1. HAEMOCYTOMETRY
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2. 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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3. 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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4. 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 .
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5. 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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6. 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.
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7. 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).
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8. COUNTING CHAMBER
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9. There are some diff. type of
counting chambers :-
1.Old neubauer counting chamber
2.Improved neubauer counting chamber
3.Burker counting chamber
4.Fuch’s rosenthal counting chamber.
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10. OLD central platform is set 0.1 mm. below the
In this the
NEUBAUER CHAMBER
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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11. 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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12. 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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13. 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.
 It’s 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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14. OLD NEUBAUER CHAMBER
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15. IMPROVED NEUBAUER
CHAMBER
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16. BURKER COUNTING CHAMBER
Like the neubauer counting chamber , this has a ruled
area of 9 sq.mm. and a depth of 0.1mm. , but it’s
smaller squares are divided by double lining not by
single one.
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17. BURKER COUNTING CHAMBER
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18. FUCH’s 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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19. FUCH’s ROSENTHAL CHAMBER
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20. 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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21. 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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22. PROCEDURE
 Take 950 µl diluting fluid in a clean, dry test tube.
 Add 50 µl 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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23. As shown in the picture
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24. 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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25. InterpretationLeucocytopenia )
Decrease (
Increase (Leucocytosis)  Bacterial infections(typhoid)
 Muscular exercise  Viral infection ( Influenza )
 High temp.  Protozoal infection(Maleria)
 Severe pain  Megaloblastic anemia
 Bacterial or viral infections  Bone marrow depression as in
 Leukemia aplastic anemia , drugs ,
 Severe hemorrhage radiation etc.
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26. 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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27. 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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28. PROCEDURE
 Take 950 µl diluting fluid in a clean, dry test tube.
 Add 50 µl 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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29. 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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30. INTERPRETATION
Increase Decrease
(Thrombocytosis) (Thrombocytopenia)
 Polycythemia  Acute leukemia
 Chronic granulocytic  Pancytopenia as in
anemia aplastic anemia
 After surgery  Liver disease
 Immediately after  Metal poisoning
hemorrhage.  Megaloblastic anemia
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31. ABSOLUTE EOSINOPHIL COUNT
Diluting fluid = Dunger’s fluid
Eosin Yellow = 0.5 g.
95% acetone = 0.5ml.
40%formalin = 0.5 ml.
Distilled water = 99 ml.
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32. 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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33. PROCEDURE
 Take 950 µl diluting fluid in a clean, dry test tube.
 Add 50 µl 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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34. 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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35. INTERPRETATION
Increase in
 Allergic conditions
 Parasitic infection especially in helminths
 Hyperadrenalism
 Leukemia
 Aplastic anemia
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36. 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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37. 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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38. THANK YOU
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