Storage time affects umbilical cord blood viability.
BACKGROUND
Cryopreserved umbilical cord blood (CB) is increasingly used as a cell source to reconstitute marrow in hematopoietic stem cell transplant patients. Delays in cryopreservation may adversely affect cell viability, thereby reducing their potential for engraftment after transplantation.


METHODS
The impact of delayed cryopreservation for up to 3 days on the viability of both CD45+ and CD34+ cell populations in 28 CB donations with volumes of 58.40 ± 15.4 mL (range, 39.4-107.4 mL) was investigated to establish whether precryopreservation storage time could be extended from our current time of 24 to 48 hours in line with other CB banks. Viability was assessed on 3 consecutive days, both before and after cryopreservation, by flow cytometry using 7-aminoactinomycin D (7-AAD) and annexin V methods.


RESULTS
The results using 7-AAD and annexin V indicated the viability of CD34+ cells before cryopreservation remained high (>92.33 ± 4.11%) over 3 days, whereas the viability of CD45+ cells decreased from 86.36 ± 4.97% to 66.24 ± 7.78% (p < 0.0001) by Day 3. Storage time significantly affected the viability of CD34+ cells after cryopreservation. Using 7-AAD, the mean CD34+ cell viability decreased by approximately 5% per extra day in storage from 84.30 ± 6.27% on Day 1 to 79.01 ± 7.44% (p < 0.0057) on Day 2 and to 73.95 ± 7.54% (p < 0.0001) on Day 3. With annexin V staining CD34+ cell viability fell by approximately 7% per extra day in storage from 77.17 ± 8.47% on Day 1 to 69.56 ± 13.30% (p < 0.0194) on Day 2 and to 62.89 ± 15.22% (p < 0.0002) on Day 3.


CONCLUSIONS
This study demonstrates that extended precryopreservation storage adversely affects viability and should be avoided.
NHS Blood and Transplant, Bristol, UK.
Martin G
MG
Guttridge
Teck Guan
TG
Soh
Heli
H
Belfield
Chris
C
Sidders
Suzanne M
SM
Watt
D017304:Annexin A5 / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D001793:Blood Preservation; D002470:Cell Survival; D015925:Cryopreservation; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000032:analysis; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D013997:Time Factors
D017304:Annexin A5; D018952:Antigens, CD34; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human