A CD34+ Cell Enrichment Protocol of Hematopoietic Stem Cells in a Well-Established Quality Management System

Abstract

Allogeneic stem cell transplantation applications have improved tremendously over the past quarter of a century. The use of new immunosuppressive protocols and elimination of T cells by CD34+ cell enrichment or T cell depletion on apheresis products increases the chance of using partially matched or haploidentical grafts. This is without increasing the risk of graft-versus-host disease, which is observed as a major complication of hematopoietic stem cell transplantation. The aim of this protocol is to evaluate the results obtained from 6 different process cycles performed on 6 different days. We used the CliniMACS Plus system located in our Cell and Tissue Manufacturing Center Quality Control Unit which is already calibrated as a class D room and includes a class A microbiological safety cabinet inside. The average purity of the end products was 95.66%, excluding only one end product which was 70%; this was higher than the values in current studies in the field. Superior to the reported studies, the CD3 quantity in each end product was below the dedicated thresholds. BactecTM FX40 blood culture system test results were detected as negative for each end product. Endotoxin testing suggested the absence of endotoxin within the products. The consistent outcomes obtained from these 6 different process cycles confirmed that the CliniMACS® Plus process cycles performed in accordance with our well-defined quality management system procedure is sufficient for the routine application of high-quality and safe CD34+ enrichment processes within our clean room area.

Utility investigation of automated techniques in hematopoietic progenitor cell count and viability assessment in the Good Manufacturing Practice (GMP) setting

“…As defined in the study of Kilic et al [12], the apheresis products were transferred from Ankara University School of Medicine İbn-i Sina Hospital Therapeutic Apheresis Unit Center to our Tissue and Cell Manufacturing Center, within a sterile container, with the facility to transport at a stable temperature recorded by a data logger [12]. The records containing the results of complete blood count (CBC), CD34+ cell enumeration and viability obtained by flow cytometric evaluation were accompanied with each sample The study was approved by the Turkish Ministry of Health Turkish Medicines and Medical Devices Agency, with the approval number 2014/2 for manufacturing of human medical products.…”

“…The CD34+ cell selection technique is used to deplete T cells from collected human-based cell products before allogeneic HSC transplantation. The CD34+ cells can be separated by various devices, one of which is the CliniMACS (Miltenyi, Biotec, GmbH, Bergish, Gladbach, Germany) [9,[12][13][14]. The CliniMACS CD34+ cell enrichment process is performed within the Quality Management System of our Tissue and Cell Manufacturing Center, as described by Kilic et al [12].…”

“…The CD34+ cells can be separated by various devices, one of which is the CliniMACS (Miltenyi, Biotec, GmbH, Bergish, Gladbach, Germany) [9,[12][13][14]. The CliniMACS CD34+ cell enrichment process is performed within the Quality Management System of our Tissue and Cell Manufacturing Center, as described by Kilic et al [12]. Cell count and viability are the most critical release criteria besides other tests, such as sterility and endotoxin testine, for each CD34+-enriched end product.…”

“…Cell count and viability are the most critical release criteria besides other tests, such as sterility and endotoxin testine, for each CD34+-enriched end product. In the 8 cell count processes included in this study, we used the trypan blue exclusion technique and an automated cell counter for cell count, and for viability testing, the trypan blue exclusion technique and the 7-AAD evaluation by flow cytometry [12].…”

“…We had previously reported our local experience with the CliniMACS (magnetic-activated cell separation system) in hematologic malignancies and immune failure disease. There, we evaluated our CliniMACS CD34+ cell enrichment process by revealing absolute cell count and viability besides other parameters for the end products [12]. This study has two objectives: one is to determine whether the two automated techniques' results for cell count match each other, and whether the automated cell counter results for viability match those of the 7-AAD technique during the manufacturing processes of cellular therapy products intended for clinical use.…”