A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product.

BACKGROUND: Manufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. MATERIALS AND METHODS: The study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. RESULTS: MycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. CONCLUSIONS: MycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

Extracorporeal photopheresis as an immunomodulatory agent: Haematocrit-dependent effects on natural killer cells.

The GvHD is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) represents an alternative therapeutic strategy to immunosuppressive therapy. Although ECP is used since 1990s, the mechanism of action has not yet been completely clarified. We analyzed cells collected from 20 ECP procedures of 4 patients affected by chronic GvHD and, for comparison, Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy donors undergoing from same type of photochemiotherapy, evaluating by flow cytometry, the effects before and after photoactivation with 8-MOP. The analysis showed a significant increase in cell death after ECP in particular in CD4 T lymphocytes as described in literature correlated with haematocrit value. Most interesting data emerge from the analysis of cytotoxic activity of NK cells, using flow cytometry analysis of surface expression of CD107a in the presence of target cells (K562). In all analyzed samples it was possible to document a statistically significant reduction of the cytotoxic activity of NK cells after photoactivation. The decrease of the cytotoxic activity was related to hematocrit value of leukoapheresis: in fact, lower HCT values were associated with a more marked reduction of cytotoxic activity. The study confirms literature data about the increase of cellular mortality induce by ECP. Furthermore, for the first time it is demonstrated that the ECP exerts a marked and significant inhibitory effect on the cytotoxic activity of NK cells. Our study suggests that lower values of hematocrit are associated with better treatment outcome.

Leukapheresis for autologous stem cell transplantation: Comparative study of two different thawing methods WSCFD(®) Stem Cell Fast Thawer KW versus 37 °C thermostatic bath.

BACKGROUND: Leukapheresis for autologous stem cell transplantation represents an efficient technique for the reconstitution of haematopoietic system in patients subjected to a high-dose chemotherapy for the treatment of haematological malignancies. The current regulations emphasise first steps of leukapheresis procedure but do not recommend methods for thawing, only suggesting that it must be performed as soon as possible in a 37 °C thermostatic bath. AIM OF THE STUDY: We compared the classic method of thawing with an innovative and fully traceable method that uses WSCFD(®) Stem Cell Fast Thawer KW. MATERIALS AND METHODS: The first part of the study was focused on the thermodynamic process of the two methods, thawing 6 "simulated" leukapheresis (buffy coats of healthy donors cryopreserved with saline solution, 5% HSA and 5% DMSO) and analysing the thawing curve obtained, by using an inside probe. In the second part, we focused on the recovery of viable CD34+ cells and leukocytes, thawing 20 real leukapheresis from paediatric patients. In this phase we also analyse final core bag temperature, time of procedure, cellular recovery with ISHAGE single platform flow cytometry assay and clonogenic potential performing a CFU assay. RESULTS: We found no significant differences between the two methods, both for thermodynamic aspect and cellular recovery. Thawing curves were similar and the paired Student's-t test used for statistical analysis showed a CD34+ cells recovery of 92.2% ± 11.4 using WSCFD(®) versus 90% ± 11.1 of thermostatic bath. Data were similar even for leukocytes recovery (80.8% ± 9.5 with WSCFD(®) and 79.2% ± 14.4 with thermostatic bath). All thawed products never exceeded the core temperature of 30 °C and no differences were found about the post-thaw clonogenic potential (614 × 10(4) ± 98.3 total CFU using WSCFD(®) versus 592 × 10(4) ± 78.5 using thermostatic bath). The only difference observed was about the thawing time: WSCFD method requires a slightly longer time but, on the other hand, it correlates with reduced mean increase in temperature per minute, as a result of a more linear thawing curve. CONCLUSIONS: WSCFD(®) can replace the 37 °C thermostatic bath thawing procedure for leukapheresis, providing more security and fully traceable process data.

Risk analysis of hematopoietic stem cell transplant process: failure mode, effect, and criticality analysis and hazard analysis critical control point methods integration based on guidelines to good manufacturing practice for medicinal product ANNEX 20 (February 2008).

The collection and handling of hematopoietic stem cells (HSCs) must meet high quality requirements. An integrated Quality Risk Management can help to identify and contain potential risks related to HSC production. Risk analysis techniques allow one to "weigh" identified hazards, considering the seriousness of their effects, frequency, and detectability, seeking to prevent the most harmful hazards. The Hazard Analysis Critical Point, recognized as the most appropriate technique to identify risks associated with physical, chemical, and biological hazards for cellular products, consists of classifying finished product specifications and limits of acceptability, identifying all off-specifications, defining activities that can cause them, and finally establishing both a monitoring system for each Critical Control Point and corrective actions for deviations. The severity of possible effects on patients, as well as the occurrence and detectability of critical parameters, are measured on quantitative scales (Risk Priority Number [RPN]). Risk analysis was performed with this technique on manipulation process of HPC performed at our blood center. The data analysis showed that hazards with higher values of RPN with greater impact on the process are loss of dose and tracking; technical skills of operators and manual transcription of data were the most critical parameters. Problems related to operator skills are handled by defining targeted training programs, while other critical parameters can be mitigated with the use of continuous control systems. The blood center management software was completed by a labeling system with forms designed to be in compliance with standards in force and by starting implementation of a cryopreservation management module.

Key role of staff competencies for patient and donor safety in a bone marrow transplantation unit: design and implementation of an accredited training and self-assessment program.

Human resources represent at the moment the most critical factor in an hospital setting characterized by a high rate of staff turnover. It is important to ensure a consistent level of expertise and knowledge of professionals who work in health care facilities to provide quality services and simultaneously support the implementation of strategies for patient safety. Unfortunately, the development of effective interventions for training newly added staff and self-evaluation of skills possessed by trained staff are closely related to understanding critical aspects of the organization. At the new Center for Bone Marrow Transplantation and Blood Transfusion Service in Meyer Hospital, during the last year, a group of professional nurses and technicians completed a specific plan to train new staff and, at the same time, a program of self-assessment of skills for experienced staff. The main purpose of this project was to promote skills development by newly added as well as experienced staff, to identify areas of weaknesses, and to correct them with training (organized by the hospital, departmental, or individual) designed to improve performance.