This document describes the process for manually counting red blood cells (RBCs). It discusses that RBC count is performed to screen for health issues or assess conditions like anemia. The manual method uses a Neubauer hemocytometer chamber under a microscope to count RBCs in a diluted blood sample within a specified area. The number of RBCs counted is then used in calculations along with dilution and chamber volume/depth factors to determine the RBC concentration in units of cells per microliter. Proper technique and controls are needed to get an accurate RBC count.

1. Red Blood Cell
Count
by Ashwin George
VIT university, vellore
By Ashwin George (16BBT0174)
VIT Univeristy, Vellore
BIT1006

2. Red Blood Cell = Erythrocyte
o RBC is a flexible cell, biconcave disc in shape, and around
8 µm in diameter.
o The number of red cells per volume of blood, measured in
microliters (µL) or cubic millimeters (mm3)

3. Why do we count RBCs?
Screening:
-Health maintenance
-NSAIDs
To assess
degree of ..
Anemia and blood
loss
Follow up:
-Response to ttt
-Chronic Anemia

4. Counting of RBCs can be performed either:
❖ Automated Method: (Electronic hematology cell counter, e.g.
COULTER)
OR
❖ Manual Method: (visual using a microscope and counting
chmber), which is a cumbersome and less accurate.

5. o The solution used for red cell count is Isotonic with RBCs;
doesn’t lyse leukocytes.
o Leukocytes are normally too few which can be identified easily
and won’t be interfered with erythrocytes count.

6. o Reagents and instruments:
1. Neubauer Chamber (Haemocytometer) & coverslips.
2. RBC diluting fluid/solution.
Consists of 3.2 g of Na-citrate and 1.0 ml of formaldehyde solution
made up to 100 ml with D.W.
o Sample:
Procedure

7. Haemocytometer (Manual method)
Improved Neubauer Chamber

8. o A thick glass slide with H shaped moats in it.
o The area between two lines of H (center) is 0.1 mm in depth.
o Moat prevents mixing of 2 samples on either side of chamber.
Haemocytometer

9. Haemocytometer

10. o Prepare a plastic tube (labeled).
o Prepare 1:200 blood dilution (4 ml of diluent + 20 µl Blood).
o So, add diluent to the tube. Mix the sample (5 times); then
aspirate 20µl and transfer to the tube and mix.
o Clean the Haemocytometer and coverslip with 70% ethanol
followed by D.W &Leave to dry.
o Place a coverslip on the Neubauer chamber.
Method

11. o Then, fill the chamber with the diluent (10 microliter) in each
side.
o Leave chamber in humidity (petri-dish with wet filter paper) for
1-2 min. !?
o Condenser slightly lowered. Iris diaphragm should be almost
closed.
o Place chamber on microscope stage. Start with 10X to focus;
then with 40X count RBCs.
Method

12. How to count RBCs?
o RBCs should be counted in the central square of the
chamber.
o Select 5 small squares (One at each corner and one in the
center).

13. W
W W
W
RBC
Areas for WBCs and RBCs count

14. W
R
Areas for WBCs and RBCs count
3 mm.sq
25 small
Squares = 1
mm.sq

15. Areas for WBCs and RBCs count
WBCs
RBCs
Central large square

16. Count all cells in specified squares, and multiply by the proper conversion
factor; the number of cells per cubic millimeter can be determined.

17. o Count all cells within 16 squares and those lying on middle lines,
EXCEPT …
How to count ?

18. RBCs (1012/L) = No. of RBCs counted X Dilution X 106
Volume (µl)
o Dilution = 200
o Depth of the chamber = 0.1 mm.
o Volume of 5 small squares = 0.02 µl
So, Red cell count/ liter = N x 0.01 x 1012 i.e. RBCs= N x 1012/L
Calculations

19. o Diluent should be correct.
o No overflow in the moats.
o No air bubbles and debris in the chamber area.
o No scratches in the ruled area of the chamber.
o Pipettes used must be clean and dry.
Precautions

20. RBCs Reference values:
RBC count Age group
4.7 – 6.1 X 1012 /L Adult Male
4.2 – 5.4 X 1012 /L Adult Female
4.4 -5.8 X 1012 /L Newborn
3.8 – 5.5 X 1012 /L Infant/ children

21. Thank you