Flow cytometric monitoring of hematopoietic reconstitution in myeloablated patients following allogeneic transplantation

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Abstract
We report a routine flow cytometric (FACS) approach to quantify circulating leukocytes (NC) in myeloablated patients before and during regeneration after allogeneic transplantation of either whole bone marrow (BM) or of highly purified (> 99%) bloodderived CD34+ cells (PBSC). Blood samples were analyzed daily between infusion of the transplant and hematopoietic reconstitution. Significant differences in the composition of NC types and CD34+ cells were observed between the two CD34 sources. The detection threshold for NC was roughly 1 cell per μL blood. The cell nadir of < 100 NC/μL was reached on Day +4 (BM) and on day 0 (PBSC), when unusual CD34+ cells of recipient genotype were detected in all patients. They were not clonogenic, showed high CD34 expression, but were negative for CD45, CD38, CD33, CD50, HLA-DR and Stro-1. Between Days +5 and +16, the onset of hematopoietic reconstitution was clearly detectable in multi-parameter evaluation of the FACS data. This was a median of 3.5 days before NC increased above 200/μL blood and 4–10 days before granulocyte counts were > 500/μL. It was marked by the appearance of monocytes, immature (CD38+) granulocytes, and clonogenic donor CD34+ cells exhibited normal size and phenotype. We conclude that dynamic FACS analyses can reliably detect hematopoietic reconstitution, but also graft rejection, before a visible increase NC numbers. This may have considerable impact on clinical management strategies.