Osmotic Fragility of Erythrocytes

The osmotic fragility test (OFT) is used to measure erythrocyte resistance to hemolysis while being exposed to varying levels of dilution of a saline solution.

There is no true consensus among laboratories in terms of a reference range of values for the osmotic fragility of erythrocytes.

Incubation of erythrocytes at 37°C for 24 hours increases the test’s sensitivity.

Normal findings ^[1] :

• Hemolysis begins at 0.5% NaCl
• Hemolysis complete at 0.3% NaCl

Conditions associated with increased osmotic fragility include the following:

• Hereditary spherocytosis

• Autoimmune spherocytosis

• Poisoning

• Severe burns

The following conditions are associated with decreased fragility:

• Thalassemias

• Iron deficiency anemia

• Sickle cell anemia

Specimen: Whole blood (6 mL [2 mL minimum])

Container: Lavender top (K2 EDTA) tube

Method: Routine venipuncture

Handling instructions: Specimen must be fresh. Deliver to laboratory after collection and processing within 72 hours, preferably within 24 hours.

Description

The osmotic fragility test (OFT) is used to measure erythrocyte resistance to hemolysis while being exposed to varying levels of dilution of a saline solution. When erythrocytes are exposed to a hypotonic environment, water enters the cell and causes swelling and eventual lysis. The susceptibility of osmotic lysis of erythrocytes is a function of surface area to volume ratio.

The classic osmotic fragility test, originally described by Parpart et al (1947), ^[2] involves a small amount of fresh blood being added to a series of solutions with tonicity ranging from 0.1%-0.9%. These samples undergo centrifugation with absorbance reading at 540 nm to calculate the percentage of hemolysis for each solution. Results are then plotted against the NaCl concentrations, yielding an osmotic fragility curve that is then compared to obtained normal control values.

At times, the result of the osmotic fragility test may be expressed as the concentration of NaCl in solution that causes 50% hemolysis of erythrocytes. This is known as the median corpuscular fragility (MCF). For a normal sample of fresh blood, this is between 4 and 4.45 g/L NaCl.

The osmotic fragility test can be performed on freshly drawn blood (within 2 hours from collection), but some laboratories incubate collected samples at 37°C for 24 hours to improve the sensitivity of the test, since a greater extent of osmotic lysis is noted for abnormal erythrocytes than normal ones.

Indications/Applications

The osmotic fragility test is used to gauge the level of hemolysis in a collected sample of a patient’s blood, and this is compared to a control sample. ^{[3, 4]}

In a disease such as hereditary spherocytosis, erythrocytes have a smaller ratio of surface area to volume and are thus more susceptible to osmotic stress, as opposed to the increased resistance characteristic of thalassemia, iron deficiency anemia, or any other condition that would cause an increased surface area–to–volume ratio.

The osmotic fragility test is mainly used in helping with the diagnosis of hereditary spherocytosis but it is also used in some countries as a method to screen for beta thalassemia, especially where laboratory resources are limited.