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Single and double biotin-avidin-peroxidase immunocytochemical methods in conjunction with an anti-trophoblast monoclonal antibody 18B/A5 and an anti-HLA-A,B,C monoclonal antibody W6/32 were used to study various human trophoblast populations. Several combinations of peroxidase substrates were tried in the double-labeling procedure. It was concluded that the use of 4-chloro-1 naphthol to develop the primary sequence peroxidase and of 3-amino-9-ethyl carbazole for the second sequence peroxidase was the most suitable. The significant findings were: Monoclonal antibody 18B/A5 proved to be a useful marker for villous as well as nonvillous trophoblast, which facilitated the identification of these cells particularly in the placental bed. The expression of MHC Class I antigens was not confined to extravillous trophoblast but these antigens were also demonstrable on the villous cytotrophoblast proliferating to form new primary villi. Double labeling revealed that many of these cells, particularly those furthest away from the mesenchymal core, expressed both trophoblast and HLA antigens as shown by a mixing of the colors produced by the two reaction products. A large number of these HLA-A,B,C, positive trophoblast cells were found to infiltrate deep into the uterine myometrium. The hypothesis was put forward that these fetal cells could be the ones that are responsible for maternal sensitization.

Human cytotrophoblast populations studied by monoclonal antibodies using single and double biotin-avidin-peroxidase immunocytochemistry.