Evaluation of ‘out-of-specification’ CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products

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Background

Immunomagnetic selection of CD34+ hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited.

Methods

In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units–granulocyte-macrophage (CFU-GM).

Results

Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 × 106 CD34+ cells to 1000 × 106 CD34+ cells with similar recovery of CD34+ cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures.

Discussion

Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

Introduction

Transplantation of positively selected CD34+ hematopoietic progenitor cells (HPC) is part of an established treatment modality for adult and pediatric patients with hematologic malignancies. Immunomagnetic selection of CD34+ HPC provides a graft profoundly depleted of T, B and natural killer (NK) cells, which reduces the incidence of both acute and chronic graft-versus-host disease (GvHD) after allogeneic stem cell transplantation (SCT) 1., 2., 3.. In autologous SCT, selection of CD34+ cells is used both to reduce the tumor cell contamination of the graft in CD34 malignancies, which may decrease relapse risk [4], and to deplete T cells in grafts used in autoimmune diseases [5, 6].

Several studies have shown that the CliniMACS device is a reliable system for clinical-scale selection of large amounts of CD34+ cells, yielding high purities and good recoveries 7., 8., 9., 10., 11.. The capacity of the disposable tubing sets and the separation capacity of 1 vial of CD34 reagent determine the number of selection procedures required and the total cost of the processing procedure. According to the manufacturer's current specifications, the maximum capacity of a normal-scale tubing set is 600 × 106 CD34+ cells and 60 × 109 total nucleated cells, whereas that of the large-scale tubing set is 1200 × 106 CD34+ cells and 120 × 109 total nucleated cells. The specified separation capacity of 1 vial of CliniMACS CD34 reagent is 600 × 106 CD34+ cells out of 60 × 109 total nucleated cells. Single or pooled HPC-apheresis (HPC-A) products from the same donor frequently exceed one of the limits of the tubing set and/or 1 vial of CD34 reagent. Therefore, many stem cell-processing laboratories in Europe regularly overload tubing sets and/or use less CD34 reagent than recommended to reduce the number of selection procedures, processing time and total cost.

In this study, we retrospectively evaluated the performance, in terms of CD34+ cell recoveries and purities and T-cell depletion efficiencies, of the CliniMACS system in a large number of ‘out-of-specification’ CD34-selection procedures performed in eight stem cell-processing laboratories.

Section snippets

Selection of CD34+ cells

Six-hundred and forty-three HPC-A products were collected from healthy donors (n=609) and patients with solid tumors or autoimmune disease (n = 34). Almost all the HPC-A products were processed within 24 h of harvest. Some HPC-A products harvested from unrelated healthy donors abroad were processed between 24 and 48 h after harvest. Positive selection of CD34+ cells was performed using the CliniMACS® device (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's standard

CD34+ cell recovery of overloaded normal- and large-scale tubing sets

In the eight participating stem cell-processing laboratories, 643 CliniMACS CD34-selection procedures were performed on HPC-A products. Two-hundred and fifty-three of these procedures were outside the limits of the tubing set capacity specified by the manufacturer. This allowed us to evaluate the effect of overloading both types of tubing sets with either excess amounts of total nucleated cells or excess amounts of CD34+ cells or both. Therefore we divided the CD34-selection procedures based on

Discussion

In this study, we evaluated a large number of out-of-specification CliniMACS CD34 selection procedures of HPC-A products for CD34+ cell recovery and purity, and compared the results with those of within-specification procedures. Our study supports three main conclusions. First, overloading normal- or large-scale tubing sets with excess numbers of nucleated cells does not hamper the recovery and purity of CD34+ cells. Secondly, overloading normal- or large-scale tubing sets with excess numbers

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