Total Red Blood Cell (RBC) Count by manual method, Lecture notes of Medical Sciences

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RED BLOOD CELL (RBC) 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.

 Each RBC is a biconcave disc having a diameter of 7.2 microns and a thickness of 2.2 microns  These cells contain the pigment hemoglobin which enables them to transport oxygen around the circulation. They also contain the enzyme carbonic anhydrase which enables them to carry CO2.

AIM OF THE EXPERIMENT RBC count is done to determine whether there is an adequate number of RBC in the circulation or not.

MANUAL RED BLOOD CELL COUNT MATERIAL AND INSTRUMENTS 

Anticoagulated whole (using EDTA or heparin

as an anticoagulant) or capillary blood can be

used.



Hayem’s solution (diluting fluid) composed of:



Hgcl2 0.05 g



Na2so4 2.5 g



Nacl 0.5 g



Distilled water 100 ml

 RBC pipette which is composed of a stem & a mixing chamber with a red bead, it is function is to mix blood with the substance and for differentiation from the WBC pipette.  Haemocytometer “ Neubauer” chamber is counting chamber with a cover slip. The same counting chamber is used also for counting total white blood cells.  Microscope  Lancet  Alcohol 70%  Cotton

 Wipe off any blood adhering to its outer side. If the blood gets beyond 0.5 marks tap the tip gently till the blood is exactly at the mark. Never allow the blood to clot inside the pipette. If the blood clots in the pipette blow the sample out, clean the pipette and begin all over again.  Aspirate diluting Hayem’s solution to the 101 mark, thus making 1:200 dilution of blood.  Hold the pipette horizontally and role it with both hands between finger and thumb.

 Blow out a quarter of the content to remove the pure diluting fluid in the stem.  Prepare the counting chamber and cover it with a cover slip. Hold the pipette 45 & touch its tip gently on the surface of the counting platfrom where it projects beyond the cover slip and a small amount of solution will be drawn under the cover slip.  Place the Neubauer chamber on the stage of the microscope and allow 2 minutes for the cells to settle.

NEUBAUER HEMOCYTOMETER

Count the cells in 5 medium squares of 16 small squares i.e. 80 squares, one at each corner and one in the center. Hemocytometer Chamber

CALCULATION

 Count the number (N) of cells in 80 small squares located in 5 middle-sizes squares (four located at the four corner and one in the middle). The size of 80 small squares in which “N” number of cells are found is: 1/20 x 1/20 x 1/10 x 80 = 1/50 mm Where 1/20 mm:- is the slid line of the square. 1/10 mm:- is the depth of the counting chamber between cover slip and the ruling. 80:- is the number of small squares used to count.

MEDICAL CONSIDERATIONS Pathological conditions:-  Polycythemia is a disease of unknown origin that results in an abnormal increase in red blood cells due to over production of red blood cells in the bone marrow not caused by physiologic need (primary polycythemia vera), while secondary polycythemia vera occur in response to hypoxia.

 Anemia: is a general term that refers to a decrease in red blood cells. Anemia can occur from either a decrease in the number of red blood cells, a decrease in the hemoglobin content, or both. A lower than normal RBC can result from a number of causes, including:  Massive RBC loss, such as acute hemorrhage  Abnormal destruction of RBC  Lack of substances needed for RBC production  Chemotherapy or radiation side effect from treatment of bone marrow malignancies such as leukemia can result in bone marrow suppression.