Comparative Analysis of Cell Dose and Viability of Cord Blood Units at Cryopreservation and at Thaw/Infusion for Unrelated Stem Cell Transplantation in Adult Recipients

Cell dose is a major criterion for cord blood unit (CBU) selection for allogeneic stem cell transplantation (allo-SCT). The aim of this study was the characterization of CBU cellular composition after thaw, and comparison with corresponding values at cryopreservation as reported by cord blood (CB) banks.

The study included 87 CBUs, that were thawed for infusion in the context of single (n=3) or dual-unit (n=42) allo-SCT in adults with hematologic malignancies, from 8/2006 to 6/2013. Upon thawing, the cryoprotective solution (DMSO 10%) was either removed by centrifugation/washing (38 CBUs) or diluted in a less hypertonic solution of Dextran 40/Human Albumin 2.5% (49 CBUs). Total nucleated cells (TNC) were measured with a hematology analyzer, while enumeration of CD34+ stem cells was performed by single-platform flow cytometry, according to ISHAGE guidelines. In 49 units, TNC and CD34+ cell viability was evaluated by addition of 7-AAD dye and sequential Boolean gating strategy.

TNC counts after thawing were lower compared to their values at freezing (Wilcoxon test, p <10^-4), and the difference was more pronounced in the units that were washed prior to infusion (Tables 1 and 2). Total cell viability was low (mean value, 42.6%), but this was mainly due to neutrophils. Regarding CD34+ cells, there was a significant difference between absolute counts at cryopreservation and at thaw (p<10^-4). Despite reduction post-thaw, the counts of both TNC and CD34+ cells did correlate with the corresponding values at cryopreservation by Spearman's analysis. Of note, washing seemed slightly advantageous in terms of CD34+ recovery (Tables 1 and 2). CD34+ cells retained high viability after thaw, with 90% of CBUs (44 out of 49 tested) demonstrating CD34+ viability ≥80%. Viability of <50% was noticed in only one CBU that failed to engraft.

In conclusion, CB cellular content and especially the CD34+ cell count is frequently shown to be inferior at thaw compared to cryopreservation. This probably reflects both the lack of standardization of CD34+ cell measurement and the effect of thawing procedure. Therefore, CD34+ cell viability may be a more meaningful marker for determining CBU quality.

Table 1
		TNC Mean±SD (range)	CD34+ Mean±SD (range)
Total cells	Pre-thaw	190.8±52.2 (70-359), x107	8.8±5.1 (1.3-28), x106
	Post-thaw	171.3±53.9 (49.1-414.8), x107	6.7±3.6 (1.3-21.2), x106
Per kg BW	Pre-thaw	2.7±0.8 (1.1-5.7), x107	1.2±0.7 (0.2-4), x105
	Post-thaw	2.4±0.8 (0.9-6.9), x107	0.9±0.5 (0.3-3.6), x105
Viability (%)		42.6±13.1 (19-71.4)	91±10.9 (47.8-100)

Table 2
	TNC			CD34+
	Washed		Diluted	Washed	Diluted
Difference (%)   between pre- and post-thaw cell numbers Mean±SD (range)	16.1±10.2 (0.7-43.8)		9.5±6.4 (0.2-26.8)	22.6±14.6 (0.8-57.9)	26±15.5 (0.2-76)
Mann-Whitney score	0.0001			0.0621
Spearman's rho	0.8661	0.9487		0.8578	0.8079