Flow cytometric monitoring of hematopoietic reconstitution in myeloablated patients following allogeneic transplantation.
BACKGROUND
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%) blood-derived CD34(+) cells (PBSC).


METHODS
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 w L blood.


RESULTS
The cell nadir of < 100 NC/ microL 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/ w L blood and 4-10 days before granulocyte counts were > 500/ microL. It was marked by the appearance of monocytes, immature (CD38(+)) granulocytes, and clonogenic donor CD34(+) cells exhibited normal size and phenotype.


CONCLUSIONS
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.
Fritsch
G
G
Children's Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria.
Witt
V
V
Dubovsky
J
J
Matthes
S
S
Peters
C
C
Buchinger
P
P
Printz
D
D
Handgretinger
R
R
Lion
T
T
Gadner
H
H
D000293:Adolescent; D000328:Adult; D018952:Antigens, CD34 / Q000096:biosynthesis; D016026:Bone Marrow Transplantation / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002648:Child; D002675:Child, Preschool; D005434:Flow Cytometry / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007223:Infant; D007962:Leukocytes / Q000166:cytology; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D014184:Transplantation, Homologous / Q000379:methods*
D018952:Antigens, CD34