Heamocytometery

RBC Counting
• Counting of RBC’s using haemocytometer
• Anaemia: Decreased RBC count
• Polycythaemia: Increased RBC count
• Requirements
• Haemocytometer
• Microscope
• Diluting fluid
• Blood sample, preferably anticoagulant added
 Dilution Fluids
• Maintains isotonicity
• Many fluids can be used
• Hayem’s Fluid
• Grower’s Fluid
• Formal Citrate Solution
• Physiological Saline
• Physiological Saline is used mostly because of availability
 Compositions
• Hayem’s Fluid
• Mercuric Chloride 0.5 g
• Sodium Sulphate (anhydrous 2.2 g)
5g
• Sodium chloride 1 g
• Distilled water 200 ml
• Grower’s Fluid
• Sodium Sulphate 12.5 g
• Glacial acetic acid 33.3 ml
• Distilled Water 200 ml
• Formal Citrate Solution
• Tri sodium citrate 31.2 g
• Distilled water 100 ml
• Formalin 10% 10 ml
• Physiological Saline
• Sodium chloride 0.89 g
• Distilled water Q.S. to make
100 ml
 Procedure
• Focus counting chamber under
microscope using 4x objective
lens
• At 4x whole counting area will
be visible
• At 10x, a single primary will
cover the visible field
• At 40x, a single secondary will
cover visible field
• RBC counting is performed
using 40x objective lens
• Take blood up to 0.5 graduation in RBC pipette
• Dilute the blood by taking dilution fluid up till 101 mark
• Shake the pipette gently, do not let the fluid drip or waste
• Waste first 2-3 drops from pipette
• After that, charge the haemocytometer (add a drop to
haemocytometer) and place a coverslip on it
• Let it settle, and then observe using 10x objective lens, then
move to 40x objective lens
• Count in 5 secondary squares
 Leave left lower
Read right upper
• Any cell present on the left and
lower border of a tertiary
square, will be not be included
• Any cell present on right and
upper border of tertiary square
will be counted
 Calculations
Area of 1 primary square = 1 mm 2

We are counting in 5 squares out of 25

So we are counting in 1/5 part of primary square
th

So, area of counting can be written as 1/5 area of primary or 1/5 mm

th 2

Multiplying this with depth → 1/5mm x 1/10mm = 1/50 mm

2 3

No. of cells counted in 1/50mm = N 3

Multiplying both sides with 50 → 1/50 x 50 = N x 50

No. of cells counted in 1 mm = N x 50
3

Adding dilution factor Normal Value of RBC's

No. of cells counted in 1mm = N x 50 x 200

3 4.1 to 5.1 millions per mm cube in Women
4.5 to 5.9 millions per mm cube in Men

So,
N x 50 x 200 = No. of RBC’s /mm of blood 3

Where N is the number of RBC counted in 5 secondary squares of RBC counting area