[Separation of monocytes and their use in a cytotoxicity test]

Observations of acute rejections in uremic patients transplanted with Kidney grafts from Human Leucocyte Antigens (HLA) identical or compatible donors have suggested the importance of the role play by antigens not codified by the Major Histocompatibility Complex (MHC). Some works detected anti-monocytes antibodies that could not be absorbed with platelets (HLA-ABC) or with B lymphocytes (HLA-DR) or granulocytes but only with endothelial cells. Since endothelial cells and monocytes share not HLA surface determinants. Methods to isolate monocytes and endothelial cells have been attempted. We isolated monocytes by Percoll gradients. We checked the purity of our cell population by using monoclonal monocytes. From 30 ml of whole blood we obtained a mean of 50 X 10(6) (min. 12, 6 X 10(6) max 14 X 10(-6] mononuclear cells. After centrifugation of Percoll gradients we recovered a mean 7, 8 X 10(6) cells (Low 1, 3 X 10(6); max. 15 X 10(6] in the monocytes interface. By testing with monoclonal antibodies we found that a mean of 68, 95% (min. 58%; max. 82%) of these cells were living monocytes. We used the cell suspension to detect anti-monocyte antibodies in sera from 171 politransfused uremic patients with long incubation NIH cytotoxicity test.