Flow cytometry for CD34 determination in hematopoietic grafts

CD34 is a type I transmembrane protein that is expressed on lympho-hematopoietic progenitor and endothelial cells and has a potential adhesion function. Various monoclonal antibodies, whether characterized by glycosylated epitopes or not, are utilized to recognize different subsets of hematopoietic progenitors. Coexpression of membrane markers involving CD34 is an approach to the definition of those subpopulations of cells previously characterized by using in vitro cultures. Thus, in the autologous transplantation procedure, flow cytometry determination of CD34+ cells in the grafts themselves, especially as concerns cytapheresis products, was enhanced with CFU-GM enumeration. This methodology required a standardized protocol with regard to the choice of the monoclonal antibody, had to be to data acquisition and to computer analysis. Within the framework of a multicentric trial, various strategies had to be evaluated. Special attention was paid to obtaining a sensitivity level of 0.1 %. New, standardized approaches are currently in the planning stage, in particular with a view to determining absolute count on the basis of readings generated by the flow-cytometer.