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

Abstract

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 histoincompatibility in such transplants, 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 most effective methods of preventing GVHD. Thus far, individual centers have used custom-developed procedures to deplete the graft of T cells that are responsible for alloreactivity, often employing relatively impure, nonstandardized 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 and validate the results between institutions, and this has limited direct comparison of data between centers. 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 OKT3 antibody. T-cell depletion was performed on PBPCs from six haploidentical donors. CD34+ cell recovery was 47 percent (range, 31-63%) with a median purity 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 challenging to accurately quantitate the remaining T cells, two highly sensitive flow 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. With the Isolex 300i and a MoAb directed against T cells, a high degree of T-cell depletion is obtained. Sensitive, accurate, and reproducible assays have now been developed for T-cell enumeration in these highly purified cell populations.