POSITIVE SELECTION OF CD34(-MARROW FOR INDIRECT PURGING OF NON-HODGKINS-LYMPHOMA CELLS() CELLS FROM HUMAN BONE)

Positive selection of CD34(+) cells has been proposed as an alternativ e to ex-vivo purging of tumour cells in autologous bone marrow transpl antation. In this study, we evaluate the ability of CD34(+) cell selec tion techniques to indirectly purge Non-Hodgkin's lymphoma cells from bone marrow. A batch-wise pre-enrichment step was used, followed by fl uorescence activated cell sorting (FAGS). CellPro-CEPRATE(c) immunoads orption columns (Bothwell, WA) were compared to high gradient magnetic separation (HGMS) as a pre-enrichment step. Cell suspensions were ass ayed at each separation step for hemopoietic colony-forming cells, lym phoma colony-forming cells, CD34(+) cells, CD19(+) cells and the prese nce of bcl-2 rearrangement by PCR. Separations were performed on bone marrow samples from lymphoma patients as well as on suspensions of nor mal marrow spiked with limited numbers of DHL-4 B-cell lymphoma cells. With the DHL-4 lymphoma model system, the CellPro columns produced a pre-enriched fraction that was purer (77 +/- 4% CD34(+)) than that pro duced by HGMS separation (61 +/- 2% CD34(+)) but fewer hemopoietic col ony forming cells CFC were recovered (43 +/- 9% versus 93 +/- 11%). Th e sorting step produced a cell suspension which was 97-99% CD34(+) wit h recovery of 74 +/- 13% of the CFCs. The overall. depletion of lympho ma cells was 4.8 log for the HGMS/sort method and 5.1 log for the Cell Pro/sort method. Separations of patient marrow samples achieved an ave rage of 4.1 log depletion of CD34 negative cells with recovery of CFCs ranging from 24% (CellPro/sorting) to 39% (HGMS/sorting). The level o f lymphoma cell depletion obtained in these small scale experiments ma y be useful for autologous bone marrow transplantation. However, the t ime required to sort (FACS) currently acceptable numbers of cells for clinical use even after batchwise pre-enrichment of CD34(+) cells woul d be approximately 10 hrs.