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Red Blood Cell Count
By: Shefaa Adel Hejazy.
Umm Al-Qura University, Makkah.Faculty of Medical Sciences. Hematology Dept.
1st Semester 1433/2012
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)
Why do we count RBCs?
Screening:-Health maintenance -NSAIDs
To assess degree of ..Anemia and blood loss
Follow up:-Response to ttt-Chronic Anemia
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.
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.
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
Haemocytometer (Manual method)
Improved Neubauer Chamber
H
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
Haemocytometer
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
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
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).
W
W W
W
RBC
Areas for WBCs and RBCs count
W
R
Areas for WBCs and RBCs count
3 mm.sq
25 smallSquares = 1 mm.sq
Areas for WBCs and RBCs count
WBCs
RBCs
Central large square
Count all cells in specified squares, and multiply by the proper conversion factor; the number of cells per cubic millimeter can be determined.
o Count all cells within 16 squares and those lying on middle lines, EXCEPT …
How to count?
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
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
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
Interpretation of Results
Decreased value ( RBCs)
1. Anaemia: due to Blood loss, production of cells, destruction of cells, and dietary insufficiency.
2. Diseases which affect the Bone Marrow such as:
i. Leukaemiaii. MMiii. Hodgkin’s Lymphoma
1. Subcutaneous bacterial endocarditis
2. Rheumatic fever
Increased value ( RBCs)
1. Polycythaemia vera
2. Secondary polycythaemia, e.g. smokers, high altitude, cyanotic heart defects, and COPD.
3. Dehydration
4. Acute poisoning
5. Severe diarrhea
Interpretation of Results
