Cell studies: Spherocytosis and osmotic fragility

Hereditary spherocytosis is a genetic disease characterized by the production of red blood cells with an abnormally spherical shape. The disease is inherited in an autosomal dominant fashion and is most prevalent in individuals of Northern European ancestry (frequency estimated at 1 in 2000 individuals in the population). Spherical erythrocytes are broken down in the systemic circulation (hemolysis), and thus patients with spherocytosis often show signs of jaundice (yellowing of skin and sclera) and anemia, which leads to shortness of breath with exertion.

Given that spherical erythrocytes are more susceptible to osmotic stress, the osmotic fragility test can be used to identify patients who have spherocytosis. When normal red blood cells are placed in a dilute saline solution, the biconcave disk stretches and assumes a spherical shape. With increasing osmotic stress, hemoglobin begins to appear in the surrounding solution. As Figure 1 illustrates, the erythrocyte membrane remains intact when placed in an isotonic 0.9% saline solution. With increasingly dilute saline solutions, hemoglobin starts to be detected in the surrounding solution. Eventually, erythrocytes burst from osmotic stress and all hemoglobin exits the cell.

Figure 1: Progressive cell membrane disruption seen in the erythrocyte osmotic fragility test

The amount of hemoglobin that has exited the cell and entered the solution can be measured with spectroscopy; in this method, the percent of hemolysis is calculated by dividing the measured absorbance by the absorbance at 100% hemolysis. Sample hemolysis curves are shown in Figure 2.

Figure 2: Hemolysis curves for the osmotic fragility experiment

Visual analysis of experimental test tubes can also be used to compare erythrocyte samples.

Figure 3: Graphical representation of test tube coloration from the osmotic fragility experiment