White Blood Cells Counting and calculation

1. Dr. UmmeHabiba

2.  Increase in total leukocyte count of more than 10,000/cu mm (μl) is known as
leukocytosis
 decrease of less than 4 000 cu mm (μl) as leukopenia.

3.  Pathological
 It is common for a transient period in infections. The degree of rise in leukocytes
depend on the type and severity of the infection and the response of the body. The
infection may be
 1) bacterial
 2) viral
 3) protozoal (malaria) or
 4) parasitic (filaria, hookworm infection).
 Leukocytosis is also observed in severe hemorrhage and in Leukemia.

4.  Physiological
 Age: At birth the total leukocyte count is about 18,000/cu mm (μl). It drops gradually
to adult level.
 Pregnancy: At full term‘ the total count tends to be about 12,000 -15,000/cu mm
(μl). It rises soon after delivery and then gradually returns to normal.
 High temperature.
 Severe pain.
 Muscular exercise

5.  Infections
 Bacterial (typhoid. paratyphoid, tuberculosis, etc.)
 Viral (hepatitis, influenza, measles, etc.)
 Protozoal (malaria)
 Some cases of Leukemia.
 Primary bone marrow depression (Aplastic anemia).
 Secondary bone marrow depression (due to drugs, radiation, etc.)
 Anemia (Megaloblastic etc)

6.  Adults : 4,000-10,000/cu mm (μl)
 At birth : 10,000-25,000/cu mm (μl)
 1 to 3 years : 6,000-18,000/cu mm (μl)
 4 to 7 years : 6,000-15,000/cu mm (μl)
 8 to 12 years: 4,500-13,500/cu mm (μl)

7.  Specimens
1. Double oxalated or EDTA blood
2. Capillary blood (specimen need not be a fasting sample).
 Requirements
1. Microscope
2. Improved Neubauer Chamber
3. WBC pipette
4. WBC diluting fluid:

8.  WBC diluting fluid:
 It is prepared as follows:
 1) Glacial acetic acid: 2.0 ml
 2) 1 % (w/v) gentian violet: 1.0 ml
 3) Distilled water: 97 ml
 This solution is stable at room temperature (25°C ± 5°C).
 A pinch of thymol may be added as preservative.

9.  The glacial acetic acid lyses the red cells while the gentian violet slightly stains the
nuclei of the leukocytes. The blood specimen is diluted 1:20 in a WBC pipette with the
diluting fluid and the cells are counted under low power of the micro scope by using a
counting chamber. The number of cells in undiluted blood are reported per cu mm (μl)
of whole blood

10. 1. Draw blood up to 0.5 mark of a WBC pipette.
2. Carefully, wipe excess blood outside the pipette by using cotton. Draw diluting fluid up to 11 mark.
3. Mix the contents in the pipette and after five minutes by discarding few drops, fill the counting
chamber and allow the cells to settle for two to three minutes.
a. Since Bulb pipettes are not recommended following procedure is performed
b. Make a 1:20 dilution of blood by adding 20 μl of blood to (1 38 ml of diluting fluid in a glass tube
(10 * 75 mm)
c. Cork the tube tightly and mix the suspension by rotating in a cell-suspension mixer for at least 1
minute.
d. Fill the Neubauer counting chamber by means of a Pasteur pipette or glass capillary.


11. 4. Focus on one of the W‘ marked areas (each having 16 small squares) by turning objective
to low power. (10 X).
5. Count cells in all four W marked corners.
6. Calculations
Number of white cells/cu mm (μl) of whole blood = (number of white cells counted * dilution) /
(area counted * depth of fluid)
Where: Dilution = 20
Area counted 4 * 1 sq.mm = 4 sq.mm
Depth of fluid = 0.1 mm (constant)
Hence no. of wbc/ cu.mm (μl) of whole blood = (No. of cells counted * 20) / (4 * 0.1)
= No of cells counted * 50
No. of leukocyte per liter of blood = No.of cells per cu mm(ml) * 106
WBC count / liter = [No. of cells counted (per 1mm2 area) / volume counted (μl)] * dilution * 106

12.  Note: The precautions taken are exactly the same as for REC counting technique. The
sources of error are also same as for RBC counting technique.
 However; in the case of WBC counting extra care is taken during the preparation and
storage of WBC diluting fluid.
 It should be perfectly free from dust particles and yeast cells, otherwise falsely high
counts are obtained due to the presence of yeast cells and dust particles.

13.  1) The inherent distribution error = λ1/2, here λ = total number of cells in each area.
 2) The error as high as 20% may make difference between 5.0 and 6.0 x 109 cells per
liter, which is of little practical significance.
 3) The error can be reduced by counting more cells. If 400 cells are counted the error
is reduced to 5%.
 4) Error may also be caused due to dirt clumped RBC debris or due to clumping of
leukocytes