HEMOCYTOMETRY 11/04/2022

The Erythrocyte Count

 The determination of the number of
erythrocytes in 1 cu.mm. of blood.

Glasswares/Reagents/Materials needed:
o Counting Chamber
o Pipettes
o Diluting fluid

PROCEDURE:
(refer to the laboratory protocol)

CALCULATION:

No. of cells x Dilution x Volume = no. of

Counted factor correction cells in
(5R sections) factor 1cumm.

A. Cell Counts of the 5 ‘R’ squares

1 sq. 110 WBC RBC
2sq. 100 Pipettes

3sq. 105
4sq. 103
5sq. 108
526
 The difference between the highest and
lowest counts should not be greater than 10
cells.
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B. Dilution Factor

0.5 mark blood

101.0 mark diluted fluid

Total Volume = .5 (blood) + 99.5 d.f.

Original Vol. .5 (blood)
of blood
= 100 parts
.5

Dilution Factor = 200

1.0 mark blood

101mark dil.fluid

Solve for the dilution factor if blood was drawn

to the 0.7 mark.
Examples of blood cells counted in a
representative area

0.5 parts blood

99.5 parts diluted fluid
100.0 parts in mixing

Chamber (1:200)

 1 part does not take part in the actual

dilution
Dilution = 1:100
Dilution Factor = 100

C. Volume Correction Factor

Number of cells: The volume of 1 “R” section is found as follows:

2,1,2,2,1,2,2,3,2,2,1,2,2,3,2,2 a.
Area of 1 x Depth of 1 = volume of 1 R
Corresponding number of cells in the small R section R section section
squares (follow arrow)
2,1,2,2
0.04sq.mm. x .1mm = 0.004cu.mm.
1,2,2,3
2,2,1,2
2,3,2,2
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b. Red Cell Count

Number of x Vol.of = Total Vol. of ‘R’ sections Significance of Results
‘R’ sections 1 R sec.
◉ Together with the hematocrit and
5 x 0.004cumm = 0.02 cu.mm. hemoglobin values, it can be used to
calculate the red cell indices which provide
a valuable guide to the classification of
anemias and diagnosis of polycythemia.
 When we counted the cells in the 5 ‘R’
sections, we counted the cells in 0.02cu.mm.
 But we must report the number in 1.0cu.mm.

c.
Volume Correction factor = Vol.desired
Vol. used

=1.0 cu.mm.
.02 cu.mm.

= 50

THEREFORE,
Number of cells Number of Cells
In 5 ‘R’ sections x D.F. x VCF = per cu.mm.

526 x 200 x 50 = 5,260,000/cu.mm.

Normal Values: (S.I.)

Men : 4.5 – 6.0 M/cu.mm. 4.5-6.0x1012/L
Women : 4.0 - 5.5 M/cu.mm. 4.0-5.5x1012/L
Children : 5.0 - 6.5 M/cu.mm. 5.0-6.5x1012/L

(Please review your laboratory mathematics in the

conversion of values to S.I. units)

Conditions, Terms / diluting fluids & composition

 HEMOCYTOMETRY 11/04/2022

Leukocyte Count e.g. 1:10 dilution

 The determination of the number of 1.0 mark blood 1.0 part blood
leukocytes in 1 cu.mm. of blood. 10.0 mark dil.fluid 9.0 parts dil. Fluid
PROCEDURE: Dilution = 1:10
Dilution Factor = 10
(refer to laboratory protocol)
Total Volume = 1.0 + 9.0 = 10 = 10
Original Vol. 1.0 1
CALCULATION:
No. of cells x Dilution x Volume = no. of C. Volume Correction Factors
Counted factor correction cells in
(4W sections) Factor 1 cu.mm. a. Volume of 1 ‘W’ section

1 sq.mm. x 0.1mm depth = 0.1 cu.mm.

A. Cells counted in 4 ‘W’ squares 0.1 cu.mm. x 4 W sections = 0.4 cu.mm.
1W sq. 40
Volume desired = 1 = 2.5
2W sq. 50 Volume Used 0.4
3W sq. 50
Number of cells No. of Cells
4W sq. 50 In 4 ‘W’ sections x D.F. x VCF = per cu.mm.
190 = TOTAL CELL COUNT
Normal Values: S.I.
Men : 5,000 – 10,000/cu.mm.
B. Dilution Factor Women: 5.0-10.0 x109/L
e.g. 1: 20 dilution

.5 mark blood 0.5 part blood The terms used when the conditions is increased
11.0 mark dil.fluid 9.5 parts diluting fluid and decreased in count.
10.0 parts in mixing What are the diluting fluids used?
Chamber
(1:20) = Total Volume

 1 part does not take part in the actual

dilution

Total Volume = 10 = 20 (Dilution Factor)

Original Vol. .5
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INTERPRETATION OF RESULTS:
Leukocytosis
 Acute infections 
 Inflammation and tissue necrosis
 Metabolic disorders
 Leukemias and myeloproliferative disorders
Leukopenia
The main causes of a reduced WBC count are:
o Viral
o Bacterial
o Parasitic infections
 Drugs e.g.
o Chloramphenicol
o Phenylbutazone
o Ionizing radiation
 Production failure as in aplastic anemia

SOURCES OF ERROR IN MANUAL BLOOD COUNTS

 Incorrect measurement of blood due to
poor technique or using a wet or chipped
pipette.
 When using anticoagulated blood, not
mixing
 the blood sufficiently or not checking the
sample for clots.
 Inadequate mixing of blood with diluting
fluid.
 Not checking whether the chamber and
cover glass are completely clean.
 What else?