Manual RBC Count PDF

Manual RBC’s Count. 1  Objectives 1. Use the hematocytometer for RBC’s count. 2. Estimate the number of circulating RBC’s /mm³ of blood. 3. Understand the physiological and pathological conditions associated with the variation of RBC’s count. 4. List some fac...

Manual RBC’s Count. 1  Objectives 1. Use the hematocytometer for RBC’s count. 2. Estimate the number of circulating RBC’s /mm³ of blood. 3. Understand the physiological and pathological conditions associated with the variation of RBC’s count. 4. List some factors that cause source of error in RBC’s count. 2  Manual RBC’s Count. RBC’s count : is the count of total # of erythrocyte in a volume of blood. Expressed as RBC’s/1mm³ blood. Erythrocyte (RBCs) are : – biconcave in appearance. – Have no nucleus. The interior of the cell contains red pigment called Hemoglobin which is responsible for red color of blood. Life span 120 days. 3  Manual RBC’s Count. The function of erythrocytes: is to combine with O2 & to lesser extent CO2 & transport them through blood vessels. Normal range of RBC’s: for healthy adult male: 4.5 - 6 million cell/mm³. for healthy adult female: 4 - 5.5 million cell/mm³. 4  Manual RBC’s Count. In this experiment you are going to determine the # of RBCs in mm³, and because this # is very large, its practical to dilute a sample with an Isotonic Solution (Hayem’s solution), then multiply the counted # by correction factor (dilution factor). 5  Manual RBC’s Count. Materials 1. Blood sample (EDTA anticoagulated blood or capillary blood). 2. RBC’s diluting pipette. 3. Diluting solution (Hayeme’s solution). 4. Hemocytometer. 5. Microscope. 6  Manual RBC’s Count. RBC’s pipette Consist of : - Stem. - Bulb.(mixing chamber) It has 3 marks (0.5,1,and 101) and usually has Red bead. 7 Manual RBC’s Count. 8 Manual RBC’s Count. 9 Manual RBC’s Count. 10 Manual RBC’s Count. 11 Manual RBC’s Count. 12  Manual RBC’s Count. The Hemocytometer : - Contains two square ruled areas separated by an h-shaped moat. - Depth = 0.1mm. - Each square has total area of 9 mm². - These squares are divided into 9 primary squares with an area of 1mm² for each one of them. - The four corner primary squares are used when counting WBC’s. 13  Manual RBC’s Count. - Each one of these 4 large corner squares, contain 16 smaller secondary (medium) squares. - The central primary square is used for RBC’s count. its consist of 25 secondary (medium) squares. we count the red blood cells in the four corner medium squares & the central medium square from the central primary square. - Each of these 25 square has a total area of 0.04 mm². also each one of them is further divided into 16 tertiary ( smallest ) squares. 14  Manual RBC’s Count. Procedure: With a safety bulb draw blood up to 0.5 marks on RBC’s pipette and complete to 101 with RBC’s diluting solution (hayem’s solution). 2. Mix for 2-3 minute. 15  Manual RBC’s Count. 3. Charge hemocytometer : load the counting chamber with diluted blood as follows: Discard the first 4-5 drops. Place tip of the pipette at edge of the central platform of hemocytometer slide and let a drop of diluted blood run between the hemocyto- meter slide and cover slip by capillary. 16  Manual RBC’s Count. 4. Let the hemocytometer to stand on the bench for 3-5 minute so the cells are settled down. 17  Manual RBC’s Count. 5. Count and calculate: An erythrocytes count is performed with neubauer hemocytometer as follow: A- Place the hemocytometer on the microscope stage. Using 40X magnification lens , count the RBC’s in the four (4) corner squares and the one in the center of the large center square of the counting chamber.( starting from the upper left medium square). and follows the pattern indicated by the arrow in the next figure. count all cells that touch any of the upper and left lines, do not count any cells that touches a lower or right line. 18 19 20 21 Manual RBC’s Count. 22 23  Manual RBC’s Count. Get the total # of cells counted in all five medium squares. Variation between two R squares shouldn’t be more than 20 cells. 24  calculation RBC’s count : is the count of total # of erythrocytes in 1mm³ of undiluted blood. But we counted RBC’s in a volume less than 1mm³ of diluted blood. So, we need two correction factor. 25  Manual RBC’s Count. B- Calculation: Number of RBCs/mm³ = counted cells in 5 squares(80 small squares) X diluting factor X volume correction factor. The dilution factor = total volume / sample volume. DF = (101 – 1) / 0.5 = 200 Volume correction factor = Desired volume / counted volume in 5 squares. VCF = 1 mm³ / counted volume 26 Manual RBC’s Count. 27  Manual RBC’s Count. counted volume = The total volume of the 5 Medium squares = Volume x # of medium squares = (width x length x depth )x 5 = (1/5mm x 1/5mm x 1/10 mm) x 5 = 0.2 X 0.2 X 0.1 X 5 = 0.02 VCF = 1 mm³ / counted volume = 1 / 0.02 = 50 28  Manual RBC’s Count. Example: If total # of RBC’s in 5 medium squares is 423 Then the # of RBCs in 1mm³= 423 x 200 x 50 = 4,230,000 So, number of cells/mm³ = n x200 x 50 = n x 10000 29  Manual RBC’s Count. Significance of the test : The purpose of a RBC’s count is to determine the # of circulating RBC’s per mm³ of blood. RBC’s carry O2 to all tissues; thus a drastic reduction in the RBC’s count will cause immediate reduction in the available O2. 30  Manual RBC’s Count. Anemia: a decrease in number of RBC’s below normal range Polycythemia: Increase in number of RBC’s above normal range Physiological Polycythemia :(up to 8 million cells/mm³) due to: – Age: at birth RBC’s count is 8-10 million cells/mm³ – High altitude 31  Manual RBC’s Count. Pathological Polycythemia: due to Primary Polycythemia: RBC’s over 14 million cells/ mm³, occur in bone marrow malignancy. Secondary Polycythemia: RBC’s over 8 million cells/ mm³,due to: » Respiratory disease » Heart disease » Chronic carbon dioxide poisoning 32  Manual RBC’s Count. Sources of error 1. Flooding of chamber with excess sample. 2. Failing to count all the cells in the squares or conversely including artifacts in the count. 33  videos https://youtu.be/sDhsyh2wKxc https://youtu.be/0f9p9JX4qJk 34