However, there was a significant correlation (P=0.02) between CLP and γδ T cells reconstitution at Day 90 in Group 1 patients (Fig1C). In these preliminary results, the different distribution of CMP, GMP, MEP and MPP did not impact on the hematopoietic/immune recovery. However, the frequency of HSC in the BM at Day 30 is statistically correlated (P=0.027) with the PLT at Day 90 (Fig1B). No significant correlation was observed between the HSC frequency in the graft and the hematopoietic/immune recovery. Notably, while CMP and GMP were very consistent across the 6 grafts, the frequency of HSC, CLP, MEP and MPP showed a 2-fold range of variation (Fig1A). Despite consistent levels of αβT-cell depletion and CD34 enrichment, the frequency of the HSPC subsets varied between the grafts.
#Issues with backgating flowjo 10 software
At least 5x10 5 events were acquired and analyzed using FlowJo software (BD). FACS analyses were performed on either fresh or frozen cells on Becton Dickinson (BD) Aria II flow cytometer. Mononuclear cells were isolated from PBSC, PB and BM by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation. Aliquots of αβhaplo-HSCT grafts were cryopreserved for later analyses. All patients were enrolled in the Stanford IRB approved BMT Protocols 179/351/361 and had peripheral blood (PB) and bone marrow (BM) evaluated at Day 30, 60 and 90 post HSCT for the primitive CD34+ Lin- HSPC subsets: HSC (CD38-CD45RA-CD90+), MPP (CD38+CD45RA+), CMP (CD38+CD45RA-CD123+), GMP (CD38+CD45RA+CD123+), MEP (CD38+CD45RA-CD123+) and CLP (CD38+CD127+). See Table 1 for details about patients and graft composition. All patients were transplanted for acute leukemia and received a myeloablative TBI-based conditioning regimen. The patients were divided in two groups: 3 patients had a robust and sustained hematopoietic recovery (Group 1) while 3 patients experienced mild cytopenia after Day 60 (Group 2).
#Issues with backgating flowjo 10 serial
Additionally, we correlated the HSPC graft composition with the distribution of the same HSPC subsets in serial post-HSCT bone marrow aspirates performed at Days 30, 60, and 90, with the peripheral blood counts and with the immune recovery (CD3+, CD3+CD4+, CD3+CD8+, αβT, γδT, NK cells) at the same time points. We have performed the first analysis of HSPC graft composition in 6 αβ T-cell/CD19 B-cell depleted haploidentical (αβhaplo) HSCT. In fact, the biological basis for different dynamics of hematopoietic/immune recovery, the risk of infection, and graft-versus-host disease (GvHD) is not fully understood. While total CD34 counts in PBSC graft products have correlated with overall likelihood of hematopoietic recovery after allogeneic hematopoietic stem cell transplantation (HSCT), analyses of the HSPC composition and its relationship to relevant post-transplant clinical outcomes are lacking.