2 OSMOTIC FRAGILITY TEST DEFINITION- it is a test that measures the resistance to hemolysis of red blood cells (RBC) exposed to hypotonic solutionsRBC are exposed to a series of saline (NaCl) solutions with increasing dilutionThe sooner hemolysis occurs, the greater is osmotic fragility of RBC
3 Isotonic (physiological) solution – 0.9 % NaCl RBC burst in hypotonic (< 0.9 % NaCl), and shrink (crenate) in hypertonic solutions (> 0.9 % NaCl)In hypotonic medium a membrane rupture occurs, allowing hemoglobin (Hb) to exit from the cellsBy measuring Hb concentration, the % of hemolysis at different NaCl concentrations can be calculated
5 INCREASED OSMOTIC FRAGILITY Hereditary spherocytosisAcquired spherocytosisHereditary spherocytosis is a disorder characterized by a defective RBC membrane and decreased surface-to-volume ratioCharacteristic round cells (spherocytes) are seen in blood smear and they are more fragile and break open in less hypotonic solutions than normal red blood cells
6 In hypotonic solutions water enters red blood cells Therefore, normal RBC with a biconcave shape swell and expand their volumeOn the other hand, spherocytes cannot absorb much extracellular liquid and break very easily
9 PROCEDUREWe use human erythrocytes that were washed with physiologic solution and thus do not contain plasmaLabel 5 eppendorf microtubes 1–5 and pipette 1 ml of the following solutions into them:tube 1 – physiological solution (non-diluted)tube 2 – physiological solution diluted with water in the ratio 3:1tube 3 – physiological solution diluted with water in the ratio 2:1tube 4 – physiological solution diluted with water in the ratio 1:1tube 5 – physiological solution diluted with water in the ratio 1:5, containing NH4Cl (NH4Cl disables remaining membrane pumps)
10 Pipette 50 μl erythrocyte suspension into each microtube, gently mix and let stand for 10 minutes. Then centrifuge 3 minutes at 3 000 × g and carefully collect the supernatants for hemoglobin assayPrepare five glass test tubes 1–5 with 2 ml Drabkin reagent. Add 50 μl of each supernatant from the previous step into the corresponding glass test tube and mix gently.Measure the absorbances of all the samples at 400 nm against blank containing the Drabkin reagent only.
11 Due to the fact that the blood sample is not fresh, we observe partial hemolysis even in test tube 1. The amount of hemoglobin determined in this test tube represents the control value and should be subtracted from the values obtained for all the tubes. The value of hemoglobin measured in test tube 5 is the maximum obtainable amount and we express it as 100% hemolysis. On this basis, calculate the percentage of hemolysis in test tubes 2–4.Using a calibration graph, express your results as the hemoglobin concentration in mg/l.