Spherical erythrocytes and hemolysis

Hereditary spherocytosis is a genetic disease characterized by the production of spherical erythrocytes instead of normal biconcave disk-shaped red blood cells. 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 (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 into 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 into an isotonic 0.9% saline solution (Case A). As erythrocytes are placed into increasingly dilute saline solutions (Case B and C), hemoglobin starts to be detected outside of the erythrocyte. Eventually, the erythrocyte bursts from the osmotic stress and all hemoglobin are now found outside of the cell and in the solution.

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 subsequently be measured with spectroscopy. Percent of hemolysis can be calculated by dividing the measured absorbance by the absorbance at 100% hemolysis (when all of the hemoglobin are outside of the burst erythrocytes). Several sample hemolysis curves are shown in Figure 2 - either Curve Y or Curve Z correctly depicts the behavior of erythrocytes taken from a patient with hereditary spherocytosis in the osmotic fragility experiment. Visual representation of coloration from experimental test tubes containing varying solutions and erythrocyte samples are shown in Figure 3.

Figure 2: Sample hemolysis curves for the osmotic fragility experiment

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