Good morning

Lab investgations in oral and maxillofacial surgery

Dr.ch.v.rao

Contents
Hematology: -Estimation of haemoglobin -Total RBC count -Total & Differential WBC count -Blood group determination

-Determination of Haematocrit -RBC indices -ESR -Absolute eosinophil and platlet count -BT/CT/PT/PTT/APTT -Blood banking techniques

 Clinical microbiology

- Various staining techniques - Culture media - Diagnostic serology

 Clinical chemistry:
- Organ function tests - Determination of serum bilirubin - Thyroid function test - Tests for diabetes - Total serum protein estimation

Routine examination of body fluids and faeces - Routine examination of urine - Routine examination of feces - Examination of sputum - Examination of cavity fluids

EFFECTIVE DIAGNOSIS HAS 4 ESSENTIAL REQUISITES

1.Awareness of pattern of clinical presentation 2.Obtaining a complete history 3.Undertaking a systematic clinical examination 4.Formulating an appropriate investigative plan

 Clinical symptoms of some diseases are non specific, underlying conditions may

not be suspected from the history alone and at times the physical examination may be surprisingly unrevealing
Its often the routine investigations that

suggest the presence of underlying pathology.

NATURE OF WORK : Four major areas in most clinical laboratories.

Hematology Clinical microbiology Clinical chemistry Routine examination of body fluids and faeces

Purpose of diagnostic tests: Help to confirm a diagnosis Monitor an illness Provide valuable information about the clients response to treatment

Diagnostic testing involves three phases:Pretest The major focus of the pretest phase is client preparation The nurse must know what equipment and supplies are needed for the specific test What type of sample will be needed and how it will be collected? Does the client need to stop oral intake for a certain number of hours prior to the test?

 Does the test include administration of contrast media and if so, it is injected or swallowed? Are medications given or withheld? Is consent form required? How long is the test?

Intratest It focuses on specimen collection and performing or assisting with certain diagnostic testing. The nurses uses of special standard precautions and sterile techniques Provide emotional and physical support while monitoring the client as needed (e.g., V /S, pulse oximetry, ECG). Correct labeling, storage, and transportation of the specimen to avoid invalid test results.

Post test The focus of this phase is on nursing care of the client and follow up activities and observations. Compare the previous and current test results Modify nursing interventions as needed Report the results to appropriate health team members.

According to STEINER
The laboratory tests and profiles can be classified into three groups.

Group 1 : Those that play an integral role in confirming the diagnosis of tissue changes confronted by the clinician.
Group II : Those that bear directly on the management

of a patient during surgery Group III :Those that may have a bearing on overall health and management of patient.

Group 1 tests
Includes diagnostic procedures i.e. Biopsy Oral cytology Bacterial smears and cultures

(Group II) tests

1. Complete blood count 2. Urine analysis 3. Blood chemistry 4. Determination of BT/CT/PT/PTT

Estimation of heamoglobin

Measured commonly to obtain information about circulating RBC and the amount Of oxygen carrying substance they contain

Sahlis method: Cynomethemoglobin method: Alkaline haematin method: Haldens carboxyhaemoglobin method: Oxyhaemoglobin method: Gasometric determation method Specific gravity method Photometric method

Principle of Sahlis method: When blood is added to 0.1N HCl, Hb is converted to brown colored acid hematin. The resulting color after dilution is compared with standard brown glass reference of Sahli hemoglobinometer.

 Principle of Cynomethemoglobin method:

When blood is mixed with Drakins reagent containing Potassium Cyanide and Ferric cyanide, Hb reacts with Ferric cyanide to form methemoglobin which is converted to stable Cynomethemoglobin by the cyanide. The intensity of color is proportional to Hb concentration and it is compared with a known Cynomethemoglobin standard at 540nm (green filter.)

 Normal values Children (upto 1 yr) Children (10 -12 yrs)

Men
Women

Hb, g/dl 11.0 13.0 11.5 14.5 13 18 12 16

Clinical Significance
A decrease in Hb is an indication of anemia Definition : reduction below the normal in the volume of packed red cells as measured by hematocrit or decrease in hemoglobin concentration of blood

Classification
Most common and simple classification which is based on the morphologic characteristics of cells is: Based on size of cells: 1. Normocytic

2. Microcytic 3. Macrocytic

Based on hemoglobinisation of cells: 1. Normochromic 2. Hypochromic

 Microcytic/hypochromic anemia (decreased MCV, decreased MCHC)

Iron deficiency (common)

Thalassemia (common, except in people of

Germanic, Slavonic, Baltic, Native American, Han Chinese, Japanese descent) Anemia of chronic disease (uncommonly microcytic) Sideroblastic anemia (uncommon; acquired forms more often macrocytic) Lead poisoning (uncommon) Hemoglobin E trait or disease (common in Thai, Khmer, Burmese,Malay, Vietnamese, and Bengali groups

 Macrocytic/normochromic anemia (increased MCV, normal MCHC)

Folate deficiency (common)

B12 deficiency (common)

Myelodysplastic syndromes (not uncommon,

especially in older individuals) Hypothyroidism (rare)

 Normochromic/normocytic anemia (normal

MCV, normal MCHC)

Normoregenerative normocytic anemias

(appropriate reticulocyte response)

Immunohemolytic anemia Glucose-6-phosphate dehydrogenase (G6PD) deficiency (common) Hemoglobin S or C Hereditary spherocytosis Microangiopathic hemolytic anemia Paroxysmal hemoglobinuria

 Hyporegenerative normocytic anemias

(inadequate reticulocyte response)

Anemia of chronic disease Anemia of chronic renal failure Aplastic anemia*

Drugs and other substances that have caused aplastic anemia include the following:

 Hemoglobin concentration drops in pregnancy due to

hemodilution

Increase in Hb concentration occurs in

Hemoconcentration (reduced plasma volume) due to loss of body fluid in severe diarrhea and vomiting, prolong deprivation of water, excessive use of diuretics

High values are also observed in congenital heart

disease due to decreased O2 supply and also in polycythemia,.

Total Erythrocyte Count

Principle:
The blood is diluted as 1: 200 with the RBC diluting fluid and cells are counted under high power objective by using Neubauer counting chamber. Sodium chloride 0.5 gm Sodium sulphate 2.5 gm Murcuric chloride 0.25 gm Distilled water 100 ml

 Normal values:

Men 4.5 6 x 106 cells/ cu mm Women 4 4.5 x 106 cells/ cu mm

Clinical Significance: At birth, the total RBC count varies from 6.5 7.25 million/ cu mm. There is a steady decline after a few hours and at the end of 15 days to 1 month there is a slow rise to normal adult levels.

Increase in RBC count is observed in conditions like

Hemoconcentration due to burns, Cholera, Central cyanotic condition, Emphysema, Pulmonary fibrosis, Cor pulmonale, Living at high altitude, Certain erythropoietin secreting tumors i.e. Renal cell carcinoma and hepatocellular carcinoma

Decrease in RBC count is seen in conditions like Old age, Pregnancy, Anemic state, Leukemias, Bbone marrow suppression, Hemorrhage.

Total Leukocyte Count

Principle: The blood specimen is diluted as 1:20 in a WBC pipette with the diluting fluid (Turks fluid) and the cells are counted under low power by using Neubauer counting chamber. Turks fluid: Glacial acetic acid destroys RBCs 1% Gention violet stains WBCs Distilled water

 Normal values:

At birth: 1-3 yrs: 4-7 yrs: 8-12 yrs: Adults:

10000 25000 cells/ cu mm 6000 18000 cells/ cu mm 6000 15000 cells/ cu mm 4500 13500 cells/ cu mm 4000 11000 cells/ cu mm

Clinical Significance

Increase in total WBC count of more than 11000 / cu mm is called leukocytosis and decrease of less than 4000 / cu mm is called leukopenia.

Causes of leukocytosis: Pathological: Common for a transient period in infections, certain tumors, myloproliferative disorders, endotoxemias, hypoxia Also observed severe hemorrhage and leukaemia Physiological: High temperature, muscular exercise At birth total leukocyte count is about 18000 / cu mm At full term of pregnancy total count is about 12000 15000 / cu mm.

 Causes of leukopenia:

Certain viral infections like Hepatitis, HIV. Bone marrow depression Anemia Following treatment with: Glucocorticoids, cytotoxic drugs Autoimmune disorders and malnutrition

Differential Leukocyte Count

Principle:
The polychromic staining solutions like Leishmans contain methylene blue and eosin. These basic and acidic dyes induce multiple colors. The cytoplasm of WBCs is stained by acidic dye and the nucleus is stained by the basic dye. The neutral component of the cells are stained by both the dyes.

Normal values
Neutrophils : Eosinophils : Basophils : Lymphocytes : Monocytes : 40 - 75 % 14 % 01 % 20 45 % 2 -8 %

Clinical Significance

Differential count is useful to identify changes in the distribution of white cells and also to determine the severity of a disease and the degree of response of the body.

Neutrophilic leukocytosis Eosinophlia

Acute bact. Infections, tissue necrosis caused by MI & burns Allergic conditions: asthama, hay fever, pemphigus v.,malignancies like hodgkins and non-hodgkins lymphomas. Monocytosis TB, bact.endocarditis, malaria, SLE. Basophilia Rare often its an indicator of myloproliferative disorder like CML. Lymphocytosis TB, Brucellosis, infection caused by hepatitis A, CMV, EBV, Thyrotoxicosis.

Neutropenia

Starvation, infections and toxemias in elderly, typhoid,malaria,hepatiti s,hypersplenism,bone marrow suppression.

Eosinopenia
Lymphopenia

Typhoid, aplastic anemia, pt. on steroids

Severe bone marrow supression, immunosupressive therapy, hodgkins disease, irradiation. Aplastic anemia.

Monocytopenia

BLOOD

GROUPING

Around 30 different common antigens and hundreds of rare ones 21 different systems. Common ones are:

ABO System
Rh system Lewis system

MNS system
P system Kell system

Duffie system
Lutheran system

ABO system & Rh system

Karl Landsteiner 1900 & 1940

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Demographics
BLOOD TYPE
O A B AB

population %
40 - 65% 35 - 45% 5 - 10% 3 - 9%

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Determination of Hematocrit (PCV)

PCV is the amount of packed red blood cells following centrifugation and expressed as percentage of the total blood volume. OR Percentage of volume occupied by red blood cells in relation to total volume of blood in centrifuged capillary tube.

Principle of Macro Haematocrit method: when an anticoagulated blood is centrifuged in a haematocrit tube at high speed, the erythrocytes sediment at the bottom. The red cell column is called PCV or haematocrit

Principle of Micro Hematocrit method: Blood is centrifuged in a sealed capillary tube and PCV is determined by a special hematocrit reader.

Used : 1. Pediatric patients 2. difficulty in drawing Sufficient amount of blood

Normal values
Birth 1-2 years 8 12 years Males Females pregnancy 50 56 % 32 38 % 36 42 % 42 52 % 36 48 % 23 37 %

Clinical Significance
Decrease in hematocrit values are observed in anemias, hydremia (excessive fluids in the blood) as occurs in pregnancy.

Increase in hematocrit values are observed in polycythemia, dehydration, emphysema, congenital

Color and opacity of plasma Yellow - Jaundice

Milky Lipemia

Cloudy - Multiple myloma

Reddish - Hemolysis

Determination of RBC indices (wintrobes constant)

The main indices calculated are Mean corpuscular volume (MCV) Mean corpuscular hemoglobin (MCH) Mean corpuscular hemoglobin concentration (MCHC) Color index (CI)

Mean corpuscular volume (MCV)

Average volume of a single red blood cell and it is expressed in cubic microns i.e.(femtoliters) MCV = PCV x 10 Rbcs/Cubic mm The normal MCV is 90 cum (78 to 90 cum). When MCV is increased, the cell is known as macrocyte and microcyte means the cell with reduced volume.

Increased :

Macrocytic Anemia

Decreased :

Microcytic Anemia

Mean corpuscular Hemoglobin (MCH)

This is the quantity or amount or content of

hemoglobin present in one red blood cell.

MCH = Hb x 10 Rbcs in millions/cu mm. Expressed in microgram or picogram (pg)
Normol value 30 pg (27-32 pg)

Increases: Macrocytic anemia (may also remain normal) Decreases : Hypochromic anaemia

Mean corpuscular Hemoglobin concentration (MCHC)

Portion of average red blood cell containing hemoglobin Amount Of Hemoglobin Expressible In Relation To The Volume Of One Red Blood cell.

MCHC = Hb x 100 PCV

Most absolute important value in the diagnosis of anemia. Normal value 30% (28-38%) Decreased : Hypochromic anemia particularly in microcytic hypochromic anemia

Increased : Hereditary spherocytosis

Color index
It is an expression of the mean Hb content of a

single cell in comparison to that of an arbitrary normal cell

Color index = Hb%

Rbc% Normal is 1 (0.85 -1.2%)

Important in determining the type of anemia

Increased : Pernicious anemia Megaloblostic anemia Reduced : Iron deficiency anemia( microcytic & hypochromic anemia) Normal: Normocytic normochromic anemia

Erythrocyte Sedimentation Rate

The rate at which the red blood cells sediment to the bottom of the test tube is known as Erythrocyte sedimentation rate.

Buffy layer

Normal values (Westergrens method) Male 5 15 mm after 1 hour Female 5 20 mm after 1 hour Normal values (Wintrobe method) Male 0 9 mm after 1 hour Female 0 20 mm after 1 hour Normal values (Landau method) Male 0 5 mm after 1 hour Female 0 8 mm after 1 hour

E.S.R. determination is useful to determine the progress of the disease and not specific for diagnosis of a perticular disease except in rheumatic fever (minor criteria).

Clinical Significance
ESR is increased in any condition causing an increase in fibrinogen or globulins like rheumatic fever, multiple myeloma, kala azar, anemias, high temperature, malignancies. ESR is greater in women than in men During pregnancy ESR gradually increases after 3rd month and returns to normal in about 3-4 weeks after delivery

Low blood count tend to accelerate ESR

Drugs : Theophylline, vitamin A, Oral contraceptives

Clinical Significance
ESR is decreased in polycythemia, sickle cell anemia, hypochromic anemia, severe dehydration, gastritis, c.c.f. and in infants.

Determination of Platelet Count

Principle: The blood is diluted to as 1:200 with the
diluting fluid using the RBC pipette. After discarding the first drop, the specimen is transferred to the counting chamber. Placing the counting chamber under the petri dish with a moist filter paper allows the platelets to settle and also prevents the evaporation of diluting fluid. When observed through the microscope, platelets will appear like highly refractile particles.

Normal Range:

250000 500000 /cu mm

Clinical Significance :
Determination of platelets is done in cases of suspected bleeding disorders.

Clinical Significance
Thrombocytosis is found in polycythemia vera, following splenectomy and Acute rheumatic fever, Hemolytic anemias

Thrombocytopenia is associated with prolonged BT and poor clot retraction and also occurs in aplastic anemia, megaloblastic anemia, hypersplenism, acute and chronic leukemias, sub acute bacterial endocarditis etc.

Determination of Reticulocyte Count

Principle of Supravital staining method: Blood is mixed with the stain which enters the cells and the RNA of the cells is precipitated by staining as dark blue network or reticulum. Reticulocyte % = No. of reticulocytes counted X 100
No . of red cells counted

Normal Range:

Adults Infants

0.2 2 % 26%

Clinical Significance: The number of reticulocytes in peripheral blood is a reflection of red cell forming activity (erythropoietic activity) of bone marrow. Increase in their number indicates hyper activity of marrow called reticulocytosis seen in acute blood loss or hemolytic anemia. Low counts of reticulocytes indicates bone marrow depression seen in aplastic anemia.

Determination of Absolute Eosinophil Count

Principle : Blood is diluted with a special diluting fluid
( Hingleman solution) that removes the red cells and stains the eosinophils red. These cells are then counted using Neubauer counting chamber.

Normal Range:

40 440 / cu mm

Clinical Significance: Increase eosinophil count is associated with allergic reactions, parasitic infections, brucellosis, leukemias etc. Decrease in eosinophil count suggest hyper adrenalism or Cushings syndrome

Bleeding Time
Normal range: Dukes method: 1 5 minutes Ivy method: 5 - 11 minutes

Prolonged bleeding time is observed in Thrombocytopenia, Platelet function disorders, Vascular abnormalities, Anti-platelet medication(Aspirin), Von-willebrands disease, Leukemia, Aplastic anemia

Clotting Time
Normal range: Lee and White method: 5 - 10 minutes Capillary method: 1 7 minutes

Clinical significance
Clotting time is prolonged in case of coagulation disorders like hemophilia,von willebrands disease,afibrinogenemia.

Prothrombin time (Quick,s method)

The time required for coagulation of citrated plasma after addition of thromboplastin, calcium mixture. Now it is commonly reported with INR. Ratio of prothrombin time that adjusts for the sensitivity of thromboplastin reagent such that coagulation profile is reported as INR of 1.
Normal 10-15 sec

 Increased
Anti Coagalant therapy like heparin Obstructive jaundice

Cirrhosis of liver
Malignancy of liver Post partum hypofibrinogenemia

Congenital deficiency of I, II, V, VII, & IX

Malabsorption syndrome Early Vit k def Warfarin therapy

 If P.T. is prolonged - thrombin clotting time is

recommended. Prolonged PT & PTT defects in common pathway, ie both intrinsic and extrinsic Prolonged PT but not PTT (Partial Thromboplastin Time), defect in extrinsic pathway.

Partial thromboplastin time:

Partial thromboplastin ( Rabbits brain as

chloroform extact) is mixed with test plasma and Ca++ ions, clot formation takes place.

Normal range: 15- 35 secs.

Clinical significance
PTT is a sensitive measure for the factors

required in intrinsic pathway. This is increased in deficiency of factor I, II, V, VIII, IX, X, XI, XII also in SLE.

Activated partial thromboplastin time

Principle:
partial thromboplastin ( brain extract in chloroform ) is incubated with kaolin (factor XII, contact factor activator.) the clotting time of plasma is determined after the addition of Ca++ ions

Normal range: 35-40 sec

Clinical Significance:
Prolonged in 1. haemophilia 2. vit. K deficiency 3. liver diseases 4. presence circulating anticoagulant 5. D.I.C. Shortened in 1. malignancies except those of liver 2. immidiately after acute hemorrhage

 PLASMA THROMBIN TIME

This is the time taken for plasma to clot when commercially available thrombin is added. Measure of fibrinogen level. Also affected by excess of heparin and other anticoagulants.

Normal range: 15 20 secs

Clinical significance
Prolonged Afibrinogenemia Chronic liver diseases Heparin administration Multiple myeloma.

End of part I

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