Objective-The aim of the study was to determine whether there was evidence of elastase containing neutrophils at the materno-fetal interface in women with pregnancy-induced hypertension (PIH). Design-An observational prospective study. Setting-The Simpson Memorial Maternal Pavilion Edinburgh.Merhods-Placentas were obtained at vaginal or abdominal delivery from 5 1

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.

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Trophoblast biologists are often uncertain as to what cell types they are investigating because the mononuclear cell populations prepared from trypsinization of human first-trimester chorionic villi are morphologically very similar. In the present study, immunocytochemical and phagocytic markers have been used to distinguish cytotrophoblast populations from cell types derived from the mesenchyme of the chorionic villus. Two anti-trophoblast monoclonal antibodies generated in our own laboratory (18B/A5 and 18A/C4) were found to be very efficient in identifying cytotrophoblast, which made up 35–40% of the cells in a smear. Most cytotrophoblast cells did not stain with a monoclonal anti-HLA-A,B,C antibody but a few cells (5%) were found to express both trophoblast and HLA-A,B,C antigens by a double-labelling technique. Endothelial cells from villous capillaries could be identified by a rabbit anti-factor VIII antibody. These cells formed 28% of the population in a cytospin smear. Macrophages from the villous mesenchyme were less readily separable as neither specific monoclonal antibodies nor localization of enzymes were found to be effective. However, these cells could be identified by their ability to phagocytose carmine. About 15% of the cells in a smear consisted of macrophages. The procedure described should prove useful in judging the efficiency of isolation methods from human placental cells.