The Development of Methods for High Purity Hematopoietic Lineage Cell Isolation and Lineage Chimerism Testing

Abstract

Chimerism analysis has become an important tool for assessing the origin of hematopoietic cells after allogeneic donor stem cell transplantations. Particularly, lineage chimerism analysis provides crucial information regarding treatment progress. Donor chimerism status for certain lineages, such as T, NK and myeloid, during early stages following post-transplant is associated with long term engraftment success and the relapse risk. However, the accurate lineage chimerism result is highly contingent on obtaining lineage cell preparation purity. There are several challenges in the field relating to the isolation of high purity cell lineages, chiefly among which is extremely low cell counts during the early stages post-transplant. Our laboratory has developed a method based on a) sequential isolation of hematopoietic lineages; b) purity assessment by flow cytometry; and c) a correction algorithm substandard purity of isolated cells. First, myeloid, B, T, and NK cells are sequentially isolated by magnetic separation using lineage specific markers. Thus each lineage isolated is largely excluded from the sample as a potential contaminant in each subsequent step. A purity with >90% can be routinely achieved when isolating lineages accounting for as little as 0.5 % of total leukocytes. Second, the purity of each population is verified by Flow Cytometry using a 6-color antibody panel staining for T, B, NK, myeloid, and total leukocytes. This enables not only the determination of cell purity of each isolated lineage cells, but also the identification and quantification of any contaminating cells in each isolated lineage. Lastly, an algorithm was derived based on the assumption that total chimerism consists of the four main lineages, T, B, NK, and myeloid. This mathematical correction is reflexively applied when isolated lineage cells are of <90% purity, often occurred as the isolated lineage accounting for < 0.5% of total leukocytes in the sample. In summary, our methods enable generation of high purity of isolated lineage cells that lead to accurate and reliable single lineage chimerism results.