HEMOCYTOMETRY
[Publish Date]
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
2sq.
100
3sq.
105
4sq.
103
5sq.
108
526
 The difference between the highest and
lowest counts should not be greater than 10
cells.
WBC RBC
Pipettes
HEMOCYTOMETRY
[Publish Date]
2,3,2,2
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
Examples of blood cells counted in a
representative area
Solve for the dilution factor if blood was drawn
to the 0.7 mark.
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:
2,1,2,2,1,2,2,3,2,2,1,2,2,3,2,2
The volume of 1 “R” section is found as follows:
Corresponding number of cells in the small
squares (follow arrow)
2,1,2,2
1,2,2,3
2,2,1,2
Area of 1
R section
a.
x
Depth of 1 = volume of 1 R
R section
section
0.04sq.mm. x .1mm = 0.004cu.mm.
HEMOCYTOMETRY
[Publish Date]
b.
Red Cell Count
Number of x Vol.of = Total Vol. of ‘R’ sections
‘R’ sections
1 R sec.
Significance of Results
5
x
0.004cumm =
0.02 cu.mm.
 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
◉
Together with the hematocrit and
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.
HEMOCYTOMETRY
[Publish Date]
Leukocyte Count
 The determination of the number of
leukocytes in 1 cu.mm. of blood.
PROCEDURE:
e.g.
1:10 dilution
1.0 mark blood
10.0 mark dil.fluid
1.0 part blood
9.0 parts dil. Fluid
(refer to laboratory protocol)
Dilution = 1:10
Dilution Factor = 10
CALCULATION:
Total Volume = 1.0 + 9.0 = 10 = 10
Original Vol.
1.0
1
No. of cells x Dilution x Volume = no. of
Counted
factor
correction
cells in
(4W sections)
Factor
1 cu.mm.
C. Volume Correction Factors
a. Volume of 1 ‘W’ section
A. Cells counted in 4 ‘W’ squares
1W sq.
40
2W sq.
50
3W sq.
50
4W sq.
50
190 = TOTAL CELL COUNT
.5 mark blood
11.0 mark dil.fluid
0.5 part blood
9.5 parts diluting fluid
10.0 parts in mixing
Chamber
(1:20) = Total Volume
 1 part does not take part in the actual
dilution
=
x
x
Volume desired
Volume Used
B. Dilution Factor
e.g. 1: 20 dilution
Total Volume
Original Vol.
1 sq.mm.
0.1 cu.mm.
10
.5
= 20 (Dilution Factor)
0.1mm depth = 0.1 cu.mm.
4 W sections = 0.4 cu.mm.
=
1
0.4
=
2.5
Number of cells
No. of Cells
In 4 ‘W’ sections x D.F. x VCF = per cu.mm.
Normal Values:
S.I.
Men :
5,000 – 10,000/cu.mm.
Women:
5.0-10.0 x109/L
The terms used when the conditions is increased
and decreased in count.
What are the diluting fluids used?
HEMOCYTOMETRY
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?
[Publish Date]