A rapid micro-method for the phytohaemagglutinin-induced human lymphocyte transformation test

Abstract

A quantitative in vitro method for phytohaemagglutinin (PHA) induced lymphocyte transformation is described. This method has the following advantages.

Cultures require as few as 10⁵ lymphocytes and are performed in microtrays, over a short incubation period (48 hr).

A short pulse time of 4 hr with [³H]thymidine is utilized. This enables high levels of [³H]thymidine to be maintained throughout the labelling period, ensures maximum incorporation of thymidine into cellular DNA, and diminishes cellular damage by internal irradiation.

The extraction process has been simplified by using a communal washing procedure after drying the cultures on glass-fibre filter discs. The procedure is both quicker and more reproducible (coefficient of variation 6%) than extraction and drying on filtration manifolds (coefficient of variation 23%).

The effect of adjusting variables such as the number of cells, incubation time, concentration of [³H]thymidine, concentration of PHA, specific activity of [³H]-thymidine and duration of [³H]thymidine pulse has been studied in order to approach optimal labelling conditions for the assay system.

The reproducibility of the method has been investigated by repeated testing of normal individuals on a day-to-day basis and over extended periods. The mean coefficient of variation for samples repeated daily and over longer time intervals (weeks or months between samples) was 15%.