A novel high-yield volume-reduction method for the cryopreservation of UC blood units.
BACKGROUND
For the application of umbilical cord blood (UCB) units as hematopoietic grafts, a dose of 3.7 x 10(7) nucleated cells (NC)/kg body weight is required. NC can be lost during volume-reduction processing and during thawing. A novel modification of the double-processing protocol with the aim of minimizing NC loss is described and evaluated.


METHODS
One-hundred and fifty UCB were collected. The volume was reduced by a centrifugation step following double-processing in the presence of 2% HES 200/0.5. Pre- and post-processing cell counts and platelet parameters were measured with an automatic counter. The number of viable CD34+ hemopoietic stem cells was measured by flow cytometry. In 25 of the samples, colony-forming units (CFU) were also determined. The same samples were thawed 6 months after cryopreservation and re-evaluated.


RESULTS
The volume was reduced to 6 +/- 1.5 mL. The recovery of NC, MNC, CD34+ hemopoietic stem cells, RBC depletion and CFU following double-processing was 93.6 +/- 3.2%, 95.8 +/- 2.2%, 98.4 +/- 1.5%, 96.8 +/- 1.1% and 107.1 +/- 6.1% (for 25 samples), respectively. The post-thaw recoveries of NC, MNC, CD34+ hemopoietic stem cells and CFU (for 25 samples) were 78.6 +/- 5.4%, 90.8 +/- 4.4%, 96.4 +/- 2.5%, 89.1 +/- 4.1%, respectively. No post-thaw cell aggregation was observed. A significant (P<0.05) post-thaw loss of platelets and signs of platelet activation was observed.


CONCLUSIONS
The protocol uses non-expensive equipment and clinically approved materials and results in samples that can be used in patients with a mean weight of 32.7 kg.
Koliakos
G
G
Department of Biological Chemistry, Medical School, Aristotle University Thessaloniki, Thermi, Thessaloniki, Greece. koliakos@med.auth.gr
Alamdari
D  H
D
Tsagias
N
N
Kouzi-Koliakos
K
K
Michaloudi
E
E
Karagiannis
V
V
D018952:Antigens, CD34; D001792:Blood Platelets / Q000166:cytology; D001793:Blood Preservation / Q000379:methods*; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D005312:Fetal Blood / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D007231:Infant, Newborn
D018952:Antigens, CD34; D003451:Cryoprotective Agents