RBCs are circular, homogenous discs of nearly uniform in size, ranging from 6-8 micron , in diameter. The centre of each normal RBC is somewhat paler. They are flexible, elastic, non-nucleated, biconcave discs.

The function of the red cell is to transport oxygen and CO2 through the agency of hemoglobin.

After birth marrow is the only organ concerned in the production of erythrocytes.

The average life span of RBCs is 120 days. The effete red cells are destroyed in the spleen by R.E. cells.

The tissue oxygen tension controls erythropoiesisand the mass of the red cells in the circulation.

Other hormonal factors e.g. haematopoietic growth factors like erythropoietin also regulate the RBC production.

(I)To determine whether a person has anaemia or polycythemia and to judge their degree.

(II)RBC count is required to determine other RBC indices.

(III)RBC count is helpful in differntiating beta thalassemia trait (minor) from iron deficiency anemia, two important causes of microcytichypochromic anemia.

Blood is diluted exactly 1:200 in a Thoma’spipette, using an isotonic haem’s fluid. Diluted blood is then placed in a hemocytometer chamber. The cells in the measured volume are then counted. This figure is multiplied by an appropriate factor (10,000) to get the number of the cells per cu mm of blood.

Specimen :-

Capillary blood or EDTA or double oxalated venous blood.

Blood with anticoagulants should be thoroughly mixed by

gentle shaking.

Equipments :-

1. Thoma’s pipette with red bead (RBC pipette)

2. Neubauer chamber(Improved)

3. Diluting fluid (Haem’s solution)

4. Microscope with 10x and 40x objective

5. Hand-tally counter

Red bead

1. Sodium Sulphate -2.5gm

Anticoagulant and it lowers the surface tension

2. Sodium chloride - 0.5gm

Provides isotonicity

3. Mercuric chloride - 0.25gm

Acts as a fixative

4. Distilled water upto 100ml

1. Take blood upto 0.5 mark (or upto the mark 1.0 in case of severe anemia) in Thoma’s pipette without any air bubble inside.

2. Remove excess blood from the tip of the pipette. Immediately draw haem’s solution to fill up bulb of the pipette and reach upto101 mark, taking care that no air bubble gets in. Dilution is then 1 in 200.

3. Mix fluid in the bulb uniformly by closing the tip of the pipette with forefingers and then rolling the pipette in horizontal position for 3 min.

4. Expel the first 2 or 3 drops fluid in the capillary portion of the pipette which has not come in contact with the blood.

5.Then charge chamber properly, place the coverslip on the chamber, so that it covers the ruled area. Gently put a drop of blood near the edge of a coverslip. It should not overflow on the other side of chamber and no air bubble should get in. Allow the cells to settle for three minutes before the counting.

Counting of Erythrocytes :-

Under the high power, counting is done.

Count all the erythrocytes in the 4 corners and one central small

square. Each of the small square is bounded by double ruling and

consists of 16 smallest squares.

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1 3

2 4

The total area of the central large square is 1 sq.mm. The smallest

square has the side of 1/20mm. So that its area is 1/400 sq.mm. ( 1/20

x 1/20 = 1/400 sq.mm.)

When coverslip is in position the depth of the ruling is 1/10mm.

So the volume of a smallest square will be 1/400 x 1/10 =

1/4000cu.mm.

Now cells are counted in 80 smallest squares. The total volume will

therefore be 80/4000 cu.mm. of diluted blood.

Since the blood is diluted as 1 in 200, so the actual volume of the whole

bloodcounted will be 80/4000 x 200 = 1/10,000 cu.mm.

Therefore if the no. of cells in 80 smallest squares is N, then no. of cells

per cu.mm. is N/1/10,000 /cu.mm

= N x 10,000/cumm

Sources of Errors :-

1.Sampling error :a) Capillary blood - failure to get free flow of the blood

- local circulatory disturbances like cyanosis or oedema.

b) Venous blood - prolonged stasis due to tourniquet. - Inadequate mixture of blood with

anticoagulant. 2.Equipment errors :- Attributed to pipette or

hemocytometer. 3.Technical errors :- Improper technique, improper volume

measure, improper charging, counting wrong calculation, touching of the objective during focussing.

Normal values :-

Adult male :- 4.5 to 5.9 millions/cu.mmAdult female :- 4.5 to 5.1 millions/cu.mm

Abnormal values :-

Increased Decreased

High altitude Anemias : Exercise i. Aplastic Pregnancy ii. Hemorrhagic Polycythemia vera iii. Hemolytic Chronic heart disease iv. Nutritional Pulmonary fibrosis

Objective:The total leucocyte count is carried out to

know its variation from normal range and its pathological causes.

Principle:Blood is diluted 1:20 in a WBC pipette using

WBC diluting fluid. This is then charged on a haemocytometer and leucocytes are counted in a measured volume. This gives total leucocyte count per cubic mm of blood.

Various techniques :

1) By semiautomated cell counter :

2) By automated cell counter :

3) Manual method :

haemocytometer chamber

Thoma white pipette

WBC pipette(11 mark):

Equipment :

HaemocytometerWBC pipette with white bead.WBC diluting fluid.

Composition of WBC diluting fluid ( Turk’s fluid ):-

Glacial acetic acid ( 3 ml ) : To lyse the RBC’s and render WBC nuclerprominent.

Methylene blue ( few drops ) : Acts as a colouring agent to stain the nucleus.

Distilled water ( 97 ml ) : For dilution

Blood sample :

Venous blood is collected in EDTA bulb. Double oxalated bulb can also be used.

By pin prick method : A drop of blood is directly taken into the WBC pipette.

Procedure :

20 microlitre or 0.02 ml blood sample is diluted in 0.38 ml of diluting fluid in a test

tube ( or such dilution can be made in a WBC diluting pipette ).

Draw blood upto 0.5 mark in the WBC pipette.

Wipe out the blood adhering to the outer side of the pipette.

Draw diluting fluid exactly upto mark 11 , without allowing any air bubbles.

Allow thorough mixing of blood and diluting fluid by gentle rotation of the pipette

in horizontal position. Fluid will lyse the erythrocytes.

Expel and discard the first 3-4 drops which correspond to the WBC fluid in the

stem of the pipette and has not come in contact with blood.Charge haemocytometer by putting a drop of such fluid from the side of a coverslip on Neubauer’s chamber.Counting is done under low power objective lens with light partially cut off. This is done to make the ruling of chamber and the cells more prominent. Confirm that the cells are evenly distributed in the ruled areas.For leucocyte count, four large corner squares of improved Neubauer’s chamber are used. ( Each large square is madeup of 16 small squares ).

Calculation :

Area of one large corner square = 1 x 1 mm= 1 sq. mmDepth ( when coverslip is in position ) = 1/10 mmTherefore, Volume of one large corner square = Area x depth

= 1 x 1/10 = 1/10cmm Volume of four large corner squares = 4 x 1/10 = 4/10cmmDilution of blood is 1:20So if X is total number of cells counted in 4 large corner squares

then,Total count / cmm of blood = X x dilution factor

Volumei.e. X x 20 x 10 = X x 50 / cmm of blood.

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Sources of error : All the errors of sampling technique, equipments & uneven distribution of cell which are encountered in enumeration of erythrocyte apply to enumeration of leucocytes also.

Normal value :

Adult : 4000 – 11000/cmm of blood

Infant : 6000 – 18000/cmm of blood

Pathological variation :

Leucocytosis : Increase in WBC count above 11000/cc.

Leucopenia : Decrease in WBC count below 4000/cc.

Agranulocytosis : Usually Total WBC count less than 1000/cc. Patients with such a low count have increased chances of infection.

DUNGER’S fluid 1. Eosin 200 mg dissolved in 10 ml of DW 2. WATER 80 ml lyses RBCs and WBCs 3. Acetone 10 ml to fix eosinophils Filter and keep in refrigerator Equipments : improved Neubaurer chamber, WBC

pipette METHOD : Take blood in WBC pipette upto mark 1 Fill the pipette with Dunger’ s fluid upto mark11 so

that dilution is 1 in 10. Mix by rotatory movement Charge the chamber , wait for 5 min to settle down

cells

Count the eosinophils in 4 corner squares using low power obj.

Eosinophils are identified by bright red granules.

AEC = Nx 10

4 x 0.1

AEC = Nx 25

Normal range is 40-450 cells / cumm

Thank You