RBC Manual 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.
Mechanism of Erythropoiesis
•
•
The regulation and production of RBCs is called
erythropoiesis.
The mechanism of erythropoiesis:
1. The kidney monitors the level of oxygen in the blood.
2. If oxygen levels are low then the kidney secretes a
hormone called erythropoietin.
3. Erythropoietin enters the blood stream and travels
throughout the body. All cells are exposed to
erythropoietin, but only red bone marrow cells,
respond to the hormone.
4. Erythropoietin stimulates the production of
erythrocytes in the bone marrow.
Mechanism of Erythropoiesis
5.
6.
7.
8.
These erythrocytes leave the bone marrow and move into
the blood stream.
As the erythrocyte population increases, the oxygen
carrying capacity of the blood increases.
When the kidney senses that oxygen levels are adequate,
it responds by slowing the secretion of erythropoietin.
This negative feedback loop ensures that the size of the
erythrocyte population remains relatively constant and
that the oxygen carrying capacity of the blood is always
sufficient to meet the needs of the body.
Erythrocyte Count
• Is the number of erythrocytes per micro litter of
blood.
• Normal ranges:
– Male
– Female
– New born
4.2-5.4
3.6-5.0
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:
•
1.
2.
3.
4.
One of the following solutions may be used:
Isotonic saline:
– 0.85% sodium chloride (NaCl) in distilled water.
Hayam’s solution:
– Sodium Sulphate
10 g.
– Sodium Chloride
2 g.
– Mercuric Chloride
0.25 g.
– Distilled Water
100ml
Gower’s solution:
– Sodium Sulphate
12.5 g.
– Glacial acetic acid
33.3 ml
– Distilled water
100 ml.
Citrate-formalin solution:
– Tri-sodium Citrate
– Formalin
•
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%