A hand out on hemocytometry methods

1. Title: Hemocytometry
Author: Negash Alamin
Profession: Clinical Laboratory scientist
E-mail: drnegash@gmail.com
Date: 27/11/17

2. Principle: Hemocytometry is a process of cell counting either manually or by automated
techniques. The cells counted are the RBC, WBC and Platelets or thrombocytes. The counting of
cells is essential in research or diagnosis of ailments; sample of anticoagulated and well mixed
are drawn and prepared as necessary i.e. RBC lysed with acids like Turks diluting fluid which
consists of 2% acetic acid, 1ml of aqueous gential violet and 98 ml of distilled water to count
WBC or RBC preserved by isotonic solution like Hayem’s fluid which contains Mercuric
chloride 0.5 gm, sodium chloride 1.0 gm, sodium sulphate 5.0 gm made up with 200 ml of
distilled water1
or we can use sodium citrate 3gm plus 1ml formalin made up with 100 ml
distilled water2
.
WBC count
A WBC count estimates the total number of white cells both agranulocytes and granulocytes of
myeloid and lymphoid lineage counted indiscriminately under the improved Neubauer chamber
invented by Louis Charles Malassez (1842-1909) —which is a thick microscope slide with a
depth of 0.1 mm and has an area of 3x3 mm. Differentiation of cells is conducted in another
procedure called Differential cell count.
Figure 1 Louis C. Malassez
1
Ramnik Sood; Medical laboratory technology: 2006:205
2
Ramnik Sood; Medical laboratory technology: 2006:205

3. Dilution
The dilution factor for WBC is 1:20 and compositing that will give the proportion of blood to
diluting fluid in 1 to 20 ratios is acceptable. We can use the Thoma pippette with white bead in it
or the test tube method for dilution. 0.38 ml of diluting fluid is mixed with 0.02ml of EDTA
blood in case of test tube method or we take blood to 0.5 mark on the Thoma pippette and take
diluting fluid till 11 mark.
Materials:
1- Neubauer chamber
2- Anticoagulated blood sample.
3- Appropriate diluting fluid for the procedure.
4- Thoma pippette or test tubes (whatever suits the case for dilution)
5- Pipette for charging sample into the chamber.
6- A cover slip (special one that comes with the chamber)
7- Compound microscope for examination
8- Gloves as PPE (personal protective equipments)
Method
As said above we can use test tube method or Thoma pippette method
A. Test tube method
1-Dispense 0.38 ml of diluting fluid into the test tube
2- Add 0.02 ml of EDTA blood into the test tube
3- Mix the ingredients by sucking and ejecting through the micropipette.
4- Wait for 5 minutes for the destruction of RBC.
B- Thoma pippette method
1- Suck blood up to 0.5 mark and wipe3
the outside of the tube.
2- Now, draw diluting fluid until 11 mark
3
Helps in preventing false high WBC count

4. 3- Mix the two gently without forming bubbles by making the infinity mark.
4- After mixing wait for 2- 5 minutes for RBC destruction.
5- Now, clean the counting chamber with alcohol and place the cover slip on the grid lines
of the chamber. Apply small amount of distilled water on the grid where the cover slip
will rest so that it sticks to it and does not slide. If properly aligned you will get or see
what is called Newton’s rings on the cover slip; which is a kind of rainbow.
6- Then, finally gently charge the chamber on each side using a micropippette via 45° angle.
7- Do not over charge or let over flow; over flow or under flow will give low count— in that
case clean the chamber and charge again.
8- Using 10x objective and low light observe the even distribution of cells and count in the
four corners of WBC counting area in a zigzag fashion on by one and report per 1
microliter (see figure 2).
Figure 2 Neubauer counting chamber 3x 3 mm

5. Calculation
Calculation is performed by multiplying total number of cells counted by VCF (volume
correction factor) and DCF (dilution correction factor) or alternatively we can use this formula
below:
Example: Number cells counted on first chamber= 100
Second chamber= 108
Thus, we get
=
5200 cells per micro liter of blood.
DCF=20
VCF= = = 2.5
Correction of Leukocyte count
Correcting the WBC count may be necessary in case we find major aberrations in our count;
which may be a sign other underlying pathology like the discovery of Nucleated RBC that might
be mistaken for WBC; thus raising our count.
Corrected WBC count =

6. Issues that cause error in WBC count
1- Blood taken from unevenly mixed EDTA sample
2- The formation of bubbles in the test tube because it will affect volume.
3- Not wiping away the blood on the outside of the Thoma pippette.
4- Taking blood more than required.
5- Uneven distribution of cells on the chamber due erroneous charging, dirt and yeasts.
6- Errors in calculation
Clinical significance: Several superficial and systemic infections will raise the WBC count
because they are the defense mechanisms in the immune system of the body against foreign anti-
self. Thus, are indications of pathology when they rise in number. Inherent genetic disorders of
either the myeloid or lymphoid lineage in hematopoiesis also increase the number of WBC count
indicating malignancy. In opposite the count may also be depressed in immune-deficiency
ailments like HIV. In addition, autoimmune disorders (rheumatoid arthritis), cancers that destroy
bone marrow, radiation therapy as well as in Vitamin B12 deficiency may reduce their count.
RBC count
This procedure is not that recommended; because red blood cells are smaller and numerous than
WBC and could become arduous to enumerate to the laboratory technician. But it may be
performed in a community laboratory which does not have a machine to do the counting.
Principle
A blood sample is withdrawn from a patient diluted with a diluent in a similar fashion as in WBC
but the dilution factor is 1:200 which is more than WBC counting.
Procedure
1- Choose a diluting fluid or a reagent either:
A) Sodium citrate 3 gm (cheap and recommended)
Formalin 1 ml
Distilled water 100 ml

7. Or
B) Hayem’s fluid (not recommended)
Mercuric chloride 0.5 gm
Sodium Chloride 1.0 gm
Distilled water to 200 ml
2- Using the Thoma pippette used for RBC which has a red bead in it draw patient’s blood
till 0.5 mark.
3- Wipe the tip of the pippette with a gauze; now, draw the diluting fluid till 101 mark.
4- Alternatively if we used test tube method rather than Thoma pipettes we can draw 0.02
ml of blood and make up with 3.98 ml of diluting fluid.
Remember
The dilution in RBC of 1:200 in Thoma pippette by drawing till 0.5 mark blood and
then diluting fluid till 101 mark will give the same value of 0.005 as in the test tube
method by taking 0.02 blood and 3.98ml of diluting fluid will give you 0.005.
5- Charge the chamber and do not over flow. Let it settle for three minutes4
.
6- Using 40 x objective count the RBC cells in the 80 small squares found in the 5 RBC
counting area. Remember that each ‘R’ has 16 small squares and when you multiply that
with 5 we get 80 small boxes.
Calculation
Each 1 R (see figure 2) has an area of 0.4 mm square and its volume multiplied by the depth of
the chamber 0.1mm will give us a volume of 0.004mm cubed. Thus, 5 R squares will come up to
be 0.02 mm cubed (volume used).
VCF=
Total RBC count =
4
Ramnik Sood: Medical laboratory technology; 206:2006

8. = value of the count
Or alternatively you can just multiply the number of RBC counted with 10,000.
Platelet count
In platelet count we use 1: 20 dilution meaning WBC pippette is used. The cells are counted in
the RBC chamber with ammonium oxalate (10 gm /L) as a diluting fluid with venous blood as
the sample of choice.
Calculation
After using 10x then 40 x to identify them multiply the number of cells counted in the 5R areas
with 1000.
Note: If Platelet count comes to be less than 100,000 count all 25 squares not just the 5 R Thus,
if you count the all 25 squares which hold 16 tiny squares in side of them then your multiplying
factor will change to 200. In short multiply by 1000 if you count cells in only 5R area; multiply
cells by 200 if you have used all the 25 squares of the RBC area.
Detail formula
All 5 R have a total volume of 0.02 mm cubed (volume used) and if you used all 25 R in case of
thrombocytopenia they have a volume of 0.1 mm cubed (volume used). Thus, VCF= Volume
desired (1mm cubed)/ volume used (0.02 mm in case of normal count) = 50
VCF= volume desired (1mm cubed)/ volume used (0.1 mm cubed in case of thrombocytopenia);
we get 10.
DCF= 20