Isolation and flow cytometric analysis of T-cell-depleted CD34+PBPCs

BACKGROUND: To extend allogeneic HPC transplantation to a greater range of patients, the use of partially matched related donors is under development. Because of the inherently higher degree of histoincom-patibility in such t ransplants, there is increased risk of GVHD as well as of graft failure. Ex vivo depletion of donor-derived T-lymphocytes from PBPCs is one of the mos t effective methods of preventing GVHD. Thus far, individual centers have u sed custom-developed procedures to deplete the graft of T cells that are re sponsible for alloreactivity, often employing relatively impure, nonstandar dized reagents such as soybean agglutinin and complement. In addition, with improved methods of T-cell depletion, it has been difficult to accurately assess the number of T cells remaining. Because different centers have used different protocols to assay T cells, it has been difficult to reproduce a nd validate the results between institutions, and this has limited direct c omparison of data between centers. STUDY DESIGN AND METHODS: A standardized approach for T-cell depletion was developed by using a Good Manufacturing Practice-manufactured magnetic cell separator (Isolex 300i, Nexell Therapeutics) and commercially available OK T3 antibody. T-cell depletion was performed on PBPCs from six haploidentica l donors. RESULTS: CD34+ cell recovery was 47 percent (range, 31-63%) with a median p urity of 94 percent (range, 75-99%) and median T-cell log depletion of 4.72 (range, 3.90-5.83). Because this high degree of depletion makes it challen ging to accurately quantitate the remaining T cells, two highly sensitive f low cytometric protocols were developed, each of which accurately detects T cells with a sensitivity of 2 per 10,000 (0.02%). The purified CD34+ cells administered to the patients (dose range, 6.13-13.50 x 10(6)/ kg) provided rapid neutrophil and platelet engraftment. CONCLUSION: With the Isolex 300i and a MoAb directed against T cells, a hig h degree of T-cell depletion is obtained. Sensitive, accurate, and reproduc ible assays have now been developed for T-cell enumeration in these highly purified cell populations.