A method for measuring lymphocyte proliferation in mixed lymphocyte cultures using a nuclear proliferation antigen, Ki-67, and flow cytometry

The mixed lymphocyte culture procedure using tritiated thymidine (3H-TdR) incorporation is time consuming and labor intensive, therefore costly. With the use of a fluorescent antibody to a human nuclear proliferation antigen, Ki-67, and flow cytometry, mixed lymphocyte cultures on 20 families of renal and bone marrow transplant patients and normal controls were performed. In this method for measuring lymphocytic proliferation, previously developed by the authors, the entire culture and staining procedures are performed in microculture plates. Finally, the cell suspensions are aspirated with a microsampler to be analyzed by a flow cytometer. Excellent correlation of the percentage of Ki-67-positive cells and the counts per minute (CPM) of 3H-TdR incorporated into the DNA was obtained. This method eliminates the use of radioactive labels, is less time consuming, and yields results two to three days earlier than the radioactive method. In addition, the authors dual-labeled the lymphocyte nuclei with Ki-67 and propidium iodide (Ki-67/PI). This permitted the comparison of the appearance of nuclear antigen with the various phases of the cell cycle.