Effect of recombinant human deoxyribonuclease on the expression of cell adhesion molecules of thawed and processed cord blood hematopoietic progenitors.

Abstract

The integrity of granulocytic cells and platelets is compromised within cryopreserved stem cell transplants, and consequent DNA release during the thawing procedure can therefore lead to clotting phenomena or microaggregate formation and that in turn may cause loss of progenitor cells. To circumvent this problem a new processing protocol was introduced using recombinant human deoxyribonuclease (rhDNase) to prevent cell aggregate formation. In addition, the impact of this new processing protocol on CD34+ umbilical cord blood (UCB) cells was assessed. Fifty samples derived from 7 buffy coat (BC) volume reduced UCB units were cryopreserved, thawed, and processed with washing solutions that were supplemented with rhDNase in various concentrations. Thereafter, clotting and microaggregate formation was scored microscopically. In addition, expression of the adhesion molecules leukocyte function-associated antigen 1 (LFA-1, n = 6), intercellular adhesion molecule-1 (ICAM-1, n = 11), and L-selectin (n = 11) on CD34+ UCB cells was analyzed by flow cytometry after incubating the samples with either dimethyl sulfoxide (DMSO) 5.5%, rhDNase 10 or 50 U/mL, or a combination of DMSO 5.5% and rhDNase 50 U/mL. At a minimal concentration of 10 U rhDNase/mL, clotting or microaggregate formation could be prevented for all tested samples, whereas cell clots could be observed for concentrations up to 8 U/mL. The expression of adhesion molecules on untreated CD34+ UCB cells (L-selectin: 64.6 +/- 18.8%; LFA-1: 62.6 +/- 7.5%; ICAM-1: 14.8 +/- 4.1%) did not show any significant difference compared with cells that were incubated with up to 50 U/mL rhDNase (L-selectin: 62.2 +/- 19.3%; LFA-1: 63.1 +/- 5.9%; ICAM-1: 17.5 +/- 6.7%). However, after a combined treatment with DMSO 5.5% and rhDNase 50 U/mL, a slight but significant decrease in L-selectin expression could be observed (P < 0.03). The supplementation of rhDNase to a final concentration of 10 U/mL cell suspension proved to be effective in preventing clot formation under the conditions examined and did not lead to decreased expression levels of adhesion molecules. We therefore recommend the use of rhDNase for the prevention of clot formation and cell loss during the processing of thawed UCB transplants.