Total RBC Count PDF

TOTAL RBC COUNT DR. SHOBHA SHRIVASTAVA ASSOCIATE PROFESSOR DEPARTMENT OF ZOOLOGY PATNA WOMEN’S COLLEGE, AUTONOMOUS PATNA UNIVERSITY, PATNA TOTAL RBC COUNT INTRODUCTION HEMOCYTOMETER ENUMERATION OF TOTAL NUMBER OF RED BLOOD CELLS INTRODUCTION A red blood cell (RBC) count is a blood test that t...

TOTAL RBC COUNT DR. SHOBHA SHRIVASTAVA ASSOCIATE PROFESSOR DEPARTMENT OF ZOOLOGY PATNA WOMEN’S COLLEGE, AUTONOMOUS PATNA UNIVERSITY, PATNA TOTAL RBC COUNT INTRODUCTION HEMOCYTOMETER ENUMERATION OF TOTAL NUMBER OF RED BLOOD CELLS INTRODUCTION A red blood cell (RBC) count is a blood test that tells how many red blood cells, also known as erythrocytes, are there in the body. Red blood cells make up almost 45 % 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. Red blood cells are so packed with hemoglobin that they lack many components, including a nucleus, found in other cells.  As blood passes through body tissues, hemoglobin then releases the oxygen to cells throughout the body. The amount of oxygen that’s delivered to the body’s tissues depends on the number of red blood cells are there in the body and how well they work. An RBC count is usually carried out as part of a full blood cell (FBC) count or complete blood count (CBC). Women usually have a lower RBC count than men, and the level of red blood cells tends to decrease with age.  The red blood cell count is the number of red blood cells per unit volume of whole blood. Normal red blood cells values at various ages are: - Newborn : 4.8 - 7.2 million - Adults (males) : 4.9 - 5.5 million - Adults (Females): 4.4 - 5.0 million - Pregnancy : slightly lower than normal adult values - Children : 3.8 - 5.5 million  The normal ranges are a guide and can vary between different hospital laboratories. The results of an RBC count can be used to help diagnose blood- related conditions, such as iron deficiency anaemia (where there are less red blood cells than normal). A low RBC count could also indicate a vitamin B6, B12 or folate deficiency. It may also signify internal bleeding, kidney disease or malnutrition (where a person’s diet does not contain enough nutrients to meet their body’s needs).  A high RBC count could be caused by a number of health conditions or health-related factors, including: - smoking - congenital heart disease - dehydration (for example, from severe diarrhoea) - low blood oxygen levels (hypoxia) - pulmonary fibrosis (a lung condition that causes scarring of the lungs) - Problem with heart’s structure and function that is present at birth (congenital heart disease) RBC count will increase for several weeks in a higher altitude. HAEMOCYTOMETER A haemocytometer or hemocytometer consists of a thick glass microscope slide with a grid of perpendicular lines etched in the middle. This chamber is engraved with a laser-etched grid of perpendicular lines. The grid has specified dimensions so that the area covered by the lines is known, which makes it possible to count the number of cells in a specific volume of solution. The haemocytometer was invented by Louis-Charles Malassez. HAEMOCYTOMETER  The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. By observing a defined area of the grid, it is therefore possible to count the number of cells in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall. A well used type of hemocytometer is the Neubauer counting chamber.  The gridded area of the Improved Neubauer ruled Haemocytometer consists of nine 1 x 1 mm (1 mm2) squares. These are subdivided in three directions; - 0.25 x 0.25 mm (0.0625 mm2), - 0.25 x 0.20 mm (0.05 mm2) and - 0.20 x 0.20 mm (0.04 mm2). The central square is further subdivided into 0.05 x 0.05 mm (0.0025 mm2) squares. The raised edges of the haemocytometer hold the coverslip 0.1 mm off the marked grid, giving each square a defined volume. HEMOCYTOMETER GRID AND DIMENTIONS ENUMERATION OF TOTAL NUMBER OF RED BLOOD CELLS To enumerate the total number of Red Blood Cells (RBC) of a given blood sample by Hemocytometer (Neubauer) Counting Method: Materials required: Hemocytometer chamber Cover slip Light microscope RBC pipette RBC diluting fluid (Haeyem’s solution or Physiological saline 0.85% NaCl) [ Haeyem’s Solution or RBC Diluting Fluid Consists of the following: Sodium Chloride, Sodium Sulphate and Mercuric Chloride. NaCl = 1 G (Isotonic solution). Na2SO4 = 5 grams. It will prevent rouleux formation. HgCl2 = 0.5 G acts as an antiseptic. D. H2O = 200 mL Used for diluting Blood prior to counting Red Blood Cells with a Haemocytometer.]  Microscope Lancet Alcohol 70% Cotton Taking the blood sample: To take the blood sample, the ring finger tip is wiped with alcohol and allowed to dry. With a sterile disposable lancet a small prick is made on the finger tip. When a blood drop of reasonable size has collected, the red blood cell pipette is held slightly tilted from the vertical position, its tip is applied to the drop and blood is aspirated to the mark 0.5. Sample preparation: It uses an RBC pipette to incorporate the blood specimen with the diluent. Blood sample up to a point (0.5) is taken. Then, the RBC pipette’s tip is wiped using blotting paper. After that, RBC diluting fluid or diluent is sucked up to a mark 101, thus making 1:200 dilution of blood. The blood and diluting fluid are mixed in the RBC pipette, by horizontally rotating, by using the palms for 2 to 3 minutes (to ensure complete hemolysis of WBC). RBC PIPETTE Loading the sample over the Haemocytometer slide: A clean, grease-free Haemocytometer slide and cover glass are taken. The cover glass is placed on top of the Haemocytometer’s lined region. Before loading the RBC sample into the Haemocytometer, 1-2 drops are discarded. Then, the RBC pipette is carried at an angle (45 degrees) and a small volume (a drop of a fluid) of RBC sample is loaded towards the edge of a cover glass. In order to settle down the RBCs in the chamber, 3-5 minutes are allowed. The prepared slide is observed under the microscope to count the number of RBCs manually. RBC Count under Microscope: First, the rulings of the Haemocytometer slide using a 10X objective lens is focused. Using coarse and fine adjustment knobs, the five squares of the large central square are focused on to count the number of red blood cells under the 40X objective. The diagram represents the pattern to count RBCs in all the five medium squares of a large central square i.e., 80 squares, one at each corner and one in the center. As already discussed, each medium square possesses 16 small squares. RBC COUNT UNDER MICROSCOPE  The number of RBCs in five medium squares are manually counted via hand tally. In each square, the red blood cells located within the square are counted. In the diagram, the red lines in the upper and right corners indicate the areas not to count RBCs, whereas green lines indicate the areas to count the RBCs. Calculation of RBCs : Total RBCs/µL = Number of RBCs counted x Dilution factor / Area x Depth The number of red blood cells (N) =? Dilution factor = 1:200 or 200 A large central square is subdivided into 25 medium squares or sub squares. So, the area will be one sq. mm. The number of RBCs is enumerated in 5 squares out of 25 squares. So, the area of 5 small squares will be 5/25 or 1/5 sq. mm. Depth of the sample = 0.1 mm  Total RBCs = N x 200 / 1/5 x 0.1 = N x 200 x 50 = N x 10,000 cells/µL Suppose, N or number of RBCs in the five squares is 486, then the equation will be represented as: Total RBCs = 486 x 10,000 = 48,60,000 cells/µL Another method of calculation: 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 mm3 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. The total number of cells in 1 mm3 are = Nx50 (before diluting the sample) The actual total number of cells after dilution should be = N x 50 x 200 = N x 10000