RBC count

1. DR.ANINDITA SAHA
RBC COUNT

2. RED BLOOD CELLS
 Red blood cells, or erythrocytes, are
the most abundant cells in the
bloodstream and contains hemoglobin,
the compound that carries oxygen
through the body.
 . Any disruption of the red blood cells,
its quantity, shape, size, structure or life
cycle can therefore affect the oxygen-
carrying capacity of the blood.

3. Shape and size of RBCs
 A red blood cell is a biconcave disc. Simply it
is a round ball that is squeezed from two
opposite ends to appear, widest at the sides
and narrowest in the middle.
 A red blood cell measures about 6 to 8
micrometers in diameter (average = 7.8 um)
with an average thickness of 2 micrometers
(2.5 um at the thickest point and less than 1um
at the center). Although a red blood cell is
wider than some capillaries, its flexibility allows
it to become distorted as it squeezes through
narrow passages and then restores to its

5. METHODS OF RBC COUNT:
1) Visual haemocytometer( manual
method )
2) Automated method

6. MANUAL METHOD:
EQUIPMENTS REQUIRED:
1)Haemocytometer or improved
Neubauer’s chamber
2)RBC pipette

7. FLUIDS REQUIRED :
 RBC diluting fluid
 Blood sample (capillary blood or EDTA
anticoagulated blood can be used )

8. RBC DILUTING FLUID
 RBCs are around only 5 millions/cumm
of blood.
 Counting this much number is highly
imposible.
 Therefore the blood sample is diluted
with the help of RBC diluting fluid.
 It fixes and preserves RBCs.
 It is isotonic to RBCs.

9. NEUBAUER CHAMBER:
 The improved neubauer’s chamber has
a metalized surface and improved
neubauer rulings.
 The chamber has two ruled areas.

10.  Each ruled area:
dimension = 3mm x 3mm
consists of 9 large squares each
measuring
1mm x 1mm
Depth – 0.1 mm

11.  The central large square is divided into
25 squares.
 Each of this 25 square is further divided
into 16 small squares.
 Each group of 16 small squares is
separated by closely ruled triple lines.
 The 4 corner squares are further
divided into 16 small squares.

12. Coverslips used in neubauer’s
chamber:
 The coverslips used for the chamber are
special.
 They are thick and optically flat so that
the volume occupied by the diluted
blood in each large square is 0.1 ml
when covered by the coverslip.

17. RBC PIPETTE:
 The pippete has a bulb for dilution and
mixing and is caliberated to deliver 20ul
or 0.02 ml of blood.
 The bulb contains a red bead which
facilitates the mixing of blood with the
diluting fluid.
 Freely mobile bead indicates dry pipette.
 Markings on pipette are 0.5 , 1 , 101.

20. DILUTING FLUID
 Two types of diluting fluids are used for
RBC counting.
1) Hayem’s fluid
2) Dacie’s fluid

21. Composition of Hayem’s fluid:
a. Sodium chloride -0.5 gm
b. Sodium sulfate -2.5 gm
c. Mercuric chloride -0.25 gm
d. Distilled water – 100 ml.

22. Composition of Dacie’s fluid:
i. Trisodium citrate – 3 gm.
ii. Formalin – 1 ml.
iii. Distill water – 99 ml.
This diluting fluid is cheap and
commonly used.

23. PROCEDURE
 Assemble all the equipment; draw the blood
directly from finger or collected sample into
RBC pipette upto 0.5 mark.
 Wipe off the tip of pipette to remove extra
blood, if present. Then immediately draw up
the diluting fluid upto 101 mark.
 Now rotate the pipette gently for 2 – 3 mins
so that the diluting fluid gets mixed properly.
This will give dilution 1: 200.
 Place the cover slip in position over the
ruled area of chamber. Once again mix the
solution thoroughly by rotating the pipette.

24.  Discard first 1 – 2 drops of blood from
pipette.Now apply slight pressure on the
rubber tube of pipette, so that the third
drop of fluid is in hanging position.
 Touch the tip of pipette (hanging drop)
against the edge of cover slip. The angle
between pipette and cover slip is 45°. With
this process, chamber gets filled with the
fluid. This is known as charging of the
chamber.
 The care should be taken that, no air
bubble is present inside the chamber and
there is no over filling beyond the ruled
area.

25.  Leave the counting chamber as it,
without disturbing for about 3 min. This
will allow the settling down of RBCs.
 Place the chamber on stage of
microscope. Adjust the light and ruled
area. Make sure that, the distribution of
RBC over the chamber is uniform.

26.  Now count the RBC using 40x in Central
Square. The central square is subdivided
into 25 squares.
 Out of 25 squares, count four at each
corner and one at center. Each of these
five squares is subdivided into 16 small
squares. Thus, RBCs are counted in 16 x 5
= 80 small squares.
 In case of marginal cells, count the cells on
‘L’ line, i.e. either right and lower or left and
upper margin. Make the total of cells
counted in 5 squares.

28.  Let the no. of RBC counted in 80 small
squares = N
 Volume of area in which RBCs counted =
( 1/5 x 1/5 x 0.1 ) x 5=1/50
 No of RBCs in 1/50 mm3= N
No of RBCs in 1 mm3= Nx50
 Dilution factor = 200
 RBC count per mm3 = Nx50x200=N x 10000

29. No. of cells in neubeaur chamber=
N x dilution factor
Volume of chamber in which
cells counted
 For RBC
N x 10000

30. ERRORS OF RBC COUNT
 TECHNICAL ERRORS:
defective apparatus/defective
technique
Technical errors can be minimised by
the use of accurately caliberated
apparatus and by careful technique.
 INHERENT ERROR:
The distribution of RBCs is of an
irregular pattern in the counting
chamber.This affects the accuracy of
visual count which can be minimised by
counting a large no. of RBCs.

31. AUTOMATED METHOD
 Electronic counter is based on the
principle of aperture impendence
method .
 Anticoagulated blood is diluted with
particle free diluting fluid such as
physiological saline or phosphate buffer
saline.
 Particles passing through a chamber in
single file scatter the light and convert
by a detector into pulses proportional to
size of the cells which are then counted
electronically.

32. Advantages:
1) Easy and rapid method
2) Many thousand of cells are counted
compared to fewer cells counted in
manual method.
Disadvantages:
1)Costly equipment
2) Caliberation error
3) Altered composition of diluent causes
erroneous results.
4)Gaint platelets are counted as RBCs.
5)High WBC count alters results.

33. Reference range:
 Adult male – 4.5 to 5.5 million/cumm
 Adult female – 3.8 to 5.2 million /cumm
 At birth – 6 to 8 million/cumm

34. Conditions of decreased RBC count:
1)Anaemia
2)Pregnancy
Conditions of increased RBC count:
Physiological: 1)new borns and infants
Pathological : 1)
polycythemia(polycythemivera,kidney
tumours,high altitude)
2) dehydration
3 ) hypoxia(COPD,
congenital heart disease)

35. THANK
YOU