Rosette formation between immobilised human lymphocytes and erythrocytes sensitised with monoclonal anti-D

A rapid, reproducible and sensitive assay was developed to investigate the ability of human lymphocytes to form rosettes with erythrocytes sensitised with human monoclonal anti-D. Erythrocytes sensitised with a known number of anti-D molecules per cell were incubated with lymphocytes immobilised on plastic by poly(L-lysine), the resulting rosettes fixed, unbound erythrocytes removed by washing and the cell preparation stained. IgG1 and IgG3 anti-D-coated erythrocytes gave similar rosette formation at sensitisation levels in the range of 5000-15,000 molecules per cell, although at lower sensitisation levels IgG3 gave greater rosette formation than IgG1. A minimum of 500 IgG3 and 1000 IgG1 anti-D molecules per erythrocyte were required for rosetting.