Aerolysin assays for detection of PNH. (A) Dose-response curve showing aerolysin sensitivity of two normal (solid lines, open symbols) versus two PNH (dashed lines, solid symbols) erythrocytes. (▪) Cells that are primarily type II erythrocytes; (⧫) type III cells. Serial dilutions of activated aerolysin (1.5 × 10−6 stock) were performed in a 96-well plate and mixed with an equal volume of 0.8% erythrocytes. Absorbance at 620 nm was measured after 10 minutes at 37°C using a plate reader. (B) Kinetic analysis of aerolysin-induced hemolysis of normal erythrocytes (bottom line) and erythrocytes from 5 different PNH patients. The rate of hemolysis was determined by measuring the change in optical density of erythrocytes at 600 nm and 37°C as a measure of time using a spectrophotometer. The percentage of CD59− erythrocytes determined by flow cytometry for each patient is shown to the right of the figure. (C) Flow cytometric analysis for CD59 expression of PNH red blood cells from the patient with 8% type III PNH erythrocytes (solid line) and a normal control (dotted line). (D) Flow cytometric analysis for CD59 expression of PNH red blood cells from the patient with 80% PNH erythrocytes (solid line) and a normal control (dotted line). The majority of cells represent type II PNH erythrocytes.
