Dextran sedimentation in a semi-closed system for the clinical banking of umbilical cord blood.
BACKGROUND
The results of current processing procedures for reducing volume and recovering HPCs from umbilical cord blood (UCB) before cryopreservation vary.


METHODS
Dextran was added to bags containing UCB, followed by sedimentation for 30 minutes. The processed UCB was then frozen. RBCs, nucleated cells, MNCs, CD34+ cells, CFUs and long-term culture-initiating cells (LTC-ICs), viability, and sterility were evaluated. Fractionations in ficoll-hypaque and hydroxyethyl starch (HES) were also run in parallel for comparison.


RESULTS
The nucleated cell (NC) recovery and RBC depletion were 86.1 percent and 94.3 percent, respectively (n = 50). Sedimentation with dextran also enabled the recovery of 80.7 percent MNCs and 82.6 percent CD34+ cells (n = 30). Postsedimentation samples displayed no impairment of CFU growth (n = 42, 108.7% CFU-C, 104.6% CFU-GEMM, 107% CFU-GM, and 95.7% BFU-E). Long-term cultures on five paired samples before and after sedimentation generated similar numbers of CFU-C each week (p = 0.88). Limiting dilution analysis of 12 paired pre/postsedimentation samples showed comparable median proportions of LTC-ICs (1/6494 vs. 1/5236; p = 0.18). The cell viability of 24 samples of thawed UCB after sedimentation was 90.3 percent (77.5-96%) and the recovery of CFU-C, CFU-GEMM, CFU-GM, and BFU-E of 11 postsedimentation samples was 93.4 percent, 84.9 percent, 92.3 percent, and 83.4 percent, respectively. NC recovery was significantly higher after treatment with dextran than with ficoll-hypaque (n = 30; 88.5% vs. 29.1%; p<0.005) and HES treatment (n = 21; 88.5% vs. 76.4%; p = 0.004). However, MNCs, CD34+ cells, CFUs, LTC-ICs, and RBCs were comparable. Two cycles of dextran sedimentation recovered 93.9 percent of NCs with cell viability of 98.6 percent (96.5-100%), whereas 11.7 percent of RBCs were retained (n = 20). The final yield volume was 33.5 (28-41) mL.


CONCLUSIONS
In a semi-closed system, dextran sedimentation enabled volume reduction of UCB without significant quantitative and qualitative losses of HPCs.
Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, People's Republic of China. tsangks@ha.org.hk
K S
KS
Tsang
K
K
Li
D P
DP
Huang
A P
AP
Wong
Y
Y
Leung
T T
TT
Lau
A M
AM
Chang
C K
CK
Li
T F
TF
Fok
P M
PM
Yuen
D001771:Blood Banks; D001799:Blood Sedimentation; D002470:Cell Survival; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D015925:Cryopreservation; D003911:Dextrans; D003973:Diatrizoate; D004906:Erythrocyte Count; D004912:Erythrocytes / Q000502:physiology; D005260:Female; D005312:Fetal Blood; D005362:Ficoll; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D006895:Hydroxyethyl Starch Derivatives; D011247:Pregnancy; D013048:Specimen Handling
D003911:Dextrans; D006895:Hydroxyethyl Starch Derivatives; D003973:Diatrizoate; D005362:Ficoll