Technical aspects of the primed lymphocyte typing (PLT) technique.

The influence of different culture conditions in the primary and secondary cultures of the primed lymphocyte typing (PLT) technique was investigated with special reference to the discriminatory capacity of the PLT-cells generated. In the primary cultures, the maximal yield of PLT-cells was observed early (about day 7) and decreased thereafter, while the maximal specificity was obtained considerably later (about day 14). In the secondary cultures, the optimal culture time was in the interval 42 h - 72 h, and up to this culture length, gamma-irradiation (2,200-8,800 rad) of the secondary stimulators had no effect on the 14C-thymidine uptake of the cultures. In U-form microtiterplates, the number of PLT-cells per well should not be less than 2.5 X 10(4), and higher PLT-cell numbers (e.g. 5.0 X 10(4) per well) may confer further robustness upon the technique. The PLT-cell response and the discrimination was only slightly influenced by the number of secondary stimulator cells in the interval 5 X 10(4) to 2 X 10(5) cells per well. Freezing of the PLT-cells under controlled conditions resulted in a minor loss of viable eosin-excluding cells, while the specificity of the PLT-cells was unaffected. Even when the culture conditions are standardized, it is necessary to perform a normalization of the data in order to obtain reproducible results. The normalization procedure should include a compensation for the variation in (i) the general responding capacity of each PLT-cell and in (ii) the general stimulatory capacity of each secondary stimulator.