Using elutriation to engineer bone marrow allografts.

We have used elutriation to deplete lymphocytes from the marrow allografts of 64 patients to date. The first phase I trial (1 x 10(6) lymphocytes/kg IBW) was designed to test the procedure for potential toxicities, most notably, graft failure. Study 2 (1 x 10(6) lymphocytes/kg, no CsA) was expected to reduce potential toxicity incurred from long term immunoprophylaxis while study 3 was aimed at reducing the incidence of GVHD by further reducing lymphocyte dose (5 x 10(5)/kg). Graft lymphocyte dose was based on morphologic determination and was subsequently confirmed by limiting dilution analysis and flow cytometry. Although grafts were standardized solely by lymphocyte dose, the product was more uniform than the original harvested BM with respect to other cell populations. Nearly all study I (n = 40) and study III (n = 20) patients engrafted with a median time to ANC greater than 500/ul of 19 days. Three of the 4 patients consecutively enrolled in study II failed to engraft, thus terminating the trial. While a moderate proportion of study I patients had AGVHD (44%) with attendant morbidity, only 20% of study III patients were found to have mild AGVHD (less than or equal to stage I). To date, this cohort has no organ or chronic GVHD and no GVHD-associated morbidity. Median follow-up times for patients in studies I and III are 27 and 11 months, respectively. Overall actuarial survival (n = 60) is 42% at 38 months (38% study I, 80% study III). Good prognosis study I patients experienced 45% actuarial survival versus 9% in the poor prognostic group. While lymphocyte depletion has been effective in reducing the incidence and severity of AGVHD, new strategies are needed to address the issue of disease relapse. As with other methods, lymphocyte depletion by elutriation caused an increased rate of leukemia relapse. The actuarial probability of remaining in remission for recipients of elutriated marrow containing 1 x 10(6) and 5 x 10(5) lymphocytes/kg, respectively, were 60% and 46% at 16 months. Elutriation provides a rapid, reproducible and flexible methodology for graft manipulation which has been effective in reducing the incidence and severity of AGVHD. However, if lymphocyte depletion is to fulfill its promise as a means of reducing the overall morbidity of allogeneic BMT, new strategies may be needed to address the issue of relapse. These may include changes in marrow ablative therapy and post-graft immunosuppression. Equally as important may be the ability to further manipulate accessory cells and lymphoid populations presently excluded from the graft.