Flow cytometric monitoring of hematopoietic reconstitution in myeloablatedpatients following allogeneic transplantation

Background We report a routine flow cytometric (FACS) approach to quantify circulating leukocytes (NC) in myeloablated patients before and during rege neration after allogeneic transplantation of either whole bone marrow (BM) or of highly purified (>99%) blood-derived CD34(+) cells (PBSC). Methods Blood samples were analyzed daily between infusion of the transplan t and hematopoietic reconstitution. Significant differences in the composit ion of NC types and CD34(+) cells were observed between the two CD34 source s. The detection threshold for NC was ronghly 1 cell per mu L blood. Results The cell nadir of <100 NC/mu L was reached on Day +4 (BM) and on da y 0 (PBSC), when unusual CD34(+) cells of recipient genotype were detected in a patients. They were not clonogenic, showed high CD34 expression, but w ere negative for CD45, CD38, CD33, CD50, HLA-DR and Stro-1. Between Days 5 and + 16, the onset of hematopoietic reconsititution was clearly detectab le in multi-parameter evaluation of the FAC data. This was a median of 3.5 days before NC increased above 200/mu L blood and 4-10 days before granuloc yte counts were >500/mu L. It was marked by the appearance of monocytes, im mature (CD38(+)) granulocytes, and clonogenic donor CD34(+) cells exhibited normal size and phenotype. Discussion We conclude that dynamic FACS analyses can reliably detect hemat opoietic reconstitution, but also graft rejection, before a visible increas e NC numbers. This may have considerable impact on clinical management stra tegies.