Abstract
Background: CBU selection for transplant is based on HLA match and potency/quality determinations associated with the speed of hematopoietic engraftment. Total Nucleated Cell (TNC) count, viable (v) CD45+ and CD34+ cells and Colony Forming Units (CFU) have been used as surrogate markers of hematopoietic stem cells. However, besides TNC measurements, most of the assays still have poor standardization among laboratories.
The NCBP has manufactured over 60,000 clinical CBU (as of 07/2018). Of those, 32,413 CBU had pre-cryopreservation samples tested prospectively (period: 03/2008-04/2018) using validated assays for TNC (Sysmex XE-2100), vCD45+ and vCD34+ cells (single platform flow cytometry using ISHAGE strategy and 7-AAD for viability) and HRDI-CFU assay (High Resolution Digital Imaging CFU strategy, Albano et al, Blood 2011;118:485). Among those, 754 CBU have been shipped to US Transplant Centers (TC) for single or double unit transplants. Prior to release for clinical use, CBU had also quality/potency tests conducted prospectively in post-thaw samples from attached segments, using the same assays.
Aims and study design: 1) to evaluate the quality/potency of 754 NCBP CBU provided for transplantation (period: 03/24/2008-12/31/2017) by pre-cryopreservation characteristics, post-thaw segment evaluations and their correlations, and 2) to assess their impact on engraftment in two patient subsets after myeloablation: cohort 1, N=99 patients (pts), who received single CBU transplants, and cohort 2, N=139 pts with double unit grafts, who engrafted with the NCBP CBU. All laboratory evaluations were performed by NCBP. Clinical data were provided by CIBMTR; only pts with information on time to engraftment (ANC>500) and established donor chimerism were analyzed.
Results: The 754 CBU selected by TC had quality/potency parameters showing a strong correlation between cell numbers and function pre-cryopreservation and post-thaw (Table 1; Pearson correlation). Segment post-thaw median recoveries were 73% (SD: 21.8%) for vCD45+ cells; 69% (SD: 20.7%) for vCD34+ cells and 60% (SD: 22.9%) for CFU, with a strong correlation between pre-cryopreservation and post-thaw values (Pair T Test: 0.74; 0.87 and 0.79, respectively; all P<0.001) and excellent segment post-thaw CD34+ viability (median: 95.8%; SD: 3.5%). There were no differences between CBU released as FDA-licensed products (N=208) or those under IND (N=546). Median time from CBU collection to transplant was 2.1 years (range: 0.1-9.6). Median time to engraftment was 19 days (range: 14-205) for the pts that received myeloablative regimens (N=238 total). Cohort 1 included primarily children with average age 9 years, average weight 24 kg (range 3-116), who received median pre-cryopreservation TNC 8.6x10^7/kg and vCD34+ 3.4x10^5/kg. Median time to ANC>500 was 16 days (range: 7-45). Cohort 2 had 85% adult pts with average age 33 years, average weight 69 kg (range: 11-129) and median time to ANC>500 19 days (range: 6-68). Engrafting CBU median pre-cryopreservation TNC was 2.6x10^7/kg and vCD34+ 1.4x10^5/kg. CBU pre-cryopreservation and segment quality/potency markers showed very good correlation also (Table 1). In cohort 1 all pre-cryopreservation cell doses and segment vCD34+ and CFU correlated with neutrophil engraftment (Table 2). Pts with earlier engraftment (≤16 vs >17 days) received CBU with significantly higher doses of TNC, vCD34+, vCD45+ and CFU (pre-cryopreservation and post-thaw tests). In the recipients of double CB transplants, the engrafting CBU TNC and vCD45+ cell doses did not correlate with neutrophil engraftment, but the vCD34+ and CFU did (Table 2). Similarly, pts with earlier engraftment (≤19 vs >20 days) received CBU with higher doses of vCD34+ and CFU, but not TNC or CD45+.
Conclusions: Prospective testing of HPC, CB products manufactured by a single CB Bank over a 10-year period and provided for transplant show consistent, highly significant correlation among pre-cryopreservation graft characteristics and segment measurements. Pre-cryopreservation values were predictive of time to ANC>500 in myeloablated recipients; in those cases, segment evaluation did not improve the correlations. Notably, vCD34+ and CFU correlations are consistently maintained after cryopreservation and thaw ensuring a HPC, CB product with reliable performance for transplantation, cell therapies or use with expansion technologies.
No relevant conflicts of interest to declare.
