Determination of Red
Blood Cell Count
Dr. Anjusha. I. B
Assistant Professor
BGS GIMS
AIM
• To count the number of red blood cells present per cubic mm of your
own sample of blood
PRINCIPLE
• The subject’s blood is diluted to a known dilution (1:200) with a
suitable diluting fluid. The RBCs in a definite volume of this diluted
blood are counted, the number is then multiplied by the dilution
factor (200), so as to express the count per cu mm of undiluted blood
APPARATUS
• RBC Pipette
• RBC Diluting fluid (Hayem’s fluid)
• Improved Neubauer’s counting chamber
• Compound Microscope
• Cover slip
• Lancet
• Cotton
• Spirit
RBC Diluting Fluid (Hayem’s fluid)
Composition
• Sodium Sulphate (Na2SO4) – 2.5g : Prevent Rouleaux formation
• Sodium Chloride (NaCl) – 0.5g : Maintain isotonicity
• Mercuric Chloride (HgCl2) – 0.25g : Preserxative : antibacterial &
antifungal
• Distilled water -100 ml :Solvent
Other RBC diluting fluids
• Isotonic Saline (0.9% NaCl)
• Dacie’s fluid
Trisodium citrate
Concentrated Formalin
Distilled water
• Gower’s solution
PROCEDURE
• Ensure that the pipettes, coverslip and Neubauer’s chamber are
thoroughly clean and dry
• Take adequate RBC diluting fluid in a watch glass
• Prick the tip of the finger taking necessary precautions
• Wipe off the first drop of blood.
• Suck blood into the RBC pipette exactly upto 0.5 mark taking care that
there are no air bubbles
• Any excess blood can be removed by tapping the tip of the pipette
against the palm of your hand
PROCEDURE
• Wipe off the blood sticking to the tip of the pipette
• Immediately suck diluting fluid exactly upto 101 mark in the pipette
• Hold the pipette horizontally between the palms and roll gently for a
minute
• Place a clean coverslip over the counting grid of Neubauer’s chamber
• Discard the first few drops from the stem of the pipette as it contains
only diluting fluid and no cells
Preparation of the slide
Step 1 Step 2
Dilute
Place the coverthe sample 1:200
slip
Step 3 Step 4
Charging Step Count in the small 5 center squares for RBC
Charging the Neubauer’s chamber
• A small drop of fluid is allowed to form at the tip of the pipette and this
drop is gently brought in contact with the edge of the coverslip.
• The fluid is drawn into the chamber by capillary action
• Both sides of the chamber should be charged
• Ideally charged chamber is one that has been charged with a single
adequate sized drop which just fills the chamber without any air bubbles
• Overcharging – if the fluid overflows in to the gutter
• Undercharging – if the fluid is insufficient to fill the chamber or have air
bubbles
• In both conditions the count will be low
Charging the Neubauer’s chamber
contd-----
• Allow sometime for the cells to settle down
• Focus the charged chamber first under the low power objective
• See whether the cells are uniformly distributed, or else recharge it
• Now change to high power objective and count the RBCs in 4 medium
sized corner squares and 1 medium sized central square
• Total RBCs in 80 small squares have to be counted
Charged RBC squares under high
power
Rule for counting of cells
• Counting is carried out in a zig-zag pattern
• Any cell lying on the left and lower border of a square is counted in
that particular square
Calculation
• Length of 1 smallest square = 1/20mm
• Breadth of 1 smallest square = 1/20mm
• Area of 1 smallest square = 1/400mm2
• Depth of 1 smallest square = 1/10mm
• Volume of 1 smallest squares = 1/400 X 1/10 = 1/4000mm3
• Volume of 80 smallest square = 80/4000mm3
• Let the number of cells counted be ‘N’
• 80/4000mm3 of diluted blood contain ‘N’ cells
• Dilution factor = 1: 200
• Therefore, 1 mm3of undiluted blood contains = = N X10000cells
• = --------million cells/cu.mm of blood
REPORT
• The Total Erythrocyte (RBC) count of my blood is ------million
cells/cu.mm of blood
Precautions
• The pipette, coverslip and Neubauer’s chamber should be thoroughly clean and dry
• Finger prick should be bold enough to give free flowing blood
• Never squeeze the finger to obtain blood
• Take blood exactly upto 0.5 mark
• Wipe off the tip of the pipette before diluting the blood
• Blood in the pipette should be diluted quickly
• Suck RBC diluting fluid exactly upto 101 mark
• Before charging the chamber, the fluid in the stem of the pipette has to be
discarded
• There should not be any overcharging or undercharging of the chamber
Precautions
• After charging the chamber, allow some time for cells to settle down
but the count should be made before the fluid starts drying up
• While counting the cells, the stage of the microscope should not be
tilted
• Avoid counting the same cells twice
Normal Range
• Males : 5 – 6 million cells per cu.mm of blood
• Females : 4.5 – 5.5 million cells per cu.mm of blood
• Newborn : 6 – 8 million cells per cu.mm of blood
QUESTIONS
• How do you discard the excess blood sucked into the pipette?
• If excess of blood is sucked into the pipette, it should be gently
tapped against the palm or finger tip to discard it. Any absorbent
material should not be used as it will suck the fluid content of the
blood and will give false high blood count.
• What are the precautions taken for pipetting?
• The pipette should be cleaned thoroughly and dry it before pipetting
• The tip of the pipette should gently touch the blood drop and blood
should be drawn exactly upto 0.5 mark
• There should not be any air bubbles in the blood column
• Any excess blood should be removed by tapping the tip of the pipette
against the palm of your hand
• Blood in the pipette should be diluted quickly so as to avoid blood
clotting
• What is the composition of Hayem’s fluid? What are the functions of
its constituents?
The composition of Hayem’s fluid is
• Sodium Sulphate (Na2SO4) – 2.5g : Prevent Rouleaux formation
• Sodium Chloride (NaCl) – 0.5g : Maintain isotonicity
• Mercuric Chloride (HgCl2) – 0.25g : Preservative : antibacterial &
antifungal
• Distilled water -100 ml :Solvent
• What are the average values of RBC count?
• Males : 5 – 6 million cells per cu.mm of blood
• Females : 4.5 – 5.5 million cells per cu.mm of blood
• Newborn : 6 – 8 million cells per cu.mm of blood
• What are the conditions in which RBC count is increased or
decreased?
• The condition where RBC count increases is called Polycythemia
Physiological Pathological
Males Polycythemia vera
Infants and Newborn Chronic Smokers
High Altitude Dehydration
• The condition where RBC count decreases is called Anaemia
Physiological Pathological
Females Blood loss
Pregnancy Anaemia due to any cause
Myxoedema
THANK YOU
