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INTRODUCTION, ROLES OF

BIOCHEMICAL LABORATORY.
MECHANIZATION AND
AUTOMATION IN CLINICAL
BIOCHEMISTRY
CLINICAL BIOCHEMICAL
ANALYSES OF PROTEINS, PLASMA
PROTEIN SPECTRUM.
Introduction to Clinical biochemistry
• CLINICAL BIOCHEMISTRY (also known as clinical chemistry or
chemical pathology) is the laboratory service absolutely essential for
medical practice or branch of laboratory medicine in which chemical
and biochemical methods are applied to the study of disease.
• The results of the biochemical investigations carried out in a clinical
chemistry laboratory will help the clinicians to determine the diseases
(diagnosis) and for follow-up of the treatment/recovery from the
illness (prognosis).
Introduction to Clinical biochemistry
Introduction to Clinical biochemistry
• The use of biochemical tests:
• Biochemical investigations are involved in every branch of clinical medicine.
• The results of biochemical tests may be of use in:
• diagnosis and in the monitoring of treatment.
• screening for disease or in assesing the prognosis.
• reseach into the biochemical basis of disease
• clinical trials of new drugs
• Biochemical investigations hold the key for the diagnosis and prognosis of
diabetes mellitus, jaundice, myocardial infarction, gout, pancreatitis,
rickets, cancers, acid-base imbalance etc. Successful medical practice is
unimaginable without the service of clinical biochemistry laboratory.
Introduction to Clinical biochemistry
• In general, biochemical tests can be broadly divided into two groups:
• In discretionary or selective requesting, the tests are carried out on the
basis of an individual patient's clinical situation. The case for discretionary
requesting has been put admirably (Asher, 1954):
• 1. Why do I request this test?
• 2. What will I look for in the result?
• 3. If I find what I am looking for, will it affect my diagnosis?
• 4. How will this investigation affect my management of the patient?
• 5. Will this investigation ultimately benefit the patient?
• In contrast, screening tests are used to search for disease without there
being any necessary clinical indication that disease is present.
Introduction to Clinical biochemistry
Test selection for the purposes of discretionary testing
Category Example
To confirm a diagnosis Plasma (free T4) and (thyroid-
stimulating hormone, TSH) in suspected
hyperthyroidism
To aid differential diagnosis To distinguish between different forms
of jaundice
To refine a diagnosis Use of ACTH to localize Cushing's
syndrome
To asses the severity of disease Plasma (creatinine) or (urea) in renal
disease
To monitor progress Plasma (glucose) to follow of patients
with diabetes mellitus
To detect complications or side effects ALT measurements in patients treated
with hepatotoxic drug
To monitor therapy Plasma drug concentration in patients
treated with antiepileptic drugs
Introduction to Clinical biochemistry
• Table 1.4 Examples of tests used in case-finding programmes.
Programmes to detect diseases in Chemical investigations
Neonates:
PKA (phenylketonuria) Serum [phenylalanine]

Hypothyroidism Serum [TSH] and/or [thyroxine]


Adolescents and young adults:
Substance abuse Drug screen
Pregnancy:
Diabetes mellitus in the mother Plasma and urine [glucose]

Open neural tube defect (NTD) in the Maternal serum [a-fetoprotein]


foetus
Industry:

Industrial exposure to lead Blood [lead]

Industrial exposure to pesticides Plasma cholinesterase activity


Malnutrition Plasma [albumin] and/or [pre-albumin]

Thyroid dysfunction Plasma [TSH] and/or [thyroxine]


Introduction to Clinical biochemistry
• ADVANTAGES OF SCREENING
• First, an uncommon or unexpected disease may be found and
created. Second, the early requesting of a battery of tests might be
expected to expedite management of the patient. Most studies have
not shown this to be so.
Disease Unexpected abnormal test results

Hyperparathyroidism Raised plasma calcium

Hypothyroidism Raised plasma TSH and/or a low T4

Diabetes mellitus High random plasma glucose

Renal tract disease Raised plasma creatinine or urea

Liver disease Increased plasma ALT, AST


Introduction to Clinical biochemistry
• DISADVANTAGES OF SCREENING
• It is easy to miss significant abnormalities in the 'flood' of data coming from the
laboratory, even when the abnormalities are 'flagged' in some way. Most of the
abnormalities detected will be of little or no significance, yet may need additional time-
consuming and often expensive tests to clarify their importance (or lack of it).
• In other instances, to simplify requesting, a wide range of tests are routinely requested
on all patients in a particular category, for example, admission screening on all those
admitted through the Accident and Emergency (A&E) Department. Mention should also
be made of batteries of tests which are generally requested on a discretionary basis but
where the test group collectively provides information about an organ system (e.g. tests
for liver disease) or a physiological state (e.g. water and electrolyte status). Many
laboratories analyse and report these functional or organ-related groups. For example, a
'liver function test' group might consist of plasma bilirubin, alanine aminotransferase
(ALT), alkaline phosphatase (ALP), f-glutamyltransferase (GGT) and albumin
measurements.
Introduction to Clinical biochemistry
Introduction to Clinical biochemistry
• Clinical biochemical tests comprise over ⅓ of all hospital laboratory investigations.

• Core biochemistry: Most biochemistry laboratories provide the "core analyses", commonly
requested tests which are of value in many patients, on a frequent basis.
• Core biochemical tests:
• Sodium, potassium, chloride and bicarbonate
• Urea and creatinine
• Calcium and phosphate
• Total protein and albumin
• Bilirubin and alkaline phosphatase
• Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST)
• Glucose
• Amylase…….
Introduction to Clinical biochemistry
• Specialized tests:
• Not every laboratory is equiped to carry out all possible biochemistry requests.
• Large departments may act as reference centres where less commonly asked for tests
are performed.
• Specialized tests:
• Hormones
• Specific proteins
• Trace elements
• Vitamins
• Drugs
• Lipids and lipoproteins
• DNA analyses
Introduction to Clinical biochemistry
• The emergency lab:
• All clinical biochemistry laboratories provide facilities for urgent tests.
An urgent test is designated as one on which the clinician is likely to
take immediate action. The main reason for asking for an analysis to
be performed on an urgent basis is that immediate treatment
depends on the result.
Introduction to Clinical biochemistry
• TYPES OF LABORATORY TESTS:
• The biochemical investigations (on blood/ plasma/serum) carried out in the
clinical biochemistry laboratory may be grouped into different types.
• 1. Discretionary or on-off tests : Most common clinical biochemistry tests that
are designed to answer specific questions, e.g., does the patient have increased
blood urea/glucose concentration? Normally, these tests are useful to support
the diagnosis.
• 2. Biochemical profiles : These tests are based on the fact that more useful
information on the patients disease status can be obtained by analysing zor more
constituents rather than one e.g., plasma electrolytes (Na+, K+, Cl-, bicarbonate,
urea); liver function tests (serum bilirubin, ALT, AST).
• 3. Dynamic function tests : These tests are designed to measure the body's
response to external stimulus e.g., oral glucose tolerance test (to assess glucose
homeostasis) : bromosulphthfein test (to assess liver function).
Introduction to Clinical biochemistry
• 4. Screening tests : These tests are commonly employed to identify
the inborn errors of metabolism, and to check the entery of toxic
agents (pesticides, lead, mercury) into the body.
• 5. Metabolic work-up tests : The programmed intensive
investigations carried out to identify the endocrinological disorders
come under this category.
• The term emergency tests is frequently used in the clinical laboratory.
It refers to the tests to be performed immediately to help the clinician
for proper treatment of the patient e.g., blood glucose, urea, serum
electrolytes.
Introduction to Clinical biochemistry
• There are over 400 different tests which may be carried out in clinical
biochemistry laboratories. They vary from the very simple, such as
the measurement of sodium, to the highly complex, such as DNA
analysis, screening for drugs, or differentiation of lipoprotein variants.
Many high volume tests are done on large automated machines. Less
frequently performed tests may be conveniently carried out by using
commercially prepared reagents packaged in "kit" form. Some
analyses are carried out manually.
Introduction to Clinical biochemistry
• Specimen collection:
• The biological fluids employed in the clinical biochemistry laboratory
include blood, urine, saliva, sputum, faeces, tissue and cells,
cerebrospinal fluid, peritoneal fluid, synovial fluid, pleural fluid,
stones.
• Among these, blood (directly or in the form of plasma or serum) is
frequently used for the investigations in the clinical biochemistry
laboratory.
Introduction to Clinical biochemistry
• Identification of patients and specimens
• The correct patient must be appropriately identified on the specimen and
request form, as follows:
• 1. Patient identification data (PID). This usually comprises name plus
unique number.
• 2. Test request information. This includes relevant clinical details (including
any risk of infection hazard), the tests to be performed and where the
report is to be sent.
• 3. Collection of specimens. In the correct tube and the appropriate
preservative.
• 4. Matching of specimens to requests. Each specimen must be easily and
unequivocally matched to the corresponding request for investigations.
Introduction to Clinical biochemistry
• Some commoner causes of errors arising from use of the laboratory.
Error Consequence
Crossover of addressograph labels between This can lead to two patients each with the other's set of results. Labels between patients Where the
patients patient is assigned a completely wrong set of results, it is important to investigate the problem in case there is a
second patient with a corresponding wrong set of results

Timing error There are many examples where timing is important but not considered. Sending in a blood sample too early after
the administration of a drug can lead to misleadingly high values in therapeutic monitoring. Interpretation of
some tests (e.g. cortisol) is critically dependent on the time of day when the blood was sampled

Sample collection tube error For some tests the nature of the collection tube is critical which is why the Biochemistry Laboratory specifies this
detail. For example, using a plasma tube with lithium-heparin as the anticoagulant invalidates this sample tube
for measurement of a therapeutic lithium level! Serum electrophoresis requires a serum sample; otherwise, the
fibrinogen interferes with the detection of any monoclonal bands. Topping up a biochemistry tube with a
haematology (potassium-ethylenediamine tetraacetic acid (EDTA) sample) will lead to high potassium and low
calcium values in the biochemistry sample

Sample taken from close to the site of an The blood sample will be diluted so that all the tests will be correspondingly site of an intravenous (IV) infusion
intravenous infusion low with the exception of those tests which might be affected by the composition of the infusion fluid itself. For
example, using normal saline as the infusing fluid would lead to a lowering of all test results but with sodium and
chloride results which are likely to be raised

Analytical error Although comparatively rare, these do inevitably happen from time to time and any result which is unexpected
should lead the requesting clinician to discuss the matter further with the Laboratory. Transcription errors within
the Laboratory are increasingly less common because of the electronic download of results to the Laboratory
computer as a source of the printout or results on the VDU. Most errors generated within the Laboratory occur at
the Reception as a result of mislabelling of samples within the Laboratory
Introduction to Clinical biochemistry
• COLLECTION OF BLOOD:
• Venous blood is most commonly used for a majority of biochemical
investigations. It can be drawn from any prominent vein (usually from a
vein on the front of the elbow).
• Capillary blood (<0.2 ml) obtained from a finger or thumb, is less
frequently employed.
• Arterial blood (usually drawn under local anesthesia) is used for blood gas
determinations.

• Precautions for blood collection : Use of sterile (preferably disposable)
needles and syringes, cleaning of patients skin, blood collection in clean
and dry vials/tubes are some of the important precautions.
Introduction to Clinical biochemistry
• Biochemical investigations can be performed on 4 types of blood
specimens – whole blood, plasma, serum and red blood cells. The
selection of the specimen depends on the parameter to be estimated.
• 1. Whole blood (usually mixed with an anticoagulant) is used for the
estimation of hemoglobin, carboxyhemoglobin, pH, glucose, urea,
non-protein nitrogen, pyruvate, lactate, ammonia etc. (Note : for
glucose determination, plasma is prefered in recent years).
• 2. Plasma, obtained by centrifuging the whole blood collected with
an anticoagulant, is employed for the parameters—fibrinogen,
glucose, bicarbonate, chloride, ascorbic acid etc.
Introduction to Clinical biochemistry
• 3. Serum is the supernatant fluid that can be collected after
centrifuging the clotted blood. It is the most frequently used
specimen in the clinical biochemistry laboratory. The parameters
estimated in serum include proteins (albumin/globulins), creatinine,
bilirubin, cholesterol, uric acid, electroylets (Na+, K+, Cl-), enzymes
(ALT, AST, LDH, CK, ALP, ACP, amylase, lipase) and vitamins.
• 4. Red blood cells are employed for the determination of abnormal
hemoglobins, glucose 6-phosphate dehydrogenase, pyruvate kinase
etc.
Introduction to Clinical biochemistry
Introduction to Clinical biochemistry
• Collection and preservation of blood specimens
• Lack of thought before collecting specimens or carelessness in collection may adversely
affect the interpretation or impair the validity of the tests carried out on the specimens.
Some factors to consider include the following:
• 1. Diet Dietary constituents may alter the concentrations of analytes in blood
significantly (e.g. plasma [glucose] and [triglyceride] are affected by carbohydrate and
fat-containing meals, respectively).
• 2. Drugs Many drugs influence the chemical composition of blood. Such effects of drug
treatment, for example, antiepileptic drugs, have to be taken into account when
interpreting test results. Details of relevant drug treatment must be given when request-
ing chemical analyses, especially when toxicological investigations are to be performed.
• 3. Diurnal variation. The concentrations of many substances in blood vary considerably
at different times of day (e.g. cortisol). Specimens for these analyses must be collected at
the times specified by the laboratory, as there may be no reference ranges relating to
their concentrations in blood at other times
Introduction to Clinical biochemistry
• Care when collection blood specimens
• The posture of the patient, the choice of skin-cleansing agent and the
selection of a suitable vien (or other source) are the principal factors to
consider before proceeding to collect each specimen:
• 1. The skin must be clean over the site for collecting the blood specimen.
However, it must be remembered that alconol and methylated spirits can
cause haemolysis, and that their use is clearly to be avoided if blood
[ethanol] is to be determined.
• 2. Limbs into which intravenous infusions are being given must not be
selected as the site of venepuncture unless particular care is taken. The
needle or cannula must first be thoroughly flushed out with blood to avoid
dilution of the specimen with infusion fluid.
Introduction to Clinical biochemistry
• 3. Venepuncture technique should be standardised as far as possible to enable
closer comparison of successive results on patients.
• 4. Venous blood specimens should be obtained with minimal stasis Prolonged
stasis can markedly raise the concentrations of plasma proteins and other non-
diffusible substances (e.g. protein-bound substances). It is advisable to release
the tourniquet before withdrawing the sample of blood.
• 5. Posture should be standardised if possible When a patient's posture changes
from lying to standing, there may be an increase of as much as 13% in the
concentration of plasma proteins or protein-bound constituents, due to
redistribution of fluid in the extracellular space.
• 6. Haemolysis should be avoided, since it renders specimens unsuitable for
plasma K+, magnesium and many protein and enzyme activity measurements.
• 7. Infection hazard High-risk specimens require special care in collection, and
this danger must be clearly indicated on the request form.
Introduction to Clinical biochemistry
• Care of blood specimens after collection
• Blood specimens should be transported to the laboratory as soon as
possible after collection. Special arrangements are needed for some
specimens (e.g. for acid-base measurements, or unstable hormones)
because of their lack of stability. Most other analytes are stable for at
least 3 h in whole blood, or longer if plasma or serum is first sepa-
rated from the cells. As a rule, whole blood specimens for chemical
analysis must not be stored in a refrigerator, since ionic pumps that
maintain electrolyte gradients across the cell membrane are inactive
at low temperatures. Conversely, separated serum or plasma is best
refrigerated, to minimize chemical changes or bacterial growth.
Introduction to Clinical biochemistry
• Several changes occur in whole blood specimens following collection. The
commoner and more important changes that occur prior to the separation
of plasma or serum from the cells are:
• 1. Glucose is converted to lactate: this process is inhibited by fluoride;
• 2. Several substances pass through the erythrocyte membrane, or may be
added in significant amounts to plasma as a result of red cell destruction
insufficient to cause detectable haemolysis. Examples include K+ and
lactate dehydrogenase;
• 3. Loss of CO2 occurs, since the Pco2, of blood is much higher than in air;
• 4. Plasma [phosphate] increases due to hydrolysis of organic ester
phosphates in the red cells;
• 5. Labile plasma enzymes lose their activity.