Hematology-1: Hemoglobin, Erythrocyte Count, PCV

(HEMATOLOGY-1) (Hemoglobin, Erythrocyte Count, PCV) 1. MEASURING HEMOGLOBIN (Hb) LEVELS (Hemoglobinometry - Sahli Method) Principle Blood hemoglobin (Hb) with 0.1N HCl will change to hematin acid.Then rate visually compare the color with the standard color.sour save This be measured with TOOLS & REAGENTS 1. Sahli-Adams Hemometer, consisting of - Standard color - Hemometer tube with scale in g% and % of normal - Sahli pipette which has a volume of 20 cmm - Glass stirrer 2. Pipette 3. 0.1N HCl solution 4. Aquades WORK PROCEDURES 1) The hemometer tube is filled with 0.1N HCl solution to the 2 g% mark. 2) The blood sample is sucked with a Sahli pipette to the 20 cmm mark. 3) Clean the outer tip of the pipette with filter paper. 4) The blood is immediately blown carefully into the 0.1N Hcl solution in the hemometer tube without creating air bubbles. 5) The pipette is rinsed by blowing and sucking the 0.1N HCl in the hemometer tube several times. Also, the outside of the Sahli pipette is rinsed several times with a few drops of 0.1N HCl solution or distilled water. 6) Wait 10 minutes, giving the opportunity for hematin acid (95%) to form. 7) This hematin acid is then diluted with distilled water drop by drop while stirring until a standard color is obtained. 8) The meniscus of the solution is read and expressed in g% (g/dl). REFERENCE VALUE ♂Man : 13.5 – 18.0 g/dL ♀Woman : 12.0 – 16.0 g/dl 2. COUNTING THE NUMBER OF BLOOD CELLS (Erythrocytes, Leukocytes, Platelets) Principle The blood is diluted and colored with a certain solution, then the blood cells are counted in a counting chamber under a microscope. Tools & Reagents 1. Hemocytometer with Thoma diluent pipette (E scale: 0.5 – 101; L: 0.5 – 11; T: 0.5 – 101) 2. Microscope (Objective: E: 45X; L: 10X; T: 45X) 3. Reagents -Erythrocytes : Hayem solution with the following composition,HgCl2 ……….…………………………. 0.25 NaCl …………………………………… 0.50 NaSO4 ……………………………………. 2.50 Aquades ad …………………………. 100 ml -Leukocytes : Turk's solution with the following composition, Glacial acetic acid ………..…………….. 3 ml Gentian violet 1% (w/v) ……………….. 1 ml Aquades …………………………….. .. 100 ml -Platelets : Rees Ecker solution with the following composition, Sodium citrate …………………………. 38 g Brilliant crecyl blue ……………………. 1 g Aquades ………………………….... 1,000 ml Work Procedures 1) The Improved Neubauer counting chamber is prepared under a microscope and covered with a cover glass. 2) Suction the blood sample with a Thoma diluent pipette - 0.5 mark (E, L, T), then followed by the diluent solution, -Hayem's solution up to mark 101 - (E), -Turk's solution up to the 11 - (L) mark, -Rees Ecker's solution up to the 101 - (T) mark. 3) Shake the diluent pipette (in a figure 8 shape). 4) The first three drops are discarded, then the counting chamber is filled with the following drops as needed. 5) Allow a few minutes for the cells to settle. 6) Perform cell counting in the 4 square counting room in the boxes with codes, -ABCDE : - Erythrocytes -1,2,3,4 : - Leukocytes, : - Platelets 7) Erythrocyte count= 10,000 N/cmm Leukocyte Count= 50 N/cmm Platelet Count= 500 N/cmm 3. MEASURING HEMATOCRIT LEVELS (Hct) = Packed Cell Volume (PCV) Principle The erythrocytes are compressed using a centrifuge, then the compressed erythrocytes are read on a chart. Tools & Reagents 1. Heparinized microhematocrit tube 2. Microhematocrit centrifuge 3. Seal (night) 4. Charts Work Procedures 1) The microHct tube is filled 2/3 with blood sample. 2) One end (which is covered in blood) is sealed. 3) Place the microHct tube in the microHct centrifuge. (Note: sealed capillary pipette tip facing outwards) 4) Spin for 5 minutes, at a speed of 20,000 rpm. 5) The results are read on the chart. D. Reference Value ♂ Man : 40-50% ♀WOMAN : 38-47%