Selective loss of progenitor subsets following clinical CD34+ cell enrichment by magnetic field, magnetic beads or chromatography separation

Abstract

Summary:In this preclinical evaluation we have compared the efficacy of three clinical CD34 ؉ enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34 ؉ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88-98%), 86% (62-97%), and 49% (18-85%), and median yield of 65% (54-100%), 40% (21-74%), and 30% (8-55%), respectively. Subset analysis recognised a selective loss of CD34 ؉ /61 ؉ after enrichment, most likely due to class I-II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2-4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34 bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between biotin-avidin and insufficient physical manipulation for CD34 ؉ cell release. Finally, the data document that CD34 selection alone is an inappropriate way to eliminate tumour cells due to the uncontrolled variables and the inconsistent outcome. The only products which can be expected to be purged free of tumour cells are the ones with very minimal (Ͻ10 ؊5 ) contamination in the starting products, ie products documented tumour free with the most sensitive techniques for quantitation. If this is not the case, the optimal purging strategy may be a twostep procedure including CD34 selection and subsequent depletion of the tumour cells in question.

The excessive numbers of total nucleated cells does not affect the performance of the CliniMACS

“…Our data on the performance of the CliniMACS were comparable to that reported before with a median purity of 92.5% and recovery of 67% [1,2,4]. Overnight storing of the leukapheresis products at 4°C and processing within 24 hours had no significant effect on the purity and yield as reported before [2,5].…”

Cotransplantation of third-party mesenchymal stromal cells can alleviate single-donor predominance and increase engraftment from double cord transplantation

“…Although lineage depletion alleviates single-donor predominance, alternative strategies not requiring lineage depletion would be more desirable because the latter is frequently associated with cell loss, 35 which might itself hamper reaching the target cell dose. To circumvent this hurdle, we postulated that cotransplantating MSCs, bone marrow-derived cells that have been implicated in the suppression of allogeneic immune response, [36][37][38][39][40] could also suppress the graft-versus-graft reaction in double cord transplantation.…”

New Strategies in the Treatment of Acute Myelogenous Leukemia (AML): In Vitro Culture of AML Cells—The Present Use in Experimental Studies and the Possible Importance for Future Therapeutic Approaches

“…+ AML cells [19,20]. However, extensive ex vivo manipulation, including cell separation procedures, can induce gene expression and thereby alter the release of soluble mediators as well as the expression of membrane molecules by native AML blasts [21][22][23][24].…”

“…Additional positive/negative selection [19][20][21][22][23][24] Can be used for all patients independent of the number Functional alterations can be induced in the AML of contaminating low-density cells in the original sample.…”