound
components such as calcium, total bilirubin, lipids and thyroxin will be
falsely elevated.
3. Insufficient specimen:
o Each blood analysis requires a certain volume of specimen to enable the
test to be carried out.
32
SamplingErrors
6. Errors in timing:
o The biggest error in the measurement of some analytes may arise from
inappropriate time of sampling.
7. Incorrect specimen container:
o For many analytes, the blood must be collected into a container with
appropriate anticoagulant. If a sample is collected into wrong container,
it should never be decanted into another type of tube.
8. Inappropriate sampling site:
o Blood samples should not be taken from an intravenous drip or from
the arm into which a canula is connected.
9. Incorrect specimen storage:
o A blood sample stored overnight before being analyzed will show falsely
high potassium, phosphate and red cell enzymes such as lactate
dehydrogenase because of leakage into extracellular fluid from the
cells.
33
2.Urine
The type of urine specimen to be collected is dictated by the tests
to be performed.
Single specimens of urine (random or morning samples) are used
for ward examinations and qualitative tests.
A clean, morning, fasting specimen is usually the most
concentrated specimen performed for microscopic examinations
and for the detection of abnormal amounts of constituents such as
proteins and glucose, or of unusual compounds such as chorionic
gonadotropin (for pregnancy test).
The doublevoided specimen is the urine excreted during a time
period after complete emptying of the bladder by 15 30 min. It is
used, for example, to glucose excretion during a glucose tolerance
test. Its collection must be timed in relation to glucose ingestion.
24 hoursurine collections are preferred for quantitative assays due
to the diurnal variation in the excretion of some substances.
34
2.Urine
The 24 hoursurine collection is as follows:
a) At a suitable time (e.g. 8:00am), the patient
empties his bladder and the urine is discarded.
b) All specimens passed during the following 24 hours
are saved in an appropriate container.
c) At the same time of the next morning the patient
empties his bladder and the urine is added to the
collecting one.
o N.B.: The creatinine content of the urine sample can
be used as a rough check on the reliability of the
collection.
35
2.Urine
Instructions for collection of urine specimens for 5
hydroxyindoleacetic acid (5HIAA) measurements
should specify avoidance of bananas, plums, walnuts,
pineapples and eggplant, as well as, acetaminophen and
cough syrup containing glyceryl guaiacolate for 72 hours
before sampling.
The patients genitalia should be cleaned before urine
voiding to minimize the transfer of surface bacteria to
the urine.
Bacterial examination of the first 10 ml of voided urine is
the most appropriate to detect urethritis, whereas the
midstream specimen is the best one for investigating
bladder disorders.
36
441
2.Urine
Catheter specimens are used for microbiological examination
in critically ill patients or in those with urinary tract
obstruction, but should not normally be obtained just for
biochemical assays. The suprapubic tap specimen is a useful
alternative, because the tap is unlikely to cause infection.
Collection of a timed specimen from an infant is difficult. But
for a random sample, a plastic bag is placed around the
infants genitalia and left in place until urine has been voided.
First, the scrotal or perineal area should be cleaned and
dried, and any natural or applied skin oils should be removed.
To obtain a sterile urine specimen for culture from an infant,
a suprapubic tap is performed.
The collection of a specimen from older children is done as in
adults, using assistance from a parent when this is necessary.
37
StorageandPreservationofUrineSpecimens
It is satisfactory in most cases to use specimens collected in cool,
clean containers.
It is particularly important to use freshly voided and concentrated
urine to identify casts, RBCs, and WBCs. One should deliver
specimens to the laboratory within one hour of collection.
One of the most satisfactory forms of preservation of urine
specimens is refrigeration immediately after collection. This is
more successful when combined with chemical preservation.
Acidification to below pH 3.0 is widely used to preserve 24hours
specimens for catecholamines, vanillylmandelic acid (VMA), 5
hydroxyindole acetic acid (5HIAA), steroids, and calcium assays.
This acidification is done by adding HCl (10 ml of 6 mol/l, per 24H
excretion), sulfamic acid (10 ml/l urine) or by boric acid (5 mg/30
ml urine). This is unsuitable for measurement of uric acid that is
precipitated by acidification.
38
Sodium bicarbonate is used to adjust pH between 8.0 and 9.0 for sample
preservation of porphyrins, porphobilinogen, urobilinogen and uric acid.
Specimens for porphyrins, porphobilinogen, urobilinogen and bilirubin
are collected and stored in dark containers to avoid their degradation by
direct exposure to light.
Toluene is the organic solvent that is still used as a preservative. It acts as
a barrier between the air and surface of the specimen. Neither thymol
nor chloroform is used nowadays.
Urine may be frozen to be assessed at a later time. When repeat testing is
expected, the specimen is stored in multiple bottles to avoid specimen
degradation as a result of repeated freeze thawing of a single specimen.
39
Before a specimen is transferred into small
containers for each of the ordered tests, it must be
thoroughly mixed to ensure homogeneity because
the specific gravity, volume and composition of the
urine may all vary throughout the collection period.
Urine should not be collected at the same time for
two or more tests for which different preservatives
are required.
Urine is collected for cytological identification of
malignant cells into equal volume of 50% alcohol.
40
3.Cerebrospinalfluid(CSF)
Cerebrospinal fluid may be obtained by lumbar, cisternal or lateral
cervical puncture or through ventricular cannulas or shunts.
Up to 20 ml of spinal fluid can be safely aspirated from an adult, although
this amount is not usually required. Clinician should provide clinical
history to the laboratory. The puncture site (i.e., lumbar, cisternal, etc.)
should be noted since cytologic and chemical parameters vary at different
sites.
CSF must not be bloodstained. The first 1.0 to 2.0 ml should be
discarded.
CSF (3 4 ml) should be aspirated into 3 tubes: the first tube should be
used for chemical and/or serological tests, the second for microbiological
tests, and the third for microscopical and cytological examination.
41
3.Cerebrospinalfluid(CSF)
Tests on spinal fluid require rapid processing of the specimens to
minimize cellular degradation, which begins within 1 hour of collection.
CSF may be evaluated in the laboratory to establish a diagnosis of
infection (bacterial, fungal, mycobacterial, or amoebic meningitis),
malignancy, subarachnoid haemorrhage, multiple sclerosis or de
myelinating disorders.
Antiglycolytic agents (sodium fluoride) usually are not added to the CSF
tube for glucose measurement because rapid processing of the sample
ensures that little metabolism of glucose occurs even in the presence of
many bacteria.
Highly bloody sample for cell count should be collected in EDTA tube to
prevent formation of clot.
Glass tubes should be avoided since cell adhesion to glass affects the cell
count.
Refrigeration is contraindicated for culture specimens because organisms
like Haemophilus influenzae and Neisseria meningitidis will not survive.
42
442
4.Peritoneal,PleuralandPericardialFluids
These fluids are aspirated by paracentesis (peritoneocentesis,
thoracentesis and pericardiocentesis) which should be
performed only by skilled and experienced physicians.
Specimens are obtained for chemical (proteins and/or
enzymes), microbiological and cytological examinations.
For most chemical evaluations, no additive is used and
specimen is allowed to clot. Bacteriological and cytological
specimens may be collected in sterile EDTA or sodium
heparin containers without preservatives.
On sample processing for mycobacterium, anaerobic bacteria
or viruses, safety precautions and special handling
procedures should be applied prior to specimen collection.
43
5.Synovialfluid
Synovial fluid is obtained by arthrocentesis, which should be
performed by a physician using sterile procedures. The technique
must be modified from joint to joint depending on the anatomical
location and the size of the joint.
Sterile plain tubes should be used for culture and for glucose, uric
acid and protein measurements. An EDTA tube is needed for total
leucocytes, differential and erythrocytes count. Microscopic slides
are prepared for staining with Grams or other stains indicated, and
for visual inspection.
The physician should establish priorities for the tests to be
performed in case the available volume of synovial fluid is in
sufficient for all tests.
Normally, only a very small amount of fluid is present in any joint,
but this volume is usually very much increased in the presence of
inflammatory conditions.
44
6.Salivaandsputum
The individual is asked to rinse out his mouth with water
and then chew an inert material as a piece of rubber or
paraffin wax for five minutes. The first mouthful of saliva
is discarded and the followed saliva is collected into a
small glass bottle.
The clinical application of methods using saliva has been
limited. However, saliva can be used for drug analysis.
On the morning for 3 successive days, the fasting patient
is asked to expectorate sputum into 3 sterile containers
respectively.
The 3 specimens will be stained by ZiehlNeelsen stain
for the diagnosis of Mycobacterium tuberculosis.
45
7.Seminalfluid
Sexual abstinence should be 3 5 days before semen
analysis.
Seminal fluid should be collected by one method of the
following:
Sheath: It may be ruptured causing loss of seminal fluid.
Coitus interruptus: The semen (some or all) may be ejaculated
into the vagina or may be contaminated by vaginal secretions.
Masturbation: It is the preferred method. The individual should
not use water and soap but masturbation should be done by dry
or salivalubricated hand.
Time of ejaculation should be written on the label, and once
the sample is received it should be placed in the incubator at
37C to be examined within the next 3 hours.
46
8.AmnioticFluid
Amniotic fluid is withdrawn by amniocentesis, which
should be performed by physician.
It is performed for prenatal diagnosis of congenital
disorders (by measurement of fetoprotein), to
assess fetal maturity (by measurement of the
lecithin / sphingomyelin ratio, or to look for Rh in
compatibility (by measurement of bilirubin) or
intrauterine infection (by culture).
The specimen is collected in dark brown container,
which should immediately be placed in ice.
47
9.Stools
The investigations which can be done on stools include:
Parasitological examination for the detection of any infested parasite. The
individual is considered free from parasites when this investigation is negative
for 3 successive days.
Biochemical investigation for detection of occult blood, for measurement of
fecal fat, reducing substances and nitrogen, and for estimation of trypsin
activity in feces of children.
Microbiological studies for detection of the cause of diarrhoea by culture
(and sometimes sensitivity tests) for Salmonella, Campylobacter, Brucella,
Shigella or Cholera.
No preservative is added to feces but the container should be kept
refrigerated throughout the collection period and should be sent to the
laboratory within a short time after completing the collection.
Care should be taken to avoid contamination from urine or water.
It is preferable to avoid using stools containing liquid paraffin or barium
salts, or which have been collected after an enema has been given.
48
443
10.SolidTissue(TissueBiopsy)
The solid tissue is most often analyzed in the clinical laboratory for
malignancy. It may be used for toxicological analysis.
At least 0.5 1.0 g tissue should be removed surgically and should
be trimmed of fat and nontumor material.
The tissue (especially that from breast for estrogen and
progesterone receptors) should be quickly frozen, preferably in
liquid nitrogen or in a mixture of dry ice and alcohol. The time
between collection and freezing should be less than 20 minutes.
Tissue biopsy can be preserved in 10% formalin until analyzed
histologically.
The tissue biopsy should be accompanied by a report from the
surgeon including patients name, sex and age, date of tissue
removal, anatomical site of the biopsy and the probable diagnosis.
49
11.Swabs
A swab can be taken from a tissue or from an organ
for cytological studies or for culture and sensitivity
test.
Swabs include conjunctival swab, throat swab,
wound swab, high vaginal swab (HVS), cervical swab
and others.
If the swab or any specimen is for culture and
sensitivity test, the patient must not be under
antibiotic therapy for 3 days before sampling.
50
12.Calculi(Stones)
Description of the received stone should be
documented.
This includes the type (either renal or gall stone),
number, color, shape, surface, size and consistency
of the stone.
51
13.Investigationsdoneontheindividual
Some investigations can be done on the individual
himself e.g.:
1. Skin allergy test: The patient should not take
antihistaminics or corticosteroids for 3 days before
the test.
2. Tuberculin test for failed or nonBCG vaccinated
individuals.
52
444
Ministry of Health
Quality Control
and Assurance
445
MinistryofHealth
Graduates
Quality Control Assurance I
Key Terms plus Sensitivity & Specificity
Aims
To have an overall understanding of the key terms associated
with medical laboratory quality control & assurance (QCA)
To be familiar with the importance of sensitivity and
specificity
2
Learningoutcomes
Tohaveabriefknowledgeandunderstandingofthemain
aspectsofqualitycontrol&assuranceincludingmanagement
Tohaveaknowledgeandunderstandingofthekeyaspects&
importanceofclinicalsensitivity&specificity
3
Teachingmethods
Formallectures
Smallgroupproblembasedtodevelopteamwork
Seminarsorworkshops
Onsitelaboratoryorpracticalsessions
Whenappropriatevideopresentations
4
Keydefinitions
Quality control: checking the performance of a particular test
each time it is run; it is to ensure that the operator, reagents
& equipment are all working properly.
5
Qualityassurance
Overall term for monitoring all practices and procedures
within the laboratory to make sure that required standards
are being maintained.
6
446
Qualityassessment(internalorexternal)
Testingthestandardofthelaboratoryprocedures
usuallybytestingablindsample
Thiscanbeaninternalsampleoronesuppliedfrom
anexternallaboratory
(Inthiscasethedeptcanmeasureitsperformance
againstnationalstandards)
7
Qualitycontrol&assurance
Policies, processes & practices undertaken by a laboratory
that are necessary to ensure a test on a clinical sample gives
the correct result & that the finding is delivered to the
appropriate clinician in a timely & efficient manner
8
Quality
Quality: this is essentially about consistency
Getting the procedures right first time and every subsequent
occasion
Quality assurance: is all the planned & systemic actions
necessary to give adequate confidence that a product or
service satisfies the necessary level of quality
9
Qualityaudit
Internalcheckofthewholeprocedurefromthepatient
throughthelaboratoryandbacktothepatient
Exercisescaninvolvemembersofstaffoutsideofpathology
10
Clinicalgovernance
A system of regulation and monitoring to make sure that the
highest quality of patient care is provided within a healthcare
organisation
11
Accreditation
Procedure through which a department is given formal
recognition that they meet certain minimum standards in
their work as defined by an independent organisation
12
447
GoodClinicalPractice(GCP)
1. A standard for design, conduct, performance, monitoring, auditing, recording and
analysis in a clinical setting/laboratory
2. That provides assurance that the data & reported results are credible and accurate
in a clinical setting/laboratory
3. That the rights, integrity and confidentiality of the patient are protected in the
laboratory and hospital setting
13
Standardoperatingprocedures(SOPs)
Thesespecifyindetailhowaparticularprocedureormethodwillbecarriedoutin
clinicallaboratoryinaccordancewithgoodlaboratorypractice(GLP)andgood
clinicalpractice(GCP)
SOPsneedtobeveryaccurateandhencecanbeverydifficulttowrite
Theymustcontainclearinstructionsregardingtheoperationofspecified
equipmentandhowtousetheappropriateconsumables
Healthandsafetyshouldalsofeature
14
GuidelinesforwritingSOPs
SOPsaretheworkinstructionstoensurestandardizationof
workingpractices&compliancewithrelevantguidelines&
standards
15
Guidelines&standards(SOPsetc)
Internationalconferenceofharmonizationgoodclinical
practice(ICHGCP)
Food&drugadministration(FDA)
Controlofsubstanceshazardoustohealth(COSHH)
16
Qualitymanagementsystem
Overall system of procedures and policies intended to ensure
that defined standards are met with clear plans to address
failures to meet these standards (the documentation in this
system must be controlled)
17
QMSContinued
In the clinical laboratory setting it is essential to ensure that
associated clinicians, doctors and patients are involved in the
laboratory service
18
448
Qualitymanagementsystems(QMS)
Trainingstaff
Showingcompliancewithcurrentregulations&standards
Internal&externalqualitycontrols(QCs)
Training&competencyassessments
Audits
19
Clinicalsensitivity&specificity
Tobeeffectiveamedicaltestisexpectedtodetect
abnormalitieswithahighdegreeofconfidence
Howlikelyisitthatanindividualwhohasapositivetesthas
thedisease?
Whatarethechancesthatanindividualhasacertain
disordereventhoughthetestforitisnegative?
20
Sensitivity
The measure of the ability of a test to identify those
individuals who have a disorder after testing positive
21
Specificity
A measure of the ability of a test to identify
individuals who are healthy after testing negative
OR
Measures the proportion of negatives which are
correctly identified as such (e.g. the percentage of
healthy people who are correctly identified as not
having the condition)
22
Warning
Unfortunatelyclinicaltestsareneither100%specificnor
100%sensitive
Infactthetwofactorsshowaninverserelationshipwitheach
other
23
Importantaspectsofobjective tests
Accuracy&validity
Variability,reproducibilityorprecision
Twoindicesusedtoevaluatetheaccuracyofatest sensitivity&
specificity
Notabsolutevalues
24
449
Sensitivity&specificity
Indicesareusuallydeterminedbyadministeringthetesttoone
groupofpersonswhohavethediseaseandanothergroupwho
donothavethedisease
25
Therelationshipbetweenadiagnostictestresult&
theoccurrenceofdisease
DISEASE PRESENT ABSENT
TEST
POSITIVE TRUE
POSITIVE
a
FALSE
POSITIVE
b
NEGATIVE FALSE
NEGATIVE
b
TRUE
NEGATIVE
c
26
True&falsepositives
Thosewhohavethediseasetestpositive
Truepositives
Thosewhotestpositivebutdonothavethedisease
Falsepositives
27
True&falsenegatives
Thosewhotestnegative&donothavethedisease
Truenegatives
Thosewhotestnegativebuthavethedisease
Falsenegatives
28
Sensitivity&specificity
Sensitivity istheproportionoftrulyillpeopleinthescreened
populationwhoareidentifiedasillbythescreeningtest
Specificity istheproportionoftrulyhealthypeoplewhoareso
identifiedbythescreeningtest
29
Sensitivity
Truepositives
Sensitivity=
truepositivesplus
Falsenegatives
30
450
Sensitivity
Truepositives
Sensitivity=
Allthosewiththe
Disease
31
Sensitivity
32
Specificity
Truenegatives
Specificity=
Truenegativesplus
Falsepositives
33
Specificity
Truenegatives
Specificity=
Allthosewithout
thedisease
34
Specificity
35
Sensitivity&specificity important
Sensitivity probabilityofapositivetestinpeoplewiththe
disease
Specificity probabilityofanegativetestinpeoplewithout
thedisease
36
451
37
TEST
POSITIVE TRUE
POSITIVE
a
FALSE
POSITIVE
b
NEGATIVE FALSE
NEGATIVE
b
TRUE
NEGATIVE
c
38
Diagnostictestcharacteristics&definitions
Sensitivity(Se)=a/a+c
Specificity(Sp)=d/b+d
Prevalence(PV)=a+c/a+b+c+d
LikelihoodRatio(LR)+=a/a+c/b/b+d
LikelihoodRatio(LR) =c/a+c/d/b+d
39 40
Falsepositives&negatives
(A)alowlimitresultsinamoresensitivetest
(B)intermediatelimitresultsinminimumtotalerror
(C)ahighlimitresultsinamorespecifictest
Predictivevalue
Sensitivity&specificityprovideinformationabouttheaccuracyofthe
test
Donotprovideinformationaboutthemeaningofapositiveornegative
testresults
Predictivevalues
Positive&negative
42
452
Positivepredictivevalue
Truepositives
Positivepredictive=
Value truepositives
plusFalsepositives
43
Positivepredictivevalue
Truepositives
Positivepredictive=
Value Allthosewitha
Positivetest
result
44
PositivePredictiveValue
45
Negativepredictivevalue
Truenegatives
Negativepredictive=
Valuetruenegatives
Plus
falsenegatives
46
Negativepredictivevalue
Truenegatives
Negativepredictive=
Value Allthosewitha
Negativetest
result
47
NegativePredictiveValue
48
453
Predictivevalues important
Positivepredictivevalue probabilityofthepersonhavingthedisease
whentestispositive
Negativepredictivevalue probabilityofthepersonnothavingthe
diseasewhenthetestisnegative
49
TEST
POSITIVE TRUE
POSITIVE
a
FALSE
POSITIVE
b
NEGATIVE FALSE
NEGATIVE
c
TRUE
NEGATIVE
d
50
Diagnostictestcharacteristics&definitions
Positivepredictivevalue(+PV)=a/a+b
Negativepredictivevalue(PV)=d/c+d
51
Relationshipbetweenprevalence,sensitivity,
specificity&positivepredictivevalue(+PV)
SensitivityXPrevalence
(+PV)=
(SensitivityXPrevalence)
+(1specificity)X(1prevalence)
52
Relationshipscontinued
Moresensitiveatestis,thebetterwillbeitsnegativepredictive
value
Themoreconfidentthecliniciancanbethattheanegativetest
resultrulesoutthediseasebeingsought
53
Relationshipscontinued
Themorespecificthetestis,thebetterwillbeitspositivepredictivevalue
Themoreconfidentthecliniciancanbethatthepositivetestconfirmsor
rulesinthediagnosisbeingsought
Predictivevaluesareinfluencedbyprevalence
54
454
Whatdidyoulearn?
55
Readinglist
Burnett,D.(2002).APracticalGuideToAccreditationIn
LaboratoryMedicine.ACBVenturePublications.London
DepartmentOfHealth(2000).AnOrganisationWithMemory.
DeptOfHealth,London.
MHRA(2007).Rules&GuidanceForPharmaceutical
Manufacturers&Distributors.PharmaceuticalPress.London
Vorley,G.,Tickle,F.(2002).QualityManagement,Tools&
Technique5
th
EdtQualityManagement&Training
(Publications)Ltd,London
56
MinistryofHealth
Lecture Quality Control Assurance II
Quality Management
Graduates
Aim
Tobefamiliarwithareasassociatedwithquality
managementinaclinicalsetting
58
Learningoutcomes
Tohaveknowledgeandunderstandingofquality
managementandrelatedsystemsassociatedwithclinical
laboratoryandhospitalsetting
Tobefamiliarwiththetypesofgoodpracticeassociatedwith
andrequiredinaclinicallaboratorysetting
59
QualityManagement(QM)
Is any system where the management has direct
control of an organization with regard to quality
Quality is a never ending cycle of events that require
the support and cooperation of all the people in
the organization
455
QMApproach
Isanattitudeorculturewithinanorganisation
Allorganisationsmustensurethatthequalityofservicesthey
providearemaintainedandcontinuallyimproved
61
QMContinued
Everystageofaprocess,systemorprojectisapossible
sourceofpoorquality
Greatpotentialforpoorqualityarecomplicatedsystemslike
hospitals
Learningorganisationshavesystemsandmechanisms&
processesthatenhancetheservicestheyprovide
62
QMContinued
Hospitals should be able to adapt to their environments and
apply the results of their to achieve better results
Hence hospitals should be continually evolving and
enhancing their capabilities
Need to learn from own and others mistakes to reduce the
future risks to patients
Difficult to reduce totally adverse events totally from
complicated systems like hospitals
63
TheInternationalStandardsOrganisation(ISO)
Developsstandardsforinternationalorganisationstofollow
oradopt
DocumentISO9001:2008outlinesguidelinesforQMS
QMSisamodelthatmaybeemployedtogiveguidelinesto
theselectionofappropriateQCactivities
64
AimofQMS
Topreventnonconformityoranonconformancetoasetof
standards
65
Nonconformity
Nonconformityisanydeviationfromthatwhichisrequired
AneffectiveQMSwilltryandsatisfycustomersorconsumers
byconcentratingontheservice
Achievethisbycustomersurveysandmonitoringcomplaints
Adocumentedprocessdesignedtoachieveconsistency,
providesagoalforthesystemcontrolandmaintainingthe
requiredlevelofqualityateachstageintheprocess
66
QMS:ISO9001:2008 Definition(Eight
Requirements)
1.Customerfocus
2.Leadership
3.Involvementofpeople
4.Processapproach
5.Systemsapproach
6.Continualimprovement
7.Actualapproachtodecisionmaking
8.Mutuallybeneficialsupplierrelationships
67
Eightrequirements
FormsthebasisforanyQMSintheclinicallaboratory
Alleightcontributetoimprovingthequalityofcareto
patients
68
CustomerfocusedQM
Meetstheneedsoftheclinicianswhotreatthepatients
Clinicallaboratoriesachievethisthroughusersurveys,
monitoringcomplaintsandcompliments
Alsobyanalysingincidents
69
Leadership
Thisisprovidedbymanagementcommitmentfrom
consultantsandseniorlaboratorymanagers
Ifseniormanagementarenotinvolvedinthequalityprocess
itwillnotwork
Leadershipundertakenthroughtheprovisionofquality
policyandhealth&safetystatements
Alsothroughtheactions&attitudesofseniormanagers
70
Leadership PathologyLab
Theseniorteammayconsistof:
Clinicaldirector
Pathologyservicemanager
Headbiomedicalscientist
Orclinicalscientistforeachdepartment
71
HighQualityServiceProvision
Onlyarisesfromcommitmentofeveryoneintheorganisation
Itneedseveryonetobeinvolvedandawareofthesystemin
place
Oftenthecasethatpeopleperformingspecifictaskshavea
betterunderstandingofproblemsthanthemanager
Needawellmotivatedworkforcewhofeelvalued&take
prideintheirwork
Excellenceintheserviceprovidedistheresponsibilityof
everyoneconcerned
72
457
Aprocessapproach
Individualareasmustnotbelookedatinisolation
Thewholeprocessmustbeexamined
Changingsomethinginoneareawillhaveaknockoneffect
inotherareas
Thereforelikelytorequirecollaborationofadifferent
servicessuchaspathology,radiology&pharmacy
73
Continualefforts
Mustbemadetoimprovetheclinicallaboratoryserviceand
toimprovetheaccuracyandtimelinesofresultsasanaidto
thediagnosticprocess
Varioustoolsrequireincludingaudit,leanthinking,cause&
effect,&processredesign
Usuallynosingleimprovementtoolislikelytoprovideallthe
answerstoimproveanyonesituation
Essentialtoselectthetoolthatbestfitstheoneinhand
74
Factualapproachtodecisionmaking
Meansmakingdecisionsbasedonfactsandtheanalysisof
data
Thisshouldallowtheclinicallaboratorytomatchtheneeds
oftheserviceuserswithbusinessplanning
Example:purchaseequipmentforpresentthatwillalsomeet
futureneedsincreasedworkloads
Maketherightdecisionusing audit,incidentreporting,
statisticalanalysistopredictfuturetrends
75
Mutualbeneficialsupplierrelationship
Improvesservicesinanumberofways
Thus,ifthelaboratory&thesuppliersoflaboratory
equipment&itsmaintenance&servicecontracts,reagents
andconsumablesworktogether
Thiswillimprovethequalityofthelaboratoryprovision
76
Personnel
QMrequirestobeledbyseniormanagement
EmploypersonneltoimplementandmaintainQMs&the
clinicalgovernanceframework
Qualitymanagerorclinicalgovernancemanager
Needtoworkwiththeeightrequirements
77
Clinicalgovernance
IsaformofQMbroughtintoimprovethequalityofcarefor
patients
Safeguardhighstandardsofcare
Createanenvironmentthatallowsclinicalexcellenceto
flourish
Requiresthecreationofaculture,aswellassystems&
methodsofworkingtoensureopportunitiesforquality
improvementareidentified
Needtoprovideabetterexperienceforpatients&staff
78
Improvingthequalityofmedicallaboratories
Adversehealthcareevents
Sucheventsinhospitalsandclinicallaboratoriescanleadto
seriousharmtoapersonorevencostlives
Whendealingwithhundredsofsamplesperdayitisit
possibleforamistaketobemade
Laboratorieshaveefficientprocessesinplacethatstriveto
deliverthecorrecttestresulttotherightpatientinatimely
&efficientmanner
Thisensuresappropriatediagnosis&effectivetreatmentcan
begiven
79
Effectiveprocess
Onceestablishedthefocusshouldbeonthecontinual
improvementoftheoperationofthelaboratory
Thiscouldmeanestablishingmoreeffectivewaysofworking,
reducingcostsorimprovingtrainingopportunities
Toimprove mustlearnfrommistakes,fromanalysisofdata
&fromothers
Processofcontinualimprovement aneverendingcycle
80
Documentationintheclinicallaboratory
Possiblehierarchyofdocumentationinaquality
managementsystem
Top:qualitypolicy&qualitymanual
Middle:policies
Bottom:StandardOperatingProcedures(SOPs)
81
Documentation standards&requirements
ISO15189:2007MedicalLaboratories Particular
RequirementsForQualityAndCompetence
ISO22870:2006 PointOfCareTestingRequirementsFor
QualityAndCompetence
TheseOutlineASetOfCriteriaThatNeedToBeFulfilledBy
ClinicalLaboratories
TheseStandardsAlsoIncludeRequirementsForAQMsAs
OutlinedInISO9001qualityManagementSystems
Requirements
DocumentationMustBeProvidedToConfirmThat
LaboratoriesAreMeetingTheseStandards
82
Needtomeetrequirementsspecifiedinstandards
QMsinalaboratoryrequirequalitypolicy(QP),quality
manual&standardoperatingprocedures(SOPs)forthe
processesinplace
Therearealsoprocedurestocontrolthedocumentsandto
controlchangesmadetoanyprocesses
83
Minimumrequirements Qualitypolicies(QPs)
Areshortonepagedocumentthatoutlinesthescopeofthe
servicethelaboratoryintendstoprovide
Containsdetailsoflaboratoryscommitmenttoitsusersand
staff&outlinestheorganizationsqualityobjectives
Designedbyaseniormanager clinicaldirector
Communicated&availabletoalllaboratorystaff
Displayedonthenoticeboard
84
Qualitymanual
Documentationthatoutlineshowtheobjectivesstatedinthe
oparetobeachieved
Sectionsarelinkedtoorreferencedtostandardswithwhich
theorganisationistryingtocomply
ItalsocontainsorganisationalspecificinformationIncluding:
organisationalstructure,processesandprocedures&
resourcesneededtoimplementqualitytomeettoobjectives
describedinSOPs
85
StandardOperatingProcedures(SOPs)
SOPs arewritteninstructionsthatdescribeinaclear&
concisemannerhowproceduresandmethodsaretobe
performed
Howareindividualtaskstobecarriedout
Thatiswhatistobedone
Bywhomandwhen
86
SOPscontinued
Accuratelydescribetheprocess&methodsthataretobe
usedinthelaboratory
Containessentialinformationsuchashealth&safety
instructions
Correctuseofequipment
Correctuseofcontrols
AlwaysneeduptodateSOPs
Correctlydescribewhatistobedonetoensurenostepis
missed&thatthequalityofresultsarenotcompromised
87
Whatdidyoulearn?
88
Readinglist
Dept.OfHealth,London.
Technique5
th
89
MinistryofHealth
Lecture Quality Control Assurance III
Standards & Regulations
460
Aims
Tobefamiliarwithstandards&regulatoryrequirements
associatedwithaclinicallaboratory
91
Learningoutcomes
Tohaveaknowledgeandunderstandingofthekeystandards
andregulatoryrequirementsassociatedwithaclinical
laboratorysetting
Tofamiliarwiththetypesofgoodpracticeassociatedwith
andrequiredinaclinicallaboratorysetting
92
Standards&regulatoryrequirementsinthe
medicallaboratory
Clinicallaboratoriessubjecttorigorousinspectionsbyvarious
assessment&accreditationbodies
93
Accreditation
Accreditation is the process by which an organisation gains
recognition that its activities & products have attained a
certain level of compliance against a set of defined standards
94
UKAS
Thisbodyassessagainstnational&internationalstandardsas
publishedbytheBritishStandardsInstitute(BSI)&the
InternationalOrganisationforStandardization(ISO)
95
Standards
Arecodesofbestpracticethatimprovesafety&efficiency
ISOistheworld'slargestdeveloper&publisherof
internationalstandards(ISO9001)
TheClinicalPathologyAccreditationLtd(CPAUKLtd)which
ispartofUKAS setsstandardsrelatedtoISO15189&9001
TheHumanTissueAuthority(HTA)
TheMedicines&HealthcareProductsRegulatoryAgency
(MHRA) inspectclinicallabs
96
ClinicalPathologyAccreditation(CPA)Standards
Organisations that declare adherence to a defined standard
of practice & have their ability to attain these standards
independently confirmed that they are accredited can
reassure their users of the quality of service they provide
97
CPA
BasedonISO15189medicallaboratoriesparticular
requirementsforquality&competence
Thisaninternationalstandardthatincorporatessome
elementsofISO9001
98
Standards covereightmajorareas
1.Organisation&qualitymanagementsystem(astandards)
2.Personnel(bstandards)
3.Premises&environment(cstandards)
4.Equipment,informationsystems&materials(dstandards)
5.Preexaminationprocess(estandards)
6.Examinationprocess(fstandard
7.Postexaminationprocess(gstandard)
8.Evaluation&qualityassurance(hstandard)
99
Organisation&QMs
Needsandrequirementsofusers
QPdocumentation
Qualityobjectivesandplansforthelaboratory
Thequalitymanual
Documentcontrolprocedures,andcontrolprocessand
qualityrecord
100
Personnel
Personnelmanagement
Staffinduction
Recordsandmeetings
Stafftraining&education
101
Premises&Environment
Encompass:
Theworkareas
Staff&patientfacilities
Health&safetyinthelaboratory
102
Equipment,InformationSystems&Materials
Coversprocurement&managementofequipment,
materials,data&information
103
PreexaminationProcess
Encompasses:
Informationforusers
Specimencollection&handling
Requirementsforspecimenrequestforms
Transportofspecimens
104
Preexaminationprocess
Sets standards relating to the selection and validation of the
examination and the quality of the examination that is
performed on the specimens
105
PostexaminationProcess
Coversareassuchasfinalreport&howtheresultsare
reported
106
Evaluation&QA
Investigateshowthelaboratoryassessusersatisfaction,the
laboratoryauditprocess&qualityimprovementprocessesin
placeinthelaboratory
107
CPAAssessmentprocess
LaboratoryenrolwithCPA
Inspectedevery4yearsbyCPA
12dayexternalauditbytrainedassessors
Teamconsistsofaregionalassessorandatleasttwopeer
assessors
RegionalareemployedfulltimebyUKASandcoordinatethe
visit
Peerassessorsdrawnfromconsultantpathologists,clinical
scientists&biochemicalscientist&specificareas
(immunologyetc)
*Thereareotherbodies
108
BloodSafety&QualityRegulations(BSQR)
ChangedtwoEuropeandirectivesintoUKlaw
[directives2002/98/Ec&2004/33/Ec]
TheUKasadutytotranslatetheEuropeandirectivesinto
nationallegislation
BSQRrequireshospitalbloodbanks&bloodestablishments
tomaintainaqualitysystembasedontheprinciplesofGood
LaboratoryPractice(GLP)
109
GoodLaboratoryPractice(GLP)
GLPprovidestheframeworkwithinwhichlaboratoryworkis
performed
Themedicines&healthcareproductsregulatoryagency
(MHRA)
Assessesthecomplianceofhospitalbloodbanks&blood
establishmentstotheBSQR
110
MHRA
LaboratorysubmitsonlinecompliancereporttoMHRA
MHRAassessesonlinereportagainsttheBSQR
MHRAmaydecidetohaveanonsiteassessment/visit
Usuallyoneday&singleassessor
Assessorcheckstoseethatthereisaccurate&complete
traceabilityofbloodandbloodcomponents
111
Records
Recordskeptbythehospitallaboratoryandwardswillbe
examined
Alsocheckrecordsanddocumentsofsatellitesitesthat
receiveandstoreblood&bloodcomponents
112
Shot&Sabre
OnerequirementofBSQRisthatthelaboratorymustnotify
theMHRAandserioushazardsoftransfusion(SHOT)ofany
seriousadversebloodreactionsandevents(SABRE)
Onlinereportingsystemthatcanbeaccessedthroughthe
MHRAwebsite
ThereportingofSABRESallowsMHRA&SHOTteamsto
identifyanytrendsinbloodtransfusionincidents
113
HumanTissueAct(England2004)
Providesalegalframeworkforissuesrelatingtothetaking,
storage&useofhumantissue&organs&wholebody
donations
114
TheAct
Theactmakesmadeconsentthefoundationforthelawful
storage,anduseofhumanbodies,bodyparts,organsand
tissue&theremovaltissuefromthedeceased,&regulates
theuseoftissuesandcellsthatcanbeusedfortransplant&
stemcellresearchfromlovingdonors
115
PointofCareTesting(POCT)requirementsfor
quality&competence
POCTcanbedefinedastestingatornearthesiteofpatient
care
Increasesthelikelyhoodthatthepatientwellreceivethe
resultofaclinicaltestinatimelymanner
OftenusedinA&Edepartments
Neonatalcareunits
GPsurgeries
Thepatientshome
116
Standards
ISO22870:2006
PointofCareTesting(POCT) Requirementsforquality&
competenciesoutlinestherequirementsforPOCT
TheclinicallaboratorymustensurethatPOCTIscoordinated
effectively&efficientlyinthehospitalenvironment
Policies&proceduresmustbeinplacethatoutlinehowthe
equipmentmustbeused
Staffmustbetrainedonuseofequipment&recordsof
trainingmaintained
117
POCTcontinued
Medicolegalconsiderationsmustbetakenintoaccount
Requirementsofdataprotectionact
Laboratorymustbeinvolvedinthepurchaseofnew
equipmentforuseoutsideofthelaboratory
Mustbeinvolvedintrainingthestaffhowtouseit
Mustensurethatequipmentisservicedinaccordancewith
manufacturersrequirements
118
POCTcontinued
POCTIsnotassessedoraccreditedinisolationbutaspart
oftheoveralllaboratoryaccreditationprocess
119
Toolsandtechniquesforcontinualimprovement
Tocheckorassurethattheprocesses&methodsusedina
clinicallaboratorycomplywithrequiredstandards
Thesetoolsinclude:
Audit
RootCauseAnalysis(RCA)
120
Audit
ISO9001Definesanaudit:
Asystematic&independentexaminationtodetermine
whetherqualityactivities&relatedresultscomplywiththe
plannedarrangementsandwhetherthesearrangementsare
implementedeffectivelyandaresuitabletoobjectives
121
Auditcontinued
IntegralpartofanyQMs
Requirementforaccreditation(CPA)
Meansforcontinuallyimprovingtheserviceproducedbya
laboratory
Identifyareasforimprovementbygatheringinformation
observation/interview/sampling
Alsoidentifiesprocessesthatareworkingwell
Opportunitytoidentifygoodpracticeandtransferthedetails
122
Auditcontinued
Enablesevaluationofprocesses
Determinedeficiencies
Generateacosteffective&efficientsolutionstoproblems
123
Auditcontinued
Checkpracticesagainstprocedures&tothoroughly
documentanydifferences
Onlyasampleexercisesocannotconfirmthatallaspectsof
theprocessarebeingcompliedwithatalltimes
124
Typesofaudit
1.Vertical
2.Horizontal
3.Witnessorexamination
4.Selfassessment
125
Exampleprocess
Step1.Startofprocess skinlesionremovedfrompatient
Step2.Sampletransportedtolab&receivedbysamplereception#
Step3.Samplelabelled,detailsaddedtocomputer
Step5.Samplesuitablyprocessedinlab
Step6.Slidesmadefromsample
Step7.Slidesstained,coverslipsadded.&Checkedforproblems
Step8.Slidesexaminedbyconsultant&reporttyped
Step9.Reportissuedendofprocess
126
466
Vertical&Horizontal
Verticalaudit examinesasingleitemfromstarttofinish(
step1.Tostep9previousslide)
Horizontalaudit examinesoneelementinaprocessthatis
performedonmorethatoneitem(step6.EXAMINESMANY
SLIDESFORONEELEMENT;sectionquality)
127
AWitness/ExaminationAudit
Examinesthepersonundertakingatask
Step3 observingapersonlabellingsamplestocheckthat
heorshehaveread&arefollowingtherelevantsop
128
SelfAssessmentAudit
This is a careful evaluation that is usually performed by the
organisations own management which results in an opinion
or judgement as to its effectiveness & efficiency
129
Internal,External&CooperativeAudits
Internal;conductedbyclinicallaboratorystaffthemselves
Example:
1.Biomedicalscientistinhaematologycouldconductaninternal
auditinthemicrobiologylab
2.Aninternalverticalauditcouldbeconductedbythelabstaffon
aclinicalsamplesubjecttoaparticularprocessormethod
IdentifyanynoncompliancewithstandardsorinQMshence
makerecommendations
130
Externalandcooperativeaudits
External:
Conductedbyanoutsidepersonorbody
Possibleaccreditation
Anexternalhorizontalcanbeconductedonaspecific
element(stafftrainingrecords)
Cooperative:
Conductedbetweenthelab&anotherpartyformutual
benefitclinicalaudits/usersatisfaction
surveys/benchmarkingactivities
131
Auditplanorschedule
Mustbedonebeforetheauditcanbecarriedout
Whatauditsaretobeundertaken,when,where&whowill
beperformingthem
Mustbediscussed&agreedbyseniormanagementatthe
beginningoftheyear
132
467
Stagesofanaudit
Fourstages:
1.Preparetheauditchecklist
2.Undertaketheaudit
3.Writeasummaryoftheaudit
4.Undertakecorrectiveactiontoimprovethelaboratory
process
133
Auditschedule
Auditoflaboratoryequipment:
January horizontalauditonequipmentsuchas
analysers/centrifuges/microscopesusedinlab
Staffrecords:
February horizontalauditonCPDofstaff
Transportofsamples:
March verticalauditonreceiptofsamplesfromgeneral
practitionerstotheissuingofresultsofclinicaltests
134
TheAuditCycle
Aneverendingcycleofimprovements
Step1.Plan:theauditschedule&checklist
Step2.Do:undertaketheaudit,report&identifynon
compliance
Step3.Act:putinplacechanges/actionstocloseoutthe
noncompliance
Step4.Check:thatactionsareinplace&areworking
Backtostep1.
135
IncidentReporting
Majortoolforexaminingwhathappenedwhenanerroror
problemoccurred
Anappropriatesystemisrequired
Musthaveacoreofsound&representativeinformationon
whichtobaseanyanalysisoferrors/problems
Inordertomakerecommendationstopreventtheir
repetition
Valueinsystematicapproachestoreporting&recording
adverseevents
136
AdverseEvents
Allcountrieshaveorganisationsorbodiesthatrecordthese
eventsloggedincentraldatabases
Canbecausedbymedicaldevices,labequipment,exposure
tobiologicalsamples,reagentsorpharmaceuticalproducts
Incidentsassignedaspecifictypeorlevel
Outcomesofinvestigationssubjecttoformalreview
Issueguidanceofhowtoimproveprocessandreducefurther
risk
Donotignorehealthcarenearmisses report!!
137
RootCauseAnalysis(RCA)
Estimatedthatforeverymajorinjury29minoronesand300
accidentswithnoinjuryoccur
Apowerfultoolthatidentifiesrecords&visualisesthe
possiblecausesofaproblem,errororincident
Investigationintotheerrortodetermineitsbasisthatisits
rootcause
138
468
BasisofRCA
Breakdownproblemintosmallermoremanageabletasks
whichallowstheproblemtobeidentifiedanddescribed
Assistinfindingasolutionorcorrectiveactionthatwill
preventthesameproblemreoccurring
139
Example:cause&effectdiagramillustratinghowan
equipmentfailureshouldbeinvestigated
Potentialcauseoffailure:
1.Maintenance&cleaningofequipmentnotundertaken
2.Lackoflabstaffleadingtoincompleteorabsenceof
QAchecks
3.Lackofstafftraininginrecognisingproblems
4.Reagentspassedtheirexpirydates
1.To4.Effectistogiveinaccurateresultstopatients
140
Whatdidyoulearn?
141
ReadingList
Dept.OfHealth,London.
Technique5
th
142
469

Laboratory Trainer Theory

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Laboratory Trainer Theory

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The Training Program for Health Institute Graduates has been

developed for the health institute graduates of the Kingdom of

is total number of cells in the population.

Normal solution of NaOH = =

increasing blood HCO

through the kidneys

This gap can detect the renal cause of metabolic acidosis.

5. Ammonia and Glutamine:

Yes, there is a matching pattern!

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