Abstract

Introduction: Nowadays UCB represents an established source of hematopoietic stem cells for unrelated transplants in children and the employ in adults is quickly growing up. Nucleated cells (NCs) content is one of the main predictors to evaluate UCB for clinical use; however, other indicators of the hematopoietic potential, such as CD34+ cell and colony-forming cells (CFUs), have recently showed similar correlations. According to Netcord-FACT standards, it is recommended to test all the above mentioned parameters before releasing UCB to the transplant center; a segmented tubing of the UCB bag should be used but a satellite cryotube is more often available. We preliminarily report the results of quality controls performed on thawed cryovials corresponding to each of 15 units delivered by our UCB Bank.

Material and methods: in our policy, all UCBs are stored accompanied by 3 satellite cryovials, treated under the same conditions of the unit. For each of the 15 UCBs released for transplantation, one cryotube was thawed in a 37°C water bath with a gentle agitation, without washing out DMSO. NCs and mononucleated cells (MNCs) were estimated with an automated cell counter. Viability and CD34+ cell count were evaluated by flow cytometry, with a no-wash, single-platform technique and 7-aminoactinomycin D. CFU assay was performed using commercial reagents (Methocult GF H4434, StemCell Technologies) and colonies were classified after a 14 days incubation. The same parameters referring to fresh UCBs (before cryopreservation) were always available.

Results: the UCB characteristics measured after thawing a cryovial were compared with those of the fresh cord and are detailed in the table below.

fresh UCB before cryopreservationUCB cryovial after thawingYield (%)
NC (x106) 1491.3 (148–2262) 1354.2 (167.4–2119.9) 92 (83–113) 
MNC (x106) 662.7 (96.5–900.3) 638.8 (295–1238.7) 95 (63–159) 
CD 34+ cells (x106) 3.47 (0.38–10.87) 3.19 (0.4–9.27) 85 (37–118) 
Viability (%) 96 (88–100) 62 (39–77)  
Viability of CD34+ cells (%)  92 (71–99)  
CFU-GM (x104) 838.5 (57.2–3581.2) 424.11 (65.29–917.64) 69 (16–184) 
BFU-E (x104) 1709.61 (159.84–5116) 473.34 (39.69–1204.14) 41 (5–139) 
total CFU (x104) 2565.17 (217.04–8953) 911.68 (164.43–2075.22) 57 (9–160) 
fresh UCB before cryopreservationUCB cryovial after thawingYield (%)
NC (x106) 1491.3 (148–2262) 1354.2 (167.4–2119.9) 92 (83–113) 
MNC (x106) 662.7 (96.5–900.3) 638.8 (295–1238.7) 95 (63–159) 
CD 34+ cells (x106) 3.47 (0.38–10.87) 3.19 (0.4–9.27) 85 (37–118) 
Viability (%) 96 (88–100) 62 (39–77)  
Viability of CD34+ cells (%)  92 (71–99)  
CFU-GM (x104) 838.5 (57.2–3581.2) 424.11 (65.29–917.64) 69 (16–184) 
BFU-E (x104) 1709.61 (159.84–5116) 473.34 (39.69–1204.14) 41 (5–139) 
total CFU (x104) 2565.17 (217.04–8953) 911.68 (164.43–2075.22) 57 (9–160) 

Excellent yields were found for NCs, MNCs and CD34+ cells. Despite of the decrease in the overall viability, the viable CD34+ cells as percentage was always highly satisfactory. The colonies growth was found lower in the thawed sample in comparison with fresh UCB before cryopreservation.

Conclusion: in our experience, highly satisfactory evaluations of UCB content could be obtained using thawed cryotubes with regard to NC, MNC and also CD34+ cell. However, concerning the latter, the different methods employed on fresh UCBs, such as CD34+ cells detection without beads, may be advocated to explain some discrepancies in the yield range. The results of CFU assay confirmed to be poorly useful, essentially because affected by a subjective interpretation even if the reduced cell growth may be also related to the presence of DMSO as inhibiting factor.

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