Clinical evaluation of B cell and T-regulator cell function using a protein A haemolytic plaque assay

Using the protein A haemolytic plaque assay (PrA-HPA) and standardized, optimal conditions, pokeweed mitogen (PWM) stimulated peripheral blood lymphocytes (PBL) from normal donors produced 5,500-48,000 (mean 16,955) IgG plaque-forming cells (PFC) per 10(6) lymphocytes, 3,375-38,000 (mean 14,179) IgA-PFC/10(6) and 5,500-69,750 (mean 18,684) IgM-PFC/10(6). Addition of 4 micrograms/ml concanavalin A (Con A) at the start of PWM stimulation of PBL from normal donors gave 59-98% (mean 84%) suppression of IgG-PFC, 46-99% (mean 79%) suppression of IgA-PFC and 31-100% (mean 82%) suppression of IgM-PFC. When PFC/10(6) values in PWM-stimulated co-cultures of unfractionated PBL from pairs of unrelated normal donors were compared with PFC/10(6) obtained in the individual PWM-stimulated cultures, observed/expected ratios were between 0.81 and 1.24 (mean 0.99) for IgG-PFC, 0.85 and 1.23 (mean 0.98) for IgA-PFC and 0.86 and 1.25 (mean 1.03) for IgM-PFC. Cord blood lymphocytes (CBL) produced few PWM-stimulated PFC and co-culture of CBL with normal PBL gave markedly reduced observed/expected ratios, indicating hyperactivity of T-suppressor cells in cord blood. PBL from a patient with partial DiGeorge syndrome also responded poorly in the PWM-driven PrA-HPA, but co-culture of this patient's PBL with normal PBL and PWM gave rise to higher than expected values, indicating reduced T-helper cell function. These simple methods could be employed in clinical evaluation of the cellular defect in humoral immunodeficiency and autoimmune disease states.