CD34+ cell selection: focus on immunomagnetic beads and chymopapain

Abstract

The aim of the present study was to develop immunoadsorption techniques for purifying CD34+ cells from the peripheral blood (PB) and the bone marrow (BM). First, we compared two methods for isolating CD34+ cells from patients with acute non-lymphocytic leukemia. Twenty-two samples (17 PB, 5 BM) were obtained from 19 patients, were density cut and, after incubation with My10 antibody, were separated by panning or by immunomagnetic beads. Beads allowed a significantly better separation, either in terms of purification of CD34+ cells (85.5% +/- 11.1% vs 55.7 +/- 23.8%, p = 0.003) or in terms of depletion of CD34+ cells from the negative fractions (3.9 +/- 7.6 vs 30.9 +/- 25.1%, p = 0.008). In a second study, 2 BMs from healthy subjects and 1 BM and 2 PBs from chronic myeloid leukemia patients were separated using immunomagnetic beads. The results confirmed the previous study the overall frequency of CD34+ cells in the isolated positive fractions was 85.0 +/- 10.6% (with a recovery of 64.0 +/- 5.7%), while it was only 2.7 +/- 6.6% in the negative fractions. In particular the purity in the two normal BMs was respectively 86 and 97%. According to these results, the great majority of clonogenic cells was separated in the CD34+ fractions. Chymopapain, that was used to detach the beads from the cells, was shown to be non-toxic to the clonogenic cells. Positive selection of CD34+ cells with immunomagnetic beads and chymopapain is a useful method for isolating almost pure progenitors from the PB and the BM for experimental use and is under investigation for clinical applications.