Osmotic Fragility of RBCs

13  Tonicity is a measure of the effective osmotic pressure gradient  A solution is isotonic when its effective osmole concentration is the same as that of another solution. In biology, the solutions on either side of a cell membrane are isotonic if the concentration of solutes outside the cell is equal to the concentration of solutes inside the cell. In this case the cell neither swells nor shrinks because there is no concentration gradient to induce the diffusion of large amounts of water across the cell membrane.  A hypertonic solution has a greater concentration of solutes than another solution. In biology, the tonicity of a solution usually refers to its solute concentration relative to that of another solution on the opposite side of a cell membrane; a solution outside of a cell is called hypertonic if it has a greater concentration of solutes than the cytosol inside the cell. 14 RBCs in hypertonic solution: shrink due to movement of water out of the cells. BCs in hypotonic solution: water diffuses into the cells : They swell up,: finally the cell membrane cannot withstand the increased intracellular pressure : The rupturing (disruption) of erythrocytes membrane (hemolysis) occurs: release of their contents (cytoplasm) into surrounding fluid (plasma). Procedure of Manual osmotic fragility test 1. 9 test tubes with the series of NaCl solutions (different concentrations) 2. heparinized venous blood 1. 2. prepare 9 test tubes tubes contain physiological solution diluted with water in the different ratio RBC are exposed to a series of saline (NaCl) solutions 1. 2. Put a few drops of citrated blood in each test tube put the samples in the centrifuge (10min)/ leave it 1h, and read the results visually. Osmotic fragility refers to the tendency (susceptibility) of erythrocytes to hemolysis under osmosis. The osmotic fragility test is measure RBC resistance to hemolysis when exposed to a series of dilute saline solutions. OF is characterized by the highest concentration of the NaCl solution in which the hemolysis is noted. In those tubes where hemolysis didn't occur, the RBCs sink to the bottom (the RBC remain in normal condition) and the fluid portion remain clear .. In the hypotonic solution (the tubes with hemolysis) , the solution will colored because the RBC swelling and finally rupture and hemoglobin release. 18  Tube 1 : 0.9% NaCl (physiological normal saline) (isotonic solution): 9 gr NaCl dissolved 1 liter of water: osmolarity is a close approximation to plasma (about 290 mOsm/L).  Tube 2: 0.8% / Tube3 : 0.7% NaCl / Tube4 : 0.6% NaCl  Tube 5: 0.5% NaCl : start of hemolysis  Tube 7: 0.3% NaCl complete hemolysis  Tube 9: 0.1% NaCl 19 Osmotic resistance of RBCs -No cells hemolyze in solutions of 0.6% saline and above(to 0.9%). -hemolysis onset at: 0.5 % NaCl - Hemolysis becomes complete at: 0.3 % NaCl (Concentration at which complete hemolysis of erythrocytes occurs)   Normally, the highest NaCl concentrations:0.5% The sooner hemolysis occurs, the greater is osmotic fragility of RBC 20 Increased osmotic fragility: Hereditary spherocytosis:  round cells (Spherocytes) : more fragile & break open in less hypotonic solutions than normal .  characterized by a defective RBC membrane Sickle Cell Decreased osmotic fragility Sickle cell anemia Iron deficiency anemia / Thalassemia Normal red cell Sickle cells inflexible