The method of whole blood microcultures for in vitro lymphoblastogenesis by mitogens has several advantages over conventional lymphocyte techniques. It offers considerable savings in time, cost and volume of blood, and allows for more frequent sampling of given patients.
We established the optimal conditions in this reaction method using PHA-P or Con A, as follows:
Heparinized 0.05ml of peripheral blood was diluted to 1.0ml by adding RPMI 1640 media. The maximum response which was expressed by the rate of incorporation of 3H-thymidine into PHA-P or Con A-stimulated lymphocytes was obtained by culture of lymphocyte sample with 1.0ug/ml of PHA-P for 3 days and 25ug/ml of Con A for 4 days, respectively. The optimal labelling time for the incorporation of 3H-thymidine was 4 hours.
Using these methods, cellular immunity was investigated in 25 pre-treated patients with head and neck cancer. No significant relationships between the response seen with this method and the numbers of lymphocytes employed were found. Generally, these patients showed a significantly lower response than was seen in the control groups (p<0.05), thus providing evidence for the usefullness of an estimation of cellular immunity in these patients.