OD sample
OD of sample with 100% lysis X 100%
- OD is recorded and percentage of NaCl is converted to mole/L and Osmols/L.
- A graph percentage of haemolysis vs. concentration of NaCl is plotted using graph paper. (this graph is known as osmotic fragility curve)
RESULT:
*RMM of NaCl is 58.5g/mole
Since percent weight per volume is grams chemical per 100 mililiter solution, this calculation gives the grams of chemical in each liter of solution because there are 1000 ml ina liter. Therefore molarity of NaCl is equal to percentage of NaCl solution times 10 divide by RMM of NaCl solution.
*Graph should be Concentration of NaCl Vs Percentage of haemolysis
Figure: Osmotic fragility curve (concentration of NaCl VS percentage of haemolysis)
DISCUSSION:
The osmotic fragility test measures the ability of red blood cells to retain their integrity in hypotonic solutions. A solution is hypotonic if its salt concentration is lower than that found in the blood, about 0.9% by weight. The test uses around 10 different hypotonic solutions varying in salinity from 0.1% to 0.9%. Each solution has a small amount of blood added to it. In a hypotonic solution, a red blood cell takes in water, lowering the salinity in the cell until it matches the salinity of the solution. This happens through the process of osmosis. Osmosis is the movement of solvent across a membrane from a lower concentration to a higher concentration.
Figure: osmosis occurs on red blood cell.
In this case, the solvent is water, the membrane is the membrane of the red blood cell and the concentration of salt in the solution is lower than the concentration in the cell. The water can move across the membrane, but the salt cannot. The water moves to bring the solution and the cell into equilibrium. When water enters the red blood cell, the volume of the cell increases until the cell bursts. In weaker salt solutions, more water will enter the red blood cells and so more cells will burst. The percentage that burst is measured by spectrophotometer (540nm). Hemoglobin (the oxygen carrying molecule inside the red blood cell) absorbs this frequency, so the more red blood cells that have burst, releasing their hemoglobin; the more the light will be absorbed.
This test is performed to detect thalassemia and hereditary spherocytosis. Hereditary spherocytosis is a common disorder in which red blood cells are defective because of their round, ball-like (spherical) shape. These cells are more fragile than normal. Spherical cells are said to have increased osmotic fragility because they are less likely to expand and break open in salt water than normal red blood cells (which are indented or curved inward on both sides). Cells that are flatter than normal are more likely to expand, and thus have decreased osmotic fragility.
Figure: Spherocytes (black arrow)
Thalassemia is an inherited condition that affects the portion of blood (hemoglobin) that carries oxygen. Some red blood cells are more fragile than normal, but a larger number are less fragile than normal. When hereditary spherocytosis are suspected on the basis of an elevated mean corpuscular hemoglobin concentration or on examination of a peripheral blood smear, the osmotic fragility test may be used to confirm the presence of spherocytes. The test does not distinguish between spherocytes in hereditary spherocytosis and in acquired autoimmune hemolytic anemia.
The test only indicates that a proportion of the red cells have decreased surface-to-volume ratios and are more susceptible to lysis in hypo-osmotic solutions. Hereditary spherocytosis patients who are experiencing significant elevations in reticulocytes may not fall outside of the normal range. Cells with increased surface-to-volume ratios, such as occur in thalassemia and iron deficiency, may show decreased osmotic fragility. For patients with acute hemolysis, a normal red cell osmotic fragility test result cannot exclude an osmotic fragility abnormality since the osmotically labile cells may be hemolyzed and not present. Recommend testing during a state of prolonged homeostasis with stable hematocrit.
Figure: osmotic fragility test.
Osmotic fragility of red cells increased in:
- Hereditary spherocytosis.
- Acquired spherocytosis.
Osmotic fragility decreased in:
- Thalassemia.
- Iron deficiency anemia.
- Sickle cell anaemia.
QUESTION:
- A test tube with blood in it has a particular solution added to it. After several minutes, the solution is not clear anymore, but becomes red. Which solution was added to the blood to obtain this result?
- 0.9% saline
- 5% saline
- Distilled water
- A 0.8% saline solution would be __________ to the cytosol of a cell.
- Hypotonic
- Hypertonic
- Isotonic
- If you placed a peeled apple or potato in 5% salt solution, it would:
- Gain weight
- Lose weight
- Stay the same weight
CONCLUSION:
Osmotic fragility test is a test that measures the resistance to haemolysis of red blood cells (RBC) exposed to hypotonic solutions. RBC is exposed to a series of saline (NaCl) solutions with increasing dilution. The sooner haemolysis occurs; the greater is osmotic fragility of RBC. RBC remains same because it same as RBC concentration, Isotonic 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 haemoglobin to exit from the cells. By measuring haemoglobin concentration, the percentage of haemolysis at different NaCl concentrations can be calculated.