[A micro-method for PHA-induced stimulation of human lymphocytes. I. Communication: Technical considerations (author's transl)].

Abstract

A microculture system is described for PHA-induced stimulation of human peripheral lymphocytes. Following purification on Ficoll-Isopaque cells are incubated in microplates (Falcon 3040) for 66 hours in 5% CO2 using PHA-P (Difco) as a stimulant. DNA-synthesis is measured by labelling with 3H-thymidine (spec. act. 400 mCi/mmole) 16 hours prior to the end of the test. Using a multiple automatic sample harvester rapid evaluation is possible. Several variables were analyzed and optimal conditions defined for PHA- and cell-concentration, time dependence, thymidine dosage and buffer capacity of the medium. High variability and otherwise unexplained loss of lymphocyte stimulation in a population of "normal donors" are often due to acute viral infections, especially during the incubation period. On the other hand, source and concentration of serum are of equal importance since sera of healthy individuals differ greatly in their capacity to support PHA-induced stimulation of normal allogeneic lymphocytes. Using pooled or autologous serum we found the reproducibility of the technique to be good. Provided optimal conditions are employed the microtest system described herein should be ideal for in-vitro-analysis of inhibitory or stimulating factors in cancer patients' sera.