Real Time Immunophenotyping of Leukocyte Subsets Early after Double Cord Blood Transplantation Predicts Graft Function Jianqiang Li, Ian Nicoud, Joseph.

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Real Time Immunophenotyping of Leukocyte Subsets Early after Double Cord Blood Transplantation Predicts Graft Function Jianqiang Li, Ian Nicoud, Joseph Blake, David Oliver, Emily Cox, Shelly Heimfeld, Filippo Milano, Suzan Imren, Colleen Delaney Biology of Blood and Marrow Transplantation Volume 23, Issue 3, Pages 412-419 (March 2017) DOI: 10.1016/j.bbmt.2016.12.625 Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Composition of PBMCs at day +7 (A) and day +14 (B) after dCBT. Total numbers of monocytes, NK cells, and T cells were set up as 100%. Patient ID, patient's ID number; EngDay, day of engraftment; GF, graft failure. Biology of Blood and Marrow Transplantation 2017 23, 412-419DOI: (10.1016/j.bbmt.2016.12.625) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Absolute numbers of PBMCs, monocytes, and NK cells at day +14 after dCBT correlate with the kinetics of engraftment. (A) The total number of PBMCs (per µL) at days +7 and +14 was counted manually using an optical microscope after dilution in Turk's solution. The absolute numbers of CD14+ monocytes (B), CD56+ NK cells (C), and CD3+ T cells (D) at days +7 and +14 were calculated by multiplying the frequency of the corresponding subpopulations documented by flow cytometry by the total number of PBMCs at that time point. The median day to engraftment was 18 in all 39 patients. Patients were divided into 2 groups. The early-engrafting (Early Eng.) patients (n = 19; black dots and squares) achieved neutrophil recovery at ≤18 days (the median); the late-engrafting (Late Eng.) patients (n = 18; blue dots and squares) achieved neutrophil recovery at >18 days. Solid lines represent the median number of cells. Statistics were performed using the 2-tailed Mann-Whitney U test. Significant differences between groups: *P < .05; **P < .01. Biology of Blood and Marrow Transplantation 2017 23, 412-419DOI: (10.1016/j.bbmt.2016.12.625) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Rapid regeneration of monocytes and NK cells after dCBT can predict the speed of hematopoietic recovery. The median day to engraftment was compared with the absolute number of monocytes (A and B) and NK cells (C and D) in recipients at days +7 and +14 after transplantation. The Wilcoxon rank-sum test was used to compare the median engraftment day above and below the median number of the indicated subset of cells (dotted horizontal line). Correlation assays were performed using a simple linear regression. Biology of Blood and Marrow Transplantation 2017 23, 412-419DOI: (10.1016/j.bbmt.2016.12.625) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Phenotypic conversion of monocytes and NK cells occurs very early after dCBT. (A) Gating strategy for the CD16+ monocyte subset. (B) The frequency of CD16+ monocytes in CB (n = 8) and recipient PBMCs at day +7 (n = 20) and day +14 (n = 25) after dCBT. (C) Gating strategy for CD56brightCD16- NK cell subset. (D) The frequency of CD56bright NK cells in CB (n = 8) and recipient PBMCs at days +7 (n = 18) and +14 (n = 22) after dCBT. Solid lines represent the median number. P values between different groups were calculated using the 2-tailed Mann-Whitney test. **P < .01; ***P < .001; ****P < .0001. Biology of Blood and Marrow Transplantation 2017 23, 412-419DOI: (10.1016/j.bbmt.2016.12.625) Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions