Hematology Lab - 08 Osmotic Fragility Test

HEMATOLOGY LABORATORY OSMOTIC FRAGILITY TEST 4. OSMOTIC FRAGILITY TEST (OFT) • • • • Measures the ability of RBC to resist osmotic stress Traditional and non-specific test for hereditary spherocytosis Sample: Heparinized Whole Blood (L.Green/Green Top) Principle: o Red blood cells are surrounded by a selectively permeable membrane that allows the exchange of gases and electrolytes. If the RBCs are placed in a hypotonic saline solution, osmotic equilibrium will be established by drawing water into the cells, which then swell. o Utilizes tonicity principle o Surface to volume ratio is inversely proportional to osmotic fragility 5. to the number labeled on each tube (When placing drop, ensure drops should be similar in size/volume) Using a similar pipette, add distilled water to each tube. The number of drops should bring the total volume of the solution in each tube to 25. Mix thoroughly o Validation of .02% increment compensate for irregularity in RBC drop 6. Add 1 drop of freshly drawn blood into each of the tubes. o Size of blood drop must be uniform 7. Cover each tube with parafilm and mix gently 8. Allow the tubes to stand for 30 minutes at room temperature o Tubes should not be disturbed during standing 9. Centrifuge tubes after incubation o Hemolysis is observed in supernatant as RBCs settle 10. Examine each tube for hemolysis (initial and complete) RESULT • • • • • Isotonic - amount of water entering the cell is equal to the water that leaves the cell; remains normal (biconcave) o Has balanced osmotic equilibrium o Eg. 0.85% NaCl Hypertonic - fluid outside the cell has higher concentration than inside of the cell o Releases water to maintain osmotic equilibrium o Dilutes extracellular fluid o RBC become crenated o Eg. >0.85% NaCl Hypotonic - intracellular concentration is higher than extracellular concentration o Absorbs water to maintain osmotic equilibrium o Causes swelling of cells which may lead to bursting o Eg. <85% NaCl Initial Hemolysis: (tube 22) o Faintly pink supernatant with red cell sediments Complete Hemolysis: (tube 18) o Fluid is red with no red cell sediments COMPUTATION • • Griffin-Sanford Method: o Tube number x 0.02 = % Reference Value: o Initial Hemolysis: 0.44 ± 0.02% (sp. 0.45% - Rodak’s) o Complete Hemolysis: 0.32 ± 0.02% (.35-.30 - Rodak’s) MATERIALS • • • • • • • 0.5% NaCl Wassermann tubes (12) Test tube rack Gum label (for labelling) Pasteur pipette (for transferring) Parafilm (for tube covering) Timer PROCEDURE 1. 2. 3. Prepare 0.5% NaCl Solution o 0.5 g NaCl in 100 mL distilled water Arrange 12 wasserman test tubes in a rack. Label them from 25 down to 14 (left to right) Deliver 0.5% NaCl in each tube drop by drop using a Pasteur pipette. The number of drops should correspond SPHEROCYTES • • • • • RBC lacking central pallor o Due to increased Hgb concentration o Normal RBC have central pallor 1/3 of its diameter Seen in hereditary spherocytosis o Not all lead to hereditary spherocytosis Decreased surface area-to-volume ratio Increased OFT At lower hypotonic strength, sample already hemolyzes CLINICAL SIGNIFICANCE • • • Result is affected by surface area-to-volume ratio of RBC o Inversely proportional o Increased Surface area-to-volume Ratio = Decreased OFT ▪ Normal RBC - w/ central pallor; allow deformation ▪ Sickle Cells - higher surface area-to-volume ratio that RBC; can accommodate more fluid ▪ Target Cells - higher surface area-to-volume ratio that RBC; can accommodate more fluid o Decreased Surface area-to-volume Ratio = Increased OFT ▪ Spherocytes Increased Osmotic Fragility o Hereditary Spherocytosis (non-specific) o Other conditions causing hemolysis in low hypotonicity ▪ Burns / severe burns ▪ Immune Hemolytic Anemia Decreased Osmotic Fragility o Thalassemia o Target Cells o Sickle Cell Anemia o Iron Deficiency Anemia (low Hgb content) Normal Hereditary Spherocytosis Thalassemia (characterized by numerous target cells) Sickle Cell Anemia • Initial Hemolysis (%NaCl) Complete Hemolysis (%NaCl) 0.45 0.35-0.30 0.65 0.45 0.35 0.20 0.35 0.20