Progenitor cell numbers (CFU-GM, CFU-D, and CFU-Mix) and hemopoietic recovery following autologous marrow transplantation.

Forty-one consecutive patients were treated with high-dose chemotherapy with or without total body irradiation followed by autologous marrow transplantation. Four treatment regimens of varying intensity were used. Every patient's harvested marrow was evaluable for nucleated cell and progenitor cell loss during the cell separation and cryopreservation process. Of the 41 patients, 38 were evaluable for peripheral blood count recovery. Multivariate analysis of colony-forming cell assays and recovery of neutrophils and platelets showed a significant association with absolute numbers of post-thaw mixed colony-forming units (CFU-Mix) infused (p less than 0.002). Prefreeze CFU-Mix also correlated with recovery to a lesser degree, as did absolute numbers of nucleated cells. The number of diffusion chamber colony-forming units (CFU-D) prefreeze, but not post-thaw infused into the patient, was associated with recovery of neutrophils (p = 0.0001), but not platelets. When the precursor cell numbers were adjusted for body weight, post-thaw CFU-Mix showed the best correlation with recovery of both platelets and neutrophils. Prefreeze CFU-D per kg was also associated with recovery of neutrophils (p = 0.02). To some extent nucleated cells per kg predicted for recovery with neutrophils and platelets (p less than 0.05). When analyzed according to treatment regimen, cyclophosphamide-BCNU-VP16 (CBV) or cyclophosphamide-total body irradiation (CY/TBI) was associated with prolonged recovery compared to cyclophosphamide-adriamycin-vinblastine (CAV) or etoposide-cyclophosphamide (EC). In this setting only CFU-D number predicted neutrophil recovery (p less than 0.002). We conclude that determination of the number of total nucleated cells, CFU-D, and CFU-Mix, before cryopreservation of the sample is important in predicting hemopoietic reconstitution in autologous bone marrow transplantation.

Critical appraisal of complement dependent microlymphocytotoxicity assay for detecting donor-specific alloantibody pretransplant--importance of indirect immunofluorescence as a superior alternative.

The ability of complement-dependent microlymphocytotoxicity assay (CdL) to detect and discriminate between the various types of donor-specific alloantibodies was reevaluated. Data obtained with the CdL assay on purified B and T lymphocytes at warm and cold temperatures was compared to other modes of antibody-detection, i.e., indirect immunofluorescence (IF) and the noncomplement-dependent antibody-dependent cellular cytotoxicity (ADCC). Additionally, the significance of antibodies as detected by CdL and IF was ascertained by correlating with kidney transplant outcome. It became apparent that the CdL assay identified weakly reactive HLA-ABC alloantibodies as being B cell specific. Such weakly reactive HLA-ABC antibodies were also not appreciated in the presence of the cold reactive IgM antibody. Accelerated rejections were the rule in the presence of weakly reactive HLA-ABC alloantibodies indicating that their detection was highly important. The IF assay could discriminate between the antibody class, could detect weakly reactive HLA-ABC alloantibodies, and could detect noncomplement fixing antibodies (ADCC). Further, use of IF prevented us from unnecessarily denying transplants to certain recipients when a positive CdL assay resulted from an IgM antibody or poor cell viability.

Effect of cooling rate on human and murine hemopoietic precursor cell recovery.

The effect of cooling rate on recovery of human and murine hemopoietic precursor cells was studied. In the presence of 10% Me2SO, a cooling rate of 7 degrees C/min from -4 to -30 degrees C was optimal for recovery of both human and murine precursor cells which give rise to colonies in diffusion chambers implanted in mice (CFU-DG). Cooling of human marrow at a rate between 3 and 7 degrees C/min resulted in the best CFU-C recovery, although no good correlation between the cooling rate and murine CFU-C recovery was demonstrated. These data suggest that recovery of the primitive hemopoietic precursor cells can be improved by changing the standard cryopreservation programs used presently. However, improved recovery of CFU-DG does not necessarily translate into faster reconstitution of hemopoiesis. No significant difference was observed in overall recovery of bone marrow cellularity in lethally irradiated mice following injection of untreated marrow and marrow cooled at a rate of 1 and 7 degrees C/min.