HAEMOCYTOMETRY

WHAT IS
HAEMOCYTOMETRY ?
It is a technique used to

enumerate the total cell count in

the BLOOD or other Biological
body fluids. This can be done
either by using Haemocytometer
or by Electronic cell counter.
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PURPOSE
In certain pathological conditions

the value of different type of cells

may have the variation. Thus by
counting the cells in the blood or
body fluids , it can be find out if
an individual is normal or not .
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Broadly , The cell count

is done
To find out normal and abnormal

count of the cells .

To support and confirm clinical
diagnosis of the patient .
To find out the response of the
patient to the treatment .

PRINCIPLE OF CELL
COUNTING
The blood is diluted with

appropriate known volume of

diluting fluid and then
counting is done by using
haemocytometer .
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HAEMOCYTOMETER
This is an instrument used for counting the

cells in blood or fluid.

It consist of a special instrument called
counting chamber, cover glass, pipette for
diluting the blood, rubber tube with plastic
mouth piece for drawing blood or fluid in
pipette.

COUNTING CHAMBER
It is a thick glass slide, center of which has

double ruling area separated by troughs

(these four troughs are extending across the
slide and set parallel to each other. The fifth
one is separating the two ruling areas from
each other).

COUNTING CHAMBER

There are some diff. type

of counting chambers :1.Old neubauer counting chamber
2.Improved neubauer counting
chamber
3.Burker counting chamber
4.Fuchs rosenthal counting chamber.
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OLD
NEUBAUER
CHAMBER
In this the central platform is set 0.1 mm.
below the level of the two side, which giving
the chamber a depth of 0.1 mm. The ruling
covers an area of 9 sq.mm. divided into 9
squares of 1 sq.mm. each. The four corner
squares are subdivided into 16 squares , each
with an area of 1/16 of a sq.mm. The central
ruled area of 1sq.mm. is divided into 16 larger
squares by set of triple lines. These large
squares are further subdivided into 16 small
squares by single lines.
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IMPROVED NEUBAUER CHAMBER

In this the triple lines which dividing the
central large square are very much closer to
each other. The central ruled area is divided
into 25 large squares. These squares are
subdivided to form 16 smaller squares each
with an area of 1/400 of 1 sq.mm. The depth
of improved neubauer chamber is same i.e.
0.1mm.

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OLD v/s IMPROVED NEUBAUER

The space occupied by the triple lines in

old neubauer chamber being used to

produce extra large squares.
In old neubauer chamber the gap b/w
triple lines was very wide and the
rectangular space b/w them looks as
similar as the squares in which cells are
to be counted. This makes the count very
difficult and chances of error was very
high.

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Cont.
In old neubauer chamber the lines were

very dull and some times it was very

difficult to recognize them.
BUT, In improved neubauer chamber
these all faults are removed.
Its triple lining are very closer to each
other ,these are dark and can easily
recognize.
By dividing central square in 25 squares
the RBC and Platelet count is become
easy to do.

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OLD NEUBAUER
CHAMBER

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IMPROVED NEUBAUER CHAMBER

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BURKER COUNTING CHAMBER

Like the neubauer counting chamber , this
has a ruled area of 9 sq.mm. and a depth of
0.1mm. , but its smaller squares are divided
by double lining not by single one.

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BURKER COUNTING CHAMBER

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FUCHs
ROSENTHAL
CHAMBER
This chamber was originally designed for
counting cells in CSF ,but as such a
relatively large area is covered , it is
preferred by some workers for counting
blood cells. The depth of this chamber is
0.2mm. and the ruled area consist of
16mm squares divided by triple lines.
These squares are subdivided to form 16
smaller squares , each with an area of
1/16 of 1 sq.mm.

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FUCHs ROSENTHAL CHAMBER

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COVER
GLASS
A special cover glass is
used which has a very
smooth , flattened
surface and even
thickness.
Different Thicknesses
are :
0.3mm
0.4mm (most common)
0.5mm
Two sizes are common:16x22 sq. mm
22x23 sq. mm
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TOTAL WBC COUNT

Diluting fluid Tuerk fluid
Glacial acetic acid 2ml
Gentian violet about a
pinch
Distilled water 97ml

Solution is stable at
room temp.
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PROCEDURE
Take 950 l diluting fluid in a clean, dry test

tube.
Add 50 l anticoagulated blood sample and mix.
Keep for 5 min. at room temp.
Mix and fill the chamber with the help of pipette.
Count the cells using low power(10x) objective.
Cells in 4 large corner squares are to be
counted.
Count the cells touching the triple lines of the
left side and on the top of the square.
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As shown in the picture

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Calculation
TLC = No. of cell counted x dil. Factor
Volume
= n x 20/0.4=n x 20 x 10/4
= n x 50/cumm.
Normal range= 4000 to 11000 per cumm.

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Interpretation

Decrease
Increase
( Leucocytopenia )
(Leucocytosis)
Bacterial
Muscular exercise
infections(typhoid)
High temp.
Viral infection ( Influenza )
Severe pain
Protozoal
infection(Maleria)
Bacterial or viral
Megaloblastic anemia
infections
Bone marrow depression
Leukemia
Severe hemorrhage as in aplastic anemia ,
drugs , radiation etc.
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NOTE
While performing RBC count by this
technique , possibilities of error is
very high . So, this technique is now
not in use for RBC count.

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PLATELET COUNT

Diluting fluid : 1%
Ammonium oxalate.
Principle :
1% Ammonium
oxalate is isotonic to
platelets and lyses the
RBC. WBC remains but
they are less in count
so they do not interfere
in counting the
platelets.

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PROCEDURE
Take 950 l diluting fluid in a clean, dry test

tube.
Add 50 l anticoagulated blood sample and mix.
Keep for 5 min. at room temp.
Mix and fill the chamber with the help of pipette.
Keep the charged chamber in moist petridish for
5 to10 min.
After that wipe the back of chamber and count
the cells at high power (40x) objective .
Platelets are also counted in the same squares
RBC used to be count.
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CALCULATION
Area of chamber counted=5x1/25=1/5sq.mm.
Depth=0.1mm
Total volume=1/5x0.1=1/50cu.mm.
Dilution=1/20
Total no. of platelets per cumm=
=no. of platelet counted/volume x dilution
n/1/50 x 1/20=n x50x20=n x 1000 per cumm
Normal range=1.5 to 4.0x1000per cumm

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INTERPRETATION
Increase
(Thrombocytosis)
Polycythemia
Chronic granulocytic
anemia
After surgery
Immediately after
hemorrhage.

Decrease
(Thrombocytopenia)
Acute leukemia
Pancytopenia as in
aplastic anemia
Liver disease
Metal poisoning
Megaloblastic
anemia
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ABSOLUTE EOSINOPHIL COUNT

Diluting fluid = Dungers fluid
Eosin Yellow = 0.5 g.
95% acetone = 0.5ml.
40%formalin = 0.5 ml.
Distilled water = 99 ml.

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PRINCIPLE
Blood is diluted with special diluting fluid
which stains the eosinophilic granules brightly
and distinctly. At the same time it lyses the
RBC and other WBC.

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PROCEDURE
Take 950 l diluting fluid in a clean, dry test tube.
Add 50 l anticoagulated blood sample and mix.
Keep for 5 min. at room temp.
Mix and fill the chamber with the help of pipette.
Keep the charged chamber in moist petridish for
5 to10 min.
Count the eosinophil under low power(10x)with
reduced light.
Count in 4 corner squares of the Improved
counting chamber .

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CALCULATION
AEC=Total cell counted x dil. Factor/volume
=N x 20/0.4
=N x 20 x 10/4
=N x 50/cumm

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INTERPRETATION
Increase in
Allergic conditions
Parasitic infection especially in helminths
Hyperadrenalism
Leukemia
Aplastic anemia

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PRECAUTIONS
The preparation of diluting fluids must be

proper.
Always clean the chamber before and after
use.
After taking blood in pipette, clean the
outer surface of tip before diluting it in the
diluting fluid.
Always mix the dilution before filling the
chamber.
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Cont..
Avoid bubbles to come in the chamber.
Never over flow the chamber with dilution.
For decrease the error more cells must be
count.
Change the cover glass if dirty and scratched.
Calculation must be done properly.
Clean the microscope before and after every
use.

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THANK
YOU
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