Cell loss and recovery in umbilical cord blood processing: a comparison of postthaw and postwash samples.
Author(s): Laroche V, McKenna DH, Moroff G, Schierman T, Kadidlo D, McCullough J
Publication: Transfusion, 2005, Vol. 45, Page 1909-16
PubMed ID: 16371043 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of freezing and thawing, washing and post-wash storage on umbilical cord blood (UCB) cell recovery and viability.
Conclusion of Paper
Freezing and thawing cord blood specimens resulted in significant decreases in total nucleated cells (TNC), colony forming units (CFUs), acridine orange/propidium iodide (AO/PI) staining viability, and TNC viability, but only non-significant effects were observed for CD34+ cell counts. Washing UCB led to a further significant decline in TNCs and TNC viability but partially attenuated the freeze/thaw effects on CFUs and increased viability, as determined by flow cytometry but not AO/PI staining. Post-wash storage of UCB led to significant loss of TNC and a significant increase in flow cytometry viability, but CD34+ cell counts, number of CFUs, AO/PI viability, and viable TNC counts were unaffected by post-wash storage of UCB.
Studies
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Study Purpose
The purpose of this study was to determine the effects of freezing and thawing, washing and post-wash storage on UCB cell recovery and viability. UCB from 10 vaginal deliveries was collected by gravity into citrate phosphate dextrose, mixed with hydroxyethylstarch and centrifuged to remove red blood cells. DMSO and dextran 40 were added to RBC-depleted UCB which was sampled before freezing in a controlled rate freezer. UCB was stored in liquid nitrogen and sampled again immediately after thawing in a 37°C water bath, after washing in 10% dextran and 5% albumin and after 1, 2, or 5 h at an unspecified temperature post-wash.
Summary of Findings:
During processing of UCB, the TNC count decreased 18% with the largest decline noted after thaw compared to pre-freezing (-11%, p<0.001) and further declines noted with washing. The CD34+ cell count decreased non-significantly by 21% after thawing UCB but then increased non-significantly to 148.9% of initial values post-washing. CFUs in UCB were 83% lower post-thaw than pre-freeze (p<0.001), and while they increased significantly post-wash (p<0.001), they remained 36% below the pre-freeze value (p<0.001). While cell viability, as determined by AO/PI staining, decreased with thawing of UCB (p<0.001), it was unaffected by washing. However, cell viability in UCB, as assessed by flow cytometry, increased post wash (p<0.001). TNC viability was significantly decreased by freezing/thawing of UCB specimens (p<0.001) and further decreased with washing (p<0.05). Post-wash storage of UCB led to significant loss of TNC after 2 h (p<0.05), and a significant increase in flow cytometry viability was achieved after 5 h (p<0.01), but CD34+ cell counts, number of CFUs, AO/PI viability, and viable TNC counts were unaffected by post-wash storage of UCB.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Pregnant
Platform:
Analyte Technology Platform Cell count/volume Hematology/ auto analyzer Cell count/volume Flow cytometry Cell count/volume Fluorescent microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)
Storage Freeze/thaw cycling 0 cycles
1 cycle
Analyte Extraction and Purification Washing Prewash
Washed in 10% dextran 5% albumin
Storage Storage duration 0 h
1 h
2 h
5 h