The Effects of Anticoagulants on Cord Blood Viability From Collection to Processing

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells for transplantation and regenerative medicine applications. UCB collection bags must contain an anticoagulant to prevent coagulation and to maintain viability during transport to the processing facility. Although Citrate Phosphate Dextrose (CPD) has been widely used based on the commercial availability of pre-filled collection bags, heparin is also accepted as an anticoagulant for processing hematopoietic stem cells. Data is lacking on the optimal anticoagulant for UCB collections particularly in the volume range of cord blood collected for family banking purposes. CPD was originally formulated for use in whole blood collections at a dilution ratio of 1:7 CPD to blood. Commercially available UCB bags contain 35 mL of CPD and are intended to allow a maximum collection volume of 210 mL in a 250 mL bag, creating up to a 1:6 dilution. However, typical cord blood collections rarely reach this volume. Given the wide variability in UCB collection volumes and the standard volume of 35 mL CPD pre-loaded in each bag regardless of collection volume, dilution ratios for UCB collections could drop as low as 1:1 CPD to blood. Concentrated exposure to CPD could adversely impact cell viability and blood chemistry.

UCB collections from 15 individual donors were each approximately equally divided (p>0.05) between a 300 mL collection bag containing 500 units of lyophilized heparin (LH) and a 250 mL bag with 35 mL liquid CPD. Samples were placed on an orbital mixer at room temperature for a simulated shipping time of 48 hours. Aliquots of the samples were assessed for total nucleated cell (TNC) count and cell viability at time of collection, and at 24 and 48 hour time points. Viable TNC were obtained utilizing acridine orange/propidium iodide staining. Total cell counts were obtained using a Sysmex hematology analyzer. The values at each time point were compared to the previous measurement to detect changes over time.

Shown in Table 1. Samples collected in CPD showed a statistically significant decrease in TNC yield at 24 hours (p=0.004) and again at 48 hours (p=0.002). There was a significant but lesser decrease in TNC yield in samples collected in LH between hours 0 and 24 (p=0.04) and no significant difference thereafter. There was a significant decrease in TNC viability in CPD samples at both time points. The viability of samples collected in LH vs. CPD was 96.6±3.0% vs. 85.9±9.0% (p<0.001) at hour 24 and 95.9±3.0% vs. 66.9±17.8% (p<0.001) at hour 48.

The TNC yield and viability of samples collected in LH compared to CPD suggest that UCB quality was impacted by anticoagulant selection. The results of this study indicate LH was more biocompatible than CPD as measured by cell viability endpoints. During the study, it was also noted that CPD exposure typical of family cord blood banking conditions caused significant acidosis of the blood. In a family banking setting, where the time from collection to processing could be as long as 48 hours and beyond, prolonged exposure to an acidic environment could be detrimental. LH was found not to cause acidosis or other dilution related issues. Because family cord blood banking preserves genetically unique stem cells for each individual newborn and his or her family, it is important to ensure that UCB unit quality will be maintained during transport regardless of the size of the collection volume.

No relevant conflicts of interest to declare.