Differentiation of single populations in a bidirectional mixed lymphocyte culture using X and Y chromosome-specific FISH markers

Objective: To validate a new combination of a viability assay and Fluorescence in Situ Hybridsation (FiSH) techniques using X- or Y-DNA probes to assay the viability of each single population within bidirectional mixed lymphocyte cultures (MLCs). Study design: We determined the absolute viable lymphocyte counts of each population in bidirectional maternal-fetal MLCs as well as in bidirectional MLCs between male and female unrelated adults using a combination of a viability assay and additional sex discrimination by FiSH, and compared the results with the classical tritiated thymidine incorporation assay and with sex discrimination by metaphase number. Results: The results of the total viability assay correlated with those of the tritiated thymidine incorporation assay. The differentiation of each single population by FiSH showed, as in the metaphase assay, that the relationship between viable maternal and neonatal cells was shifted significantly (p < 0.05) towards the neonatal cells, while the relationship of the viable cells in the MLCs between unrelated adults was balanced. Conclusion: Our results show that X/Y discrimination by FiSH would be a useful tool for analyzing the immunologic network by bidirectional MLCs. The main advantage over sex discrimination by metaphase counting is that not only the proliferating cells but all viable cells are taken into account. An additional aspect would be the possibility to sort the cells according to surface markers (e.g. CD8+ HLA DR+) before discrimination by X and Y chromosomes. (C) 2002 Elsevier Science B.V. All rights reserved.