BOMBE PRIMARY HOSPITAL LABORATORY (BPHL) WOLAYTA,ETHIOPIA

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Title: Standard Operating Procedure for white cell count using Neubauer Chamber
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1. Purpose
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A white blood cell (WBC) count is used to investigate HIV/AIDS, infections and unexplained
fever, and to monitor treatments which can cause leukopenia. In most situations when a total
WBC count is requested it is usual to perform also a differential WBC.
2. Principle
Whole blood is diluted with a 3% acetic acid solution, which hemolyzes mature erythrocytes and
facilitates leukocyte counting. The standard dilution for leukocyte counts is 1:20. This dilution is
prepared using the leukocyte Unopette system. The dilution is mixed well and incubated to permit
lysis of the erythrocytes. Following the incubation period, the dilution is mounted on a
hemacytometer. The cells are allowed to settle and then are counted in specific areas of the
hemacytometer chamber under the microscope. The number of leukocytes are calculated per µL(x
109/L) of blood.

The enumeration of blood cells is a fundamental examination in the clinical laboratory.

Cell counts are nowadays performed by automated procedures/instruments. The classical manual
procedures are still used in many laboratories and Also used as a back up & QC for automated
methods

3. Specimen
Whole blood, anticoagulated with EDTA, or free-flowing capillary blood may be used. The
count should be performed within 6 hours (blood should not be refrigerated).
Heparin or sodium citrate anticoagulated blood must not be used.

4. Equipments and reagents

-Equipment for dilution:
a. Thoma white cell pipet
b. 20 µL micropipet (or sahli pipet) and 10 x 75 mm test tubes or
c. WBC unopette
-Microscope
-Improved Neubauer hemocytometer
-Syringe fitted with rubber tubing for aspiration of specimen and diluting fluid (home made)
-Lint free cloth
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Reagents

-Diluting fluid:
 2% Acetic acid, v/v, in distilled water
 1% HCl or
 Turk’s diluting fluid (3 ml acetic acid, 1ml aqueous gentian violet, 100 mL dH 2O)
 (Note: if WBC unopette is used, it contained premeasured amount of diluting fluid)

5. Method and Procedure

5.1. method
 The manual counting involves:
 Diluting the blood specimen
 Loading it into a special counting chamber known as a Hemocytometer
 Counting the cells

 The traditional unit of reporting was cubic millimeters (cu mm or mm 2)

 1 mm3 = 1.00003 µL is felt to be insignificant, 1 µL is considered equivalent to 1 cu mm.
Thus,1 mm3 = 1 µL = 10-6 liters
A hemocytometer consists of a thick rectangular glass slide. In the center of the upper surface there are :
ruled areas separated by moats/channels from the rest of the slide and two raised transverse bars one of
which is present on each side of the ruled area
The ruled portion may be:
 in the center of the chamber (single chamber) or
 an upper and lower ruled portion (double chamber)
The double chamber is to be recommended because it enables duplicate counts to be made rapidly.
When an optically plane cover glass is rested on the raised bars there is a predetermined gap (depth). But
the Depth varies with the type of chamber. The ruled area itself is divided by microscopic lines into a
pattern that varies again with the type of the chamber. In our lab The Improved Neubauer ruled chamber
is recommended for cell counts.
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Improved Neubauer Hemocytometer

 Each hemocytometer side has a counting chamber (two)
 Each counting chamber is 3mm x 3mm (area of 9mm2).
 Each chamber is divided into 9 squares – each large square is 1mm x 1mm (area of 1mm 2)
 Each of the four corner squares are divided into 16 smaller squares.

 The centre square (1 mm2) is divided into 25 smaller squares each bordered by double-ruled lines
 Each of the 25 small squares in centre square is 1/5mm per side (area of
1/25mm2=0.04mm2)
 Each of these 25 small squares is further subdivided into 16 smaller squares (400 tiny
squares of 0.0025 mm2)

 Counting chamber cover glasses

 Special optically plane cover glasses of defined thickness Cover Glass (20x26x0.5mm)
designed for use with hemocytometers are required.
 Other cover glasses give incorrect calculations
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 Dilution of the Sample

Can be accomplished by using
1. Thomma pipette
 small calibrated diluting pipettes designed for WBC or RBC count
 The WBC pipet has an upper numerical value of 11 while that of the RBC pipet
marked by 101.
2. Tube dilution method
 larger volumes of blood and diluting fluid are used
 greater accuracy compared with the smaller volumes used in the thomma pipette
techniques.
Counting and Calculation
The diluted cells are introduced into the counting chamber and allowed to settle and Counting
should be only in the designated area (s). Cells lying on or touching the upper or left boundary
lines are included in the count while those on the lower and right boundary lines are disregarded
(or vice versa).

Count cells in a systematic manner (zigzag pattern)

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5.2. Procedures
A. Test tube dilution method
1. Dispense 0.38 ml of diluting fluid to a test tube (you can take 0.4 ml and discard 20 ul of
diluting fluid).
2. Take 20 µl (0.02 ml, 20 cu mm) of well-mixed EDTA anticoagulated venous blood or free-
flowing capillary blood
3. Clean the outside of the micropipet tip or Sahli pipette with dry cotton/gauze with out touching
the tip
4. Dispense the blood to the diluent in the tube
5. Rinse by sucking and expelling 3-4 times to remove blood clinging in the inside wall of the
pipette
6. Mix by tapping (will become a 1:20 dilution)
1. Avoid formation of bubbles while mixing
7. Allow about 5 minutes for red cells to lyse
B. Thoma pipette method
1. Suck blood up to the 0.5 mark; wipe the outside with clean gauze without touching the tip
2. Take diluting fluid up to 11 mark
3. Detach the aspirator from the Thoma pipet by sealing the open tip by your index finger (hold
horizontally)
4. Mix systematically (like figure of 8)
5. The volume contained between 1 and 11 mark will be 10 units out of which 0.5 is blood and this
will make a 1:20 dilution.
6. Wait at least 5 min
B. Thoma pipette method
1. Suck blood up to the 0.5 mark; wipe the outside with clean gauze without touching the tip
2. Take diluting fluid up to 11 mark
3. Detach the aspirator from the Thoma pipet by sealing the open tip by your index finger (hold
horizontally)
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4. Mix systematically (like figure of 8)

5. The volume contained between 1 and 11 mark will be 10 units out of which 0.5 is blood and this
will make a 1:20 dilution.
6. Wait at least 5 min
Note
 Avoid formation of bubbles in the bulb of the pipette as this affects volume

 The long stem of the pipette should be filled with clear fluid
 The final volume should exactly be on the 11 mark

7. Clean chambers and cover slip with alcohol and dry well with lint free cloth
8. Place cover slip on hemocytometer (press cover slip on both corners until Newton’s ring (rain
bow) formation is observed)
9. Re-mix blood with diluent by inverting several times before charging on hemocytometer to
ensure even distribution of cells
10. Discard a few drops from the pipette and plate one dilution on each side of hemocytometer (to
ensure quality)*
11. Fill the chamber smoothly and don't overfill or under fill it; there should be no bubbles
N.B. if overflow cells will spill into the moat, thus falsely reducing the cell count. Under filling
also gives a falsely lower cell count
12. Allow cells to settle for 3 minutes
13. Use 10x (low power) objective with low light by lowering the condenser
14. Check for even distribution of cells
N.B. Difference in the number of cells between two corner squares should not exceed
±10%
15. Count WBCs in the four large corner squares

6. Quality control
Cell counts are performed in duplicate using two pipettes and counting both sides of the
hemocytometer.
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i. The difference between the two counts should not be more than 10%.

Additional squares should be counted if the cell count is low.

 When there are decreased and increased WBC counts, alter the dilution
 If WBC>50 x 109/L, increase dilution e.g. 1:40 (20 mL blood & 0.76 mL diluting
fluid)
 If WBC< 2 x 109/L, decrease dilution e.g. 1:10 (40 mL blood & 0.38 mL diluting
fluid)
 Verifying count with WBC estimate from May Grunwald-Giemsa or Wright’s stained
smear

How to calculate the % difference between two counts

1 Record the number of cells counted in Count 1 and Count 2.
2 Calculate:
- the difference in the number of cells counted between the two counts.
– the mean of the two counts
3 Calculate the difference of the two counts as a percentage of the mean.

Example
Cells counted in Count 1 = 88, Count 2 =76
Difference in numbers of cells between the two counts: 88–76 = 12
– Mean of Count 1 and Count 2: 88+76 / 2= 82
– Difference of the two counts as a % of the mean: 12 x 10 / 82= 14.6%

Note: When the difference between the two counts is more than 20%, repeat the counts.

7. Reporting result
Calculation:
 The white cell count is reported as the number of white cells in 1cubic millimeter (l µl) of
undiluted blood (1 mm3= 1µl)

 However we diluted the blood and we count the cells in less than 1 mm 3of blood!
Therefore we must multiply our total counted cells by two correction factors:
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 Dilution correction factor (DCF) and

 Volume correction factor (VCF)

 DCF compensates for dilution of blood. Since we diluted the blood 1:20 the dilution
factor is 20.

 VCF compensates for the volume in which the cells were counted

 Total number of White cells/ µL = No. WBCs counted x VCF x DCF

 VCF=1/V (=volume desired/volume counted)

 V= L x W X h (h=depth of the chamber)

 Dilution factor = invert dilution used (if 1:20, then DCF is 20; if 1:10, DCF is10)

Alternative formula:
Cell/mm3=Number of cells X dilution factor X 10*
Area counted
*invert 0.1 depth of chamber

Area counted =4mm2 that is ( L x W ) in 4 corners

So volume will be = Area counted x depth or ( L X W X H ), which will be 0.4mm3

Cell/mm3=Number of cells X dilution factor

Area counted (mm2) x 0.1

Since DF is 20
Cell/mm3=Number of cells X 20 number of cells X 50
4mm2 x 0.1

 Report WBC counts to nearest hundred or nearest tenth, depending on units.

 Counts higher than 50.0 x 109/l

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When a count is higher than 50.0/109/l, repeat the count using 0.76 ml of diluting fluid and 20 _l
of blood. Multiply the result by 2. Very high WBC counts are found in some forms of leukaemia.
Always examine a stained thin blood film.
 Counts lower than 2.0 x 109/l
When a count is lower than 2.0 _ 109/l, repeat the count using 0.38 ml of diluting fluid and 40 µl
of blood. Divide the result by 2.

The corrected Leukocyte count

 The white cell diluting fluids destroy only the non-nucleated red blood cells. In certain disease
states, NRBCs are present in the peripheral blood. When there are more than 10 NRBCs per 100
WBC in the blood film, correct the WBC count as follows:

Corrected WBC Count = Uncorrected WBC count x 100

100 + Nucleated RBCs*
*Number of nucleated RBCs per 100 WBC as seen in stained blood film.
Result interpretation
 Reference range vary with age, by gender, race, etc
Children at 1y 6.0-18.0 x 109/L
Children 4-7 y 5.0-15.0 x 109/L
Adults 4.0-10.0 x 109/L
Adults of African origin 2.6-8.3 x 109/L
Pregnant women up to 15 x 109/L
 Ethiopian adult WBC Reference range: 3.0-10.2 x 109/L (Tsegaye A et al Clin Diagn Lab
Immunol 1999; p410-414)

8. References
1. Cheesbrough M. District Laboratory Practice in Tropical Countries2006. 442 p.

STAFF REVIEW DOCUMENTATION:

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I have read and understand this SOP and I agree to consistently follow the procedure as described.

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