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PREPARATION OF MYELOID AND LYMPHOID CELLS FOR ENGRAFTMENT STUDIES USING A RAPID IMMUNOROSETTING TECHNIQUE.
George D. Montoya, BA, Sue Bassinger, BS, Cecily Yee, CHT, Cynthia Moehlenkamp, BS, Thomas M. Williams, MD and David S. Viswanatha, MD. Albuquerque NM, USA, University of New Mexico and TriCore Reference Labs, 87131, Genetics and Cytometry.
Following hematopoietic stem cell transplantation, molecular studies of the relative amounts of donor and recipient blood cells via microsatellite genotyping is often useful. Analysis of specific myeloid and lymphoid cell subsets may provide more refined data in these assays. However, the preparation of cell subsets by flow cytometric sorting is cumbersome. We evaluated a new approach for preparing enriched populations of myeloid and lymphoid cell for engraftment testing employing negative immunoselection of cell subsets (Rosettesep, Stem Cell Technologies). Whole blood is incubated with a cocktail of monoclonal antibodies directed against myeloid, lymphoid and red cell surface markers followed by density centrifugation with recovery of enriched myeloid or lymphoid populations. We prepared artificial mixtures of donor and recipient whole blood normalized for cell counts as shown in Table 1. Rosettesep enrichment of these mixtures yielded preparations that were >98% pure for myeloid and T-lymphoid cells, respectively, as assessed by flow cytometry. Microsatellite based engraftment assays with 9 loci yielded estimates of donor/recipient cells in the myeloid and lymphoid subsets that correlated well with the artificial mixtures prepared. These studies suggest that Rosettesep may be a rapid and attractive method for preparing cell subsets for engraftment assays without the need for traditional flow cytometric-based cell sorting.
Percent Recipient Detected in Engraftment Assays of Rosettesep Cell Subsets D:R Mixture(1) 100 95 80 20 5 0 Myeloid Subset (1) >97 89 80 25 11 <3 Lymphoid Subset (1) >97 88 72 15 8 <3 (1) % Recipient