Erythrocyte Fragility Assay Protocol

Normal erythrocyte is suspended in isotonic plasma; if placed in a hypertonic solution, the erythrocyte will crumple due to water loss. Conversely, if placed in a hypotonic solution, water will enter the erythrocytes and cause them to swell. If the ambient osmotic pressure continues to fall, the erythrocytes will rupture as they continue to swell, releasing hemoglobin, which is called hemolysis. The erythrocyte membrane is resistant to hypotonic solutions, a characteristic known as the osmotic fragility of erythrocytes. The greater the resistance of the erythrocyte membrane to hypotonic solutions, the less likely that hemolysis will occur in hypotonic solutions, i.e., the less osmotic fragility of the erythrocyte.

• Ten small test tubes are taken and 10 different concentrations of sodium chloride hypotonic solution (0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.9%) are prepared.
• The method of blood collection varies from animal to animal, but peripheral blood is usually used. Put the blood drops on the surface dish with 1% heparin and mix well (0.1 ml of 1% heparin can resist 10 ml of blood).
• Use the dropper to suck the anticoagulated blood, add one drop in each test tube, shake gently, and leave it for 1-2 h.
• Judge the erythrocyte fragility according to the difference in color and turbidity of the liquid in each tube. Test tube without hemolysis, the lower layer of liquid has a large number of erythrocyte precipitate, and the upper layer is colorless and transparent, indicating that no erythrocyte rupture. Partial hemolysis of the erythrocyte in the test tube, the lower layer of the liquid for the erythrocyte precipitation, the upper layer of transparent light red (light reddish-brown) color, indicating that part of the erythrocyte has been ruptured, known as incomplete hemolysis. Test tube with total hemolysis of the erythrocyte, the liquid turns completely transparent red and there is no erythrocyte precipitate at the bottom of the tube, indicating that the erythrocyte is completely ruptured, which is called complete hemolysis.

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• When mixing, gently pour 1 or 2 times to reduce mechanical shock and avoid artificial hemolysis.
• The anticoagulant is preferably heparin; other anticoagulants can change the permeability of the solution.
• The preparation of different concentrations of NaCl solution should strive to be accurate and error-free. The concentration gradient of the NaCl solution can be adjusted appropriately according to the actual situation of the animals.

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