Donors and cells
Peripheral blood apheresis products from G-CSF mobilized healthy volunteer donors were obtained during aphereses where favourable donor-recipient weight ratio and mobilization efficiency enabled the extraction of a large excess of cells within the 300 min apheresis duration allowed per German guidelines. Two such aphereses were performed; the second one served as starting material for split runs two and three. Donor assessment was done as described [6]. Stem cell donation for these validations required donors’ written informed consent. The protocol was approved by the ethics committee of Goethe University Medical School (#468/13) and was performed in agreement with the Helsinki declaration in its current version. Aphereses were performed with Terumo BCT Spectra Optia devices as described [7, 8]. Collection targets were the same as for clinical routine, i.e. hematocrit <4 % and maximal WBC extraction at the possible expense of higher neutrophil and platelet contents in apheresis products [7, 8]. Three products containing 37, 46 or 54 × 109 total WBC and 811, 916 or 1145 × 106 total CD34+ cells were obtained. Products contained 8–16-fold more T-cells and 3–4-fold more B-cells than CD34+ target cells. Collections and selections were done in Q3 of 2015.
Immunomagnetic selection
The CliniMACS Plus and Prodigy devices [3, 5, 9, 10], CliniMACS TS and Prodigy TS310 tubing sets, CliniMACS CD34 reagent (1 vial each) and CliniMACS PBS/EDTA buffer were obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany). NaCl 0.9 %, H2O ad. inj. and human serum albumin (HSA) were from Baxter (Unterschleißheim, Germany). Both the CliniMACS Plus and Prodigy “normal scale” CD34 selection modules’ specifications are up to 60 × 109 total leukocytes or 600 × 106 CD34+ cells, and one vial of CD34 reagent (the same reagent for both devices) is used to enrich the CD34+ cells. CliniMACS Plus selections were performed according to local SOP which are identical with manufacturer-recommended protocols; the method was previously published [9]. Briefly, platelets were depleted by successive soft-spins, cells were incubated with CliniMACS CD34 Reagent (monoclonal CD34 antibody coupled to superparamagnetic nanobeads), washed to remove free antibody and then connected to a TS tubing system which was fitted on the CliniMACS Plus device. CliniMACS Plus subsequently automatically performed the column application, wash and elution steps. Prodigy selections used the same reagent; a TS310 tubing set was installed and all liquids were connected as prompted by the device. After starting the selection, the process was completely automatically guided by a release candidate version of the “LP-34 Enrichment” process for Prodigy software version 1.2.0, including both “large scale” and “normal scale” options. In view of the information gained during evaluation of the “large-scale” CD34 selection process, [4, 5] in Prodigy software version 1.2.0 “LP-34 Enrichment” “large scale” and “normal scale” processes were modified from the version used in the referenced work (1.1.4 or prior) to further reduce non-target cell trapping in the tubing system, by increasing the intensity of the washing steps of the separation column and pre-column. The “LP-34 Enrichment” process together with Prodigy software version 1.2.0 not yet being CE marked, none of the cell products were intended for clinical use.
Assessment of selection outcomes
Leukocyte concentrations in starting population, non-target and target population were determined using the Sysmex XT1800 (Norderstedt, Germany) automatic hemocytometer. Flow cytometry was performed with FACSCalibur and LSRFortessa (Becton–Dickinson, Heidelberg, Germany). Cells were stained with the following antibodies (all from BD Biosciences unless otherwise noted): anti-CD45-FITC (2D1)/anti-CD34-PE (8G12) (BD Stem Cell Reagent), anti-CD14-V450 (MφP9), anti-CD3-APC (SK7), anti-CD4-AmCyan (SK3), anti-CD8-APC-Vio770 (BW135/80, Miltenyi Biotec), anti-CD20-APC-eFluor780 (2H7, eBioscience, Frankfurt, Germany), anti-CD56-PE-Cy7 (CMSSB, eBioscience). To assess viability, 7AAD (BD Biosciences) was added to the FACS suspension buffer. IVD grade reagents were used where possible. Three platforms each were tested on apheresis product and positive fraction, i.e. the commercial single-platform SCE-kit (BD), [11] our clinical routine single-platform residual T-cell detection panel, validated to detect 1 T-cell in 10 × 103 non-T-cells with a precision of ±20 % (0.8–1.25 T-cells per 10 × 103 non-T-cells), and a second residual cell identification panel designed for extended characterization of leukocyte subsets for the purpose of research/development studies such as this one, as previously reported [4]. Unless otherwise indicated, all cell concentrations, frequencies or numbers refer to 7AAD-negative (viable) cells only. In two split-validations, colony forming activity was assessed on the final products; aliquots of cells were plated in commercially available cytokine-replete semi-solid media (MethoCult H4434, Stem Cell Technologies, Vancouver, BC) and read after 2 weeks’ incubation under standard conditions, using an inverted microscope with 2.5× magnification, as described [11].
Goals of the study
The aims of the study were to test feasibility of CD34 cell selection with the “normal scale” CD34 selection module on Prodigy and to compare type and quantity of contaminating non-target cells in Prodigy- and concurrently generated CliniMACS Plus-products. The pre-defined pass criteria for the validation exercise as outlined in the change control process was generation of three successive Prodigy products meeting the specification of an allogeneic CD34-selected product, for which our institution holds a marketing authorization. Besides being sterile and non-infectious with a panel of blood-transmissible agents, products must contain a dose of viable CD34+ cells of ≥4 × 106/kg and ≤50 × 103 T-cells/kg of the recipient (i.e. T-cell frequency cannot exceed 1.25 % of the CD34+ cell frequency). B-cell content must be measured and declared. This specification is based on a joint position paper by the German societies for Hematology/Oncology DGHO, Pediatric Hematology/Oncology GPOH and Transfusion Medicine DGTI and thus applies to all licensed allogeneic CD34 selected transplants in Germany.
Statistics
Data were entered into Excel (Microsoft, Redmond, WA) spreadsheets from which descriptive statistics were extracted. −log T-cell depletion was calculated as the negative logarithm to base 10 of number of total T-cells in the final product divided by the number of T-cells in the apheresis product [12]. Student’s t test was used to identify statistically significant differences between CliniMACS Plus and Prodigy products; significance was assumed at p < 0.05.