1. RBC Manual Count

2. 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.

3. 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.

4. Mechanism of Erythropoiesis
The regulation and production of RBCs is called erythropoiesis.
The mechanism of erythropoiesis:
The kidney monitors the level of oxygen in the blood.
If oxygen levels are low then the kidney secretes a hormone called erythropoietin.
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.
Erythropoietin stimulates the production of erythrocytes in the bone marrow.

5. 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.

7. Erythrocyte Count
Is the number of erythrocytes per micro litter of blood.
Normal ranges:
Male /µL
Female /µL
New born /µL
Erythrocyte count increased in case of polycythemia and decreased in anemia.

8. 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.

9. Diluting fluid: One of the following solutions may be used:
Isotonic saline:
0.85% sodium chloride (NaCl) in distilled water.
Hayam’s solution:
Sodium Sulphate g.
Sodium Chloride g.
Mercuric Chloride g.
Distilled Water ml
Gower’s solution:
Sodium Sulphate g.
Glacial acetic acid ml
Distilled water ml.
Citrate-formalin solution:
Tri-sodium Citrate
Formalin

10. 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:
(Pipettes) used one of the following:
Thoma pipette (RBCs)
Micropipette –20l is the desired volume.
Improved Neubauer chamber with the cover slips.
Conventional light microscope.
Clean gauze.

11. 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.
Load the cleaned hematocytometer.
Place the hematocytometer on the microscope stage, lower the condenser.

12. Focus with x10 objective lens on the large central square
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.
Switch to 40 objective lens, and start counting in the five designated squares.

14. 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 = 0.02 cumm
Volume correction factor = 1/0.02 = 50
So, Total RBC count = N X 200 X 50 = N X

15. Discussion
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.
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.
The range of error for a manual RBC is generally about 10 to 20%

16. 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.
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.