Hematology I - Experiment 5: Hematocrit Determination PDF

HEMATOLOGY I LABORATORY MANUAL Name: Date: Section: Score: Group: EXPERIMENT 5 HEMATOCRIT DETERMINATION Hematocrit is the volume of packed red blood cells (PRBCs) after the centrifugation of a blood sample It is otherwise known as packed cell volume (PCV) or erythrocyte volume fraction (EVF)...

HEMATOLOGY I LABORATORY MANUAL Name: Date: Section: Score: Group: EXPERIMENT 5 HEMATOCRIT DETERMINATION Hematocrit is the volume of packed red blood cells (PRBCs) after the centrifugation of a blood sample It is otherwise known as packed cell volume (PCV) or erythrocyte volume fraction (EVF) and is reported in percent (%), cell volume percent (CV%), or volume percent (vol%). Hematocrit determination is one of valuable tests in the simplest, most accurate, and hematological investigation. It is more useful than RBC count in detecting cases of anemia. Red blood cell indices can be computed manually from the values of the RBC count, hemoglobin level, and hematocrit level. Objectives: 1. Measure packed cell volume using different methods 2. Correlate hematocrit levels with diseases 3. Describe the causes of increased and decreased values of hematocrit MATERIALS: EDTA blood Capillary tube (blue or red) Sealing clay Microhematocrit centrifuge Microhematocrit reader Disposable sterile blood lancet ADAM'S MICROHEMATOCRIT METHOD 1. Fill around 3/4 of the capillary tube with blood. 2. If the blood is from a skin puncture, use heparinized (red) capillary tube. A non-heparinized (blue) capillary tube is used if blood is collected with a anticoagulated tube 3. Seal one end of the capillary tube with sealing clay (about 3 mm). 4. Centrifuge the blood at 10,000 rpm for 4-5 minutes using a microhematocrit centrifuge. 5. Determine the level of packed red blood cells using a microhematocrit reader. 6. Estimation of hemoglobin and RBC is possible on the basis of the hematocrit value under normal circumstances. 1% hematocrit = 0.34 gm% hemoglobin = 107,000 RBC/mm3 Normal Value: Conversion factor - 0.01 CU (%, CV%, or Vol%) SI (L/L) Male 40-54 0.40-0.54 Female 35-49 0.35-0.49 1 HEMATOLOGY I LABORATORY MANUAL OTHER METHODS OF HEMATOCRIT DETERMINATION 1. Macro Methods A. Wintrobe Method This method utilizes a Wintrobe tube. The tube has two calibrations, 0 to 10, which is used for erythrocyte sedimentation rate (ESR), and 10 to 0, which is used for hematocrit (Fig. 5.1). The anticoagulant of choice for this determination is double oxalate. Procedure: 1. Fill the Wintrobe tube with blood using a Pasteur pipette. 2. Centrifuge the tube at 3,000 rpm for 30 minutes. 3. Read the volume of packed red blood cells. 4. Compute for the hematocrit level using the following formula. Computation: Hematocrit (%) = height of PRCBs X 100 height of whole blood used B. Haden's Modi cation The anticoagulant of choice is 1.1% sodium oxalate in distilled water. The method uses a calibrated tube. Procedure: 1. Place mL of 1.1% sodium oxalate into the tube. 2. Add 5 mL of blood. Mix well. 3. Centrifuge the mixture for 20 minutes at 3,000 rpm. 4. Read the volume of packed red blood cells. Computation Hematocrit (%) = volume of PRCBs × 20 NOTE: If less than 5 mL of blood is used, use the following formula: Hematocrit (%) = volume of PRCBs X 100 volume of whole blood used C. Van Allen Method The anticoagulant of choice is 1.6% sodium oxalate in distilled water. The method uses a tube with bulb and calibration of 1 to 10 cm or 10 to 100 mm. Procedure: 1. Fill the tube with blood up to the 10th mark. 2. Dilute the blood with the diluting uid up to the bulb, about half full. fl fi 2 HEMATOLOGY I LABORATORY MANUAL 3. Seal the tube and centrifuge (shaft end down) at 2,500 rpm for 15-30 minutes. Read the volume of PRCBs. 4. Each unit if division is equal to 1%. D. Sanford-Magath The anticoagulant of choice is 1.3% sodium oxalate; the tube is calibrated at 1mm per division. The tube is about 5 inches long and with a funnel-like mouth. Procedure: 1. Place 1 mL of anticoagulant into the tube. 2. Add 5 mL of venous blood and mix. 3. Centrifuge the mixture at high speed for 15 minutes. 4. Read the volume of PRCBs. Normal Value: Male: 2.3 mL = 46-48 vol% Female: 2.0 mL = 40-42 vol% E. Bray’s The anticoagulant of choice is heparin, and a Bray's tube is used. This tube is calibrated on both sides, similar to the Wintrobe tube. The calibration is from 10-50 mm, each division is 1 mm; and the capacity is 5 mL. Procedure: 1. Fill the tube with heparinized blood. 2. Let the tube stand at a vertical position for one hour. 3. Read the volume of RBCs from the lower right side calibration. Normal Value: Male: 2.3 mL = 47 vol% Female: 42 vol% 2. Automated Method 1. Coulter counter 2. Autoanalyzer 3 HEMATOLOGY I LABORATORY MANUAL Name: Date: Section: Group: Score: REVIEW QUESTIONS 1. Give two examples of diseases where there is: (a) decreased hematocrit; (b) increased hematocrit. 2. Give the possible variations in color of the plasma when performing hematocrit determination and state the corresponding signi cance of each. 3. Give three conditions that cause oligocythemia. 4. Why should ve to six drops of diluted blood be discarded prior to charging? fi fi 4