Osmotic fragility test

The osmotic fragility test measures the ability of red blood cells to retain their integrity in hypotonic solutions. A solution is hypotonic if it’s salt concentration is lower than that found in the blood, about 0.9% by weight (9 grams per litre).

The test uses around 15 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. 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 shining a particular frequency of green light (540nm) through the solution and seeing how much is absorbed. 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.

The results are typically presented as a graph showing the profile of bursting at different salt concentrations. The equivalent results from a control blood sample are also plotted for comparison. If the result is given as a single number, then the salt concentration that causes 50% of the red blood cells to burst is often used. This is known as the median corpuscular fragility (MCF). Alternatively, the percentage of burst red blood cells at a particular concentration is sometimes given.

Spherocytes (the spherical red blood cells that give spherocytosis its name) are less able to withstand hypotonic solutions and will burst, even in salinities that are relatively close to blood. This is partly because the red blood cell membranes are weaker, but also because the cells are already spherical and so can not increase their volume by changing shape, unlike the normal biconcave red blood cells.

Most testing laboratories will leave the blood in the salt solutions for 24 hours before measuring the amount of bursting. This improves the accuracy of the test. Not all testing laboratories do this.

There is no widely accepted set of normal results because different laboratories use different testing procedures and this can substantially affect the results. As an example, performing the test at different temperatures can have an effect. This is one reason why a control sample is tested at the same time.

Having said this, after 24 hours, normal red blood cells might burst at around 0.5% salinity whereas spherocytes might burst at around 0.7%.

The test is also used for thalassemia. For that condition, the red blood cells burst at salinity levels even lower than normal red blood cells, the opposite behaviour to the spherocytes.

The osmotic fragility test can only report the presence of spherocytes, it gives no information about why they are there. They could be caused by hereditary spherocytosis, but they could also be caused by other conditions including autoimmune spherocytosis, severe burns or poisoning.

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