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Practical hematology
Assist.Prof. Dr.Sajeda Al-Chalabi
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RBC COUNT
HEMOCYTOMETER
Hemo: blood
Cyto: cell
Meter: measurement/counter
Thus, it is an instrument used to count the blood
cells.
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It includes:
a) Neubauer’s slide
b) Cover slip
c) RBC pipette
d) WBC pipette
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NEUBAUER’S SLIDE
It is the name given to a thick glass slide. In the
centre of the slide, there is an H- shaped groove.
On the two sides of the central horizontal bar,
there are scales for counting the blood cells.
The depth of the scales is 1/10mm or 0.1mm.
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Neubauer Hemocytometer
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Hemocytometer
Chamber
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The central square is subdivided into twenty five
smaller squares and each of these smaller
squares is further subdivided into sixteen
smallest squares.
These are meant for platelet and RBC counting.
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the RBCs are counted in five small squares, four
of corners and one of center.
(total of 80 smallest squares)
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Each of the smaller squares is further
subdivided into sixteen smallest squares.
Length of one smallest square = 1/5 x 1/4
= 1/20mm
Width of one smallest square = 1/20mm
Depth of one smallest square = 1/10mm
Volume of one smallest square
= 1/20 x 1/20 x 1/10
= 1/4000mm³
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Cell Counts by Hemocytometer
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Procedure
For RBC counting
Blood is filled till mark 0.5 and Hayem’s fluid is then
filled till mark 101.Both are thoroughly mixed and then
few drops are discarded which contain just the diluting
fluid in the stem. Thus, 1 portion out of 101 is
discarded. So,
0.5 part of blood is in 100 parts of fluid or,
1 part of blood is mixed in 200 parts of fluid
Thus, dilution factor for RBC counting is
200.
You can count blood cells with as little as a drop of blood.
Because the cell density is very high, you have to dilute .
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PROCEDURE


Before starting ensure that both the hemocytometer and its
coverslip are clean by removing any dust particles with
lens paper.
make sure to first place the coverslip over the counting
surface before loading the cell suspension. Then place the
pipette tip with your sample into one of the V-shaped wells,
as in Figure, and gently expel the sample. The area under
the coverslip fills by capillary action. Enough liquid should
be introduced so that the mirrored surface is just covered,,
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PROCEDURE

The loaded hemocytometer is then placed on the
microscope stage and the counting grid is brought
into focus at low power. Allow the sample to
settle for a couple of minutes and avoid moving
the coverslip as it might introduce air bubbles
and make counting difficult.
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HAYEM'S SOLUTION

Hayem's solution contains: sodium chloride
0.5%, sodium sulphate 2.5% and mercuric
chloride 0.25%. The sodium chloride to prevent
hemolyses, the sodium sulphate discourages
clumping of the erythrocytes and the mercuric
chloride is a preservation.
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FOCUSING
4X to see the general formation
of slide.
 10X for WBC counting
 40X for RBC counting

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PROCEDURE
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COUNTING RULE

Do not count cells
touching
 Bottom line
 Right line
 Count cells touching the
upper& left lines
 This is to avoid double
counting.
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CALCULATION
 Number
of cells/mm³=
counted cells in 80 small squares ×
diluting factor ×
volume correction factor.
=N × 200 ×50=N × 10000
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NORMAL RESULTS
Normal RBC ranges are:
Male: 4.7 to 6.1 million cells /mm³
Female: 4.2 to 5.4 million cells/ mm³
What Abnormal Results Mean
Higher than normal numbers of RBCs(polycythemia)
Your RBC count will increase for several weeks when you
are in a higher altitude.
Lower-than-normal numbers of RBCs may be due to
Anemia
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THANKS
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