TOTAL LEUCOCYTE COUNTTOTAL DIFFERENTIAL COUNT

Dr Neha MahajanMD Pathology

Haematopoeisis

GRANULOPOEISIS

Type of WBC’sGranulocytes—have large granules in their cytoplasm

Neutrophils( 40 to 75%) Eosinophils(1 to 6%) Basophils(0 to 1%)

Types of WBC’s Agranulocytes—do not

have granules in their cytoplasm

Lymphocytes(20 to 40%) Monocytes( 2 to 10%)

Granuloctyes Neutrophils

Stain light purple with neutral dyes Granules are small and numerous—

course appearance Several lobes in nucleus 65% of WBC count Diapedesis,inflammation

Granulocytes Eosinophils or Acidophils:

Large, numerous granules Nuclei with two lobes 2-5% of WBC count Found in lining of respiratory and digestive

tracts Protections against infections caused by

parasitic worms and involvement in allergic reactions

Secrete anti-inflammatory substances in allergic reactions

Basophils Least found- 0.5 to 1% Contain histamine,serotonin,heparin—

inflammatory chemical

Agranulocytes Lymphocytes

Smallest WBC Large nuclei/small amount of

cytoplasm Account for 25% of WBC count Two types—T lymphocytes—attack an

infect or cancerous cell, B lymphocytes—produce antibodies

against specific antigens (foreign body)

Agranulocytes Monocytes

Largest of WBCs Dark kidney bean shaped nuclei Highly phagocytic

TOTAL LEUCOCYTE COUNT

WHITE CELL COUNT (WBC)

White cell count (WBC) is the total number of leukocytes in a volume of blood, expressed as thousands/µl.

WBC can be done by manual methods or by automated cell counters.

Normal Values:• Newborn 9.0-30.0 x 103/μl• 1 week 5.0-21.0 x 103/μl• 1 month 5.0-19.5 x 103/μl• 6-12 months 6.0-17.5 x 103/μl• 2 years 6.2-17.0 x 103/μl• Child/adult 4.8-10.8 x 103/μl

PRINCIPLE OF WBCS COUNT TEST

Free-flowing capillary or well-mixed anticoagulated venous blood is added to a diluent) at a specific volume in the thoma pipette.

The diluent lyses the erythrocytes but preserves leukocytes and stains the nuceli.

The diluted blood is added to the hemacytometer chamber.

Specimen: EDTA- anticoagulated blood or capillary blood is

preferred.

Reagents, supplies and equipment: White blood cells count diluting fluid

Turks' solution which is formed of: Glacial acetic acid 3 ml Crystal violet 1 ml 100 ml distilled water.

EQUIPMENT1. White blood cells count diluting

fluid2. Thoma white pipette3. Hemacytometer and coverslip4. Microscope5. Lint-free wipe6. Alcohol pads

haemocytometer chamber

Thoma white pipette

Rubber sucking tube

HEMACYTOMETER

The hemacytometer counting chamber is used for cell counting.

It is constructed so that the distance between the bottom of the coverslip and the surface of the counting area of the chamber is 0.1 mm.

The surface of the chamber contains two square ruled areas separated by an H-shaped moat.

HEMACYTOMETER

PROCEDURE

1. Draw the blood up to 0.5 mark in the thoma pipette.

2. Wipe the outside of the capillary pipette to remove excess blood that would interfere with the dilution factor.

3. Holding the pipette almost vertical place into the fluid. Draw the diluting fluid into the pipette slowly until the mixture reaches the 11 mark, while gently rotating the pipette to ensure a proper amount of mixing.

4. Place the pipette in a horizontal position and firmly hold the index finger of either hand over the opening in the tip of the pipette, detach the aspirator from the other end of the pipette now the dilution of the blood is completed

PROCEDURE

5. Mix the sample for at least 3 minutes to facilitate hemolysis of RBCs.

6. Clean the hemacytometer and its coverslip with an alcohol pad and then dry with a wipe.

7. Before filling the chamber, discard the first four to five drops of the mixture on apiece of gauze to expel the diluent from the stem.

PROCEDURE

8. Carefully charge hemacytometer with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.

PROCEDURE FOR COUNTING WBC’S

1. Under 10 x magnifications, scan to ensure even distribution. Leukocytes are counted in all 4 large squares of counting chamber.

2. Count cells starting in the upper left large corner square. Move to the upper right corner square, bottom right corner square, bottom left corner square and end in the middle square.

3. Count all cells that touch any of the upper and left lines, do not count any cell that touches a lower or right line.

CALCULATIONSDilution factor 20Volume= Area x depthTLC/uL= No of WBCx Correction for Volume X

dilution No of large squares (4) = N x 20 x10 4 = N x 50

Corrected TLCDiluting fluid does not lyse nucleated

RBC`s/erythroblastsFalsely counted as WBC Corrected TLC/uL= TLC x 100 NRbc/100 Wbc+ 100

WHITE BLOOD CELL DIFFERENTIAL COUNT

DEFINITION

The relative percentage of each type of white blood cells in peripheral blood.

This experiment is a part of blood routine test.

PERIPHERAL BLOOD SMEAR A properly prepared blood smear is

essential to accurate assessment of cellular morphology

The wedge smear is the most convenient and commonly used technique for making PBS

PERIPHERAL BLOOD SMEARWedge technique of

making PBS

A. Correct angle to hold spreader slide

B. Blood spread across width of slide

C. Completed wedge smear

PERIPHERAL BLOOD SMEAR Characteristics:

It is smooth without irregularities, holes, or streaks

When the slide is held up to light, the featheredge of the smear should have a “rainbow” appearance

The whole drop is picked up and spread Well-made PBS

tail body head

PERIPHERAL BLOOD SMEAR Examples of unacceptable smears

PERIPHERAL BLOOD SMEAR Examples of unacceptable smears

Observing direction:

Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction*avoid repeat or miss some cells

ROMANOWSKY STAINING

Leishman's stain : a polychromatic stain• Methanol : fixes cells to slide• methylene blue stains RNA,DNA blue-grey color • Eosin stains hemoglobin, eosin granules orange-red color • pH value of phosphate buffer is very important

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PROCEDURE• Thin smear are air dried.• Flood the smear with stain. • Stain for 1-5 min.• Experience will indicate the optimum time. • Add an equal amount of buffer solution and mix the stain by blowing

an eddy in the fluid.• Leave the mixture on the slide for 10-15 min. • Wash off by running water directly to the centre of the slide to prevent

a residue of precipitated stain.• Stand slide on end, and let dry in air.

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FEATURES OF A WELL-STAINED PBS

Macroscopically: color should be pink to purple Microscopically:

RCS: orange to salmon pinkWBC: nuclei is purple to blue

cytoplasm is pink to tan granules is lilac to violet

Eosinophil: granules orange Basophil: granules dark blue to black

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Optimal Assessment Area:1. RBCs are uniformly and singly distributed2. Few RBC are touching or overlapping3. Normal biconcave appearance

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PRINCIPLE

White Blood Cells1. Check for even distribution and estimate the number

present (also, look for any gross abnormalities present on the smear).

2. Perform the differential count. 3. Examine for morphologic abnormalities.

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MANUAL DIFFERENTIAL

Red Blood Cells, Examine for: 1. Size and shape ( Anisocytosis,Poikilocytosis2. Relative hemoglobin content. 3. Polychromatophilia. 4. Inclusions. 5.  Rouleaux formation or agglutination

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WBC ESTIMATION UNDER 40X• Using the × 40 high dry with no oil.• Choose a portion of the peripheral smear where there is

only slight overlapping of the RBCs. • To do a WBC estimate by taking the average number of

white cells and multiplying by 2000.

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PLATELET ESTIMATION UNDER 100X1. Use the oil immersion lens estimate the number of

platelets per field.2. Look at 5-6 fields and take an average.3. Multiply the average by 20,000.4. Note any macroplatelets.

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Platelets per oil immersion field (OIF)

1) <8 platelets/OIF = decreased

2) 8 to 20 platelets/OIF = adequate

3) >20 platelets/OIF = increased

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MANUAL DIFFERENTIAL COUNTS• These counts are done in the same area as WBC and platelet

estimates with the red cells barely touching.• This takes place under × 100 (oil) using the zigzag method.• Count 100 WBCs including all cell lines from immature to

mature. Reporting results• Absolute number of cells/µl = % of cell type in differential x

white cell count

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MICROSCOPIC EXAM

10× (low fold): overall smear quality, rouleaux, agglutination or parasites

100× (oil Len): WBC Diff, RBC morphology

RBC Morphology WBC- Total count Differential count Platelets Abnormal cells Parasites

Peripheral smear reporting

Normal blood smear

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NORMAL NEUTROPHIL COUNT (40 TO 75%) NEUTROPHILIA

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Absolute neutrophil count greater than 7500/uL

CausesAcute bacterial infections-

abscesses,pneumonia,meningitis,UTITissue necrosis- burns,injury,MIAcute blood lossAcute haemorrhageMyeloproliferative disordersPosisoning

NEUTROPENIA Absolute netrophil count less than 2000/uL Mild- 2000 to 1000/uL Moderate-1000 to 500/uL Severe- < 500/uLCAUSESI )Decreased or ineffective production in bone

marrow Infections- bacterial,protozoal,viral Haematologic disorders- megaloblastic

anemia,aplastic anemia,aleukemic leukemia Drugs Ionising radiation Congenital disorders

II) Increased destruction in peripheral bloodNeonatal isoimmune neutropeniaSystemic lupus erythematosusFelty`s syndromeIII) Increased sequestration in spleenHypersplenism

EOSINOPHILA

Absolute eosinophil count greater than 600/uL

CAUSES1.Allergic diseases-Asthma,rhinitis,urticaria

2.Skin diseases-Eczema,pemphigus,Dermatitis herpetiformis

3.Parasitic infections with tissue invasion- filariasis,trichinosis,echinoccocoosis

4.Hematologic disorders-MPD,Hodgkins disease,Peripheral T cell lymphoma

5.Carcinoma with necrosis6.Radiation therapy7.Lung diseases- loefflors syndrome,tropical

eosinophilia8.Hypereosinophilia syndrome

MONOCYTOSIS Increase in absolute monocyte count >

1000/uLCauses Infections-TB,SABE,Malaria,Kala Azar Recovery from neutropenia Autoimmmune disorders Hematologic diseases- MPD,Monocytic

leulemia,hodgkins disease Others-ulcerative colitis,chron`s

disease,sarcoidosis

LYMPHOCYTOSIS Absolute lymphocyte count more than upper

limit of normal for age( 4000/uL in adults,>7200/uL in adolescents,>9000/uL in children and infants)

CausesInfections-Viral- acute infectious

lymphocytosis,neoatitis,CMV,rubella,mumps,varicella

Bacterial- pertussis,TBProtozoal- toxoplasmosis

Hematological disorders-ALL, CLL, Multiple myeloma, Lymphoma Others-Serum sickness, post vaccination, drug

reactions

PLATELETS Small,1 to 3 um in diameter,purple structures

with tiny irregular projections on surface Occur in clumps Pseudothrombocytopenia Platelet sateletism

SUMMARY Normal haematopoeisis

Total leucocyte Count

Manual differential countPeripheral smearInterpretation

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