Practical Hematology Lab
- LAB 3 -
Red Blood Cell (RBCs) Count
Introduction
• Red blood cells make up almost 45 percent of the blood
volume.
• Their primary function is to carry oxygen from the lungs
to every cell in the body.
• Red blood cells are composed predominantly of a protein
and iron compound, called hemoglobin, that captures
oxygen molecules as the blood moves through the lungs,
giving blood its red color.
• As blood passes through body tissues, hemoglobin then
releases the oxygen to cells throughout the body.
• Red blood cells are so packed with hemoglobin that they
lack many components, including a nucleus, found in
other cells.
RBCs Structure
• The membrane, or outer layer, of the red blood cell is
flexible, like a soap bubble, and is able to bend in many
directions without breaking.
• This is important because the red blood cells must be
able to pass through the tiniest blood vessels
(capillaries) to deliver oxygen wherever it is needed.
• The capillaries are so narrow that the red blood cells,
normally shaped like a disk with a concave top and
bottom, must bend and twist to maneuver single file
through them.
Erythrocyte Count
• Is the number of erythrocytes per micro litter of
blood.
• Normal Ranges:
• Male
4.2 – 5.4
• Female
3.6 – 5.0
• New born
5.5 – 6.5
106/µL
106/µL
106/µL
• Erythrocyte count increased in case of
polycythemia and decreased in anemia.
Erythrocyte Count
• Principle:
• In order to facilitate RBCs count a specified
volume of blood is diluted with a specified
volume of isotonic fluid.
• Red cell diluting fluid must be:
• anti-coagulant anti-hemolysis.
• anti-aggregation.
• anti-Rouleaux.
• preserve RBC shape.
Erythrocyte Count
Diluting fluid:
•
One of the following solutions may be used:
1. Isotonic saline:
• 0.85% sodium chloride (NaCl) in
distilled water.
2.
Hayam’s solution:
•
•
•
•
Sodium Sulphate
Sodium Chloride
Mercuric Chloride
Distilled Water
10g.
2g.
0.25g.
100ml
3.
Gower’s solution:
1. Sodium Sulphate
2. Glacial acetic acid
3. Distilled water
4.
Citrate-formalin solution:
1. Tri-sodium Citrate
2. Formalin
12.5 g.
33.3 ml
100 ml.
Note:
• In certain conditions, Hayam’s solution may
cause clumping of RBCs and Rouleaux
formations, while Gower's solution prevents
these problems
Sample:
• Whole blood using EDTA or heparin as
anticoagulant. Capillary blood may also be
used.
Equipments:
1. (Pipettes) used one of the following:
Thoma pipette (RBCs)
Micropipette –20l is the desired volume.
2. Improved Neubauer chamber with the
cover slips.
3. Conventional light microscope.
4. Clean gauze.
Procedure
1. Dilution of the blood:
•
Micropipette (20) 1:201 dilution.
• Pipette 4.0ml of diluting fluid into a tube
• Pipette 20l of will mixed anticoagulated whole
blood to the tube.
•
Thoma red count pipette.
• Draw the blood up to exactly the 0.5 mark and
dilute to the 101 mark.
• Mix continuously for 2-3 minutes.
2.
Load the cleaned hematocytometer.
3.
Place the hematocytometer on the microscope stage,
lower the condenser.
Procedure
4. Focus with x10 objective
lens on the large central
square. This square is ruled
into 25 small squares, each
of which is further divided
into 16 smaller squares, of
the 25 squares, only the four
corner squares, and one
middle square are used to
count RBCs.
5. Switch to 40 objective lens,
and start counting in the five
designated squares.
Calculations
•
Total RBC Count =
•
N x Dilution Factor x Volume Correction Factor
Where:
•
•
•
N = the total number of red cells counted in the counting
chamber.
Dilution 1: 200
•
Dilution Factor = 200
Counted Volume:
•
•
•
Each counted square has a volume of 0.2 X 0.2 X 0.1 =
0.004
5 squares volume = 5 X 0.004 = 0.02 cumm
Volume correction factor = 1/0.02 = 50
So,
•
Total RBC count =
•
N X 200 X 50 = N X 10.000
Discussion
1. In certain conditions, such as polycythemia, the
red blood cell count may be extremely high,
which makes it difficult to obtain an accurate
count. In this instance, make a larger dilution
of blood. For a 1:301 dilution, add 20L of
whole blood to 6.0mL of diluting fluid.
2. For a patient who has severe anemia and in
whom the RBC is low, make a 1:101 dilution
by adding 20L of whole blood to 2.0mL of
diluting fluid.
Discussion
3. Make certain the pipettes, hemacytometer, and
cover glass are free from dirt, lint, and dried
blood. Ensure that the diluting fluid is free from
blood and other contamination.
4. RBC takes longer to perform than a WBC
because of the larger number of cells.
Therefore, proceed as quickly as possible once
the cells have settled. Drying of the dilution in
the counting chamber causes inaccuracies in the
final cell count.
5. The range of error for a manual RBC is
generally about 10 to 20%