Background: the European Federation of Immunogenetics excellence accreditation awarded to the Transfusion Medicine Complex Structure of the “SS. Antonio and Biagio e Cesare Arrigo” Hospital in the year 2000, requires all laboratories working in the field of hematopoietic stem cell transplantation to meet specific standards for guaranteeing a good level of service quality. Objectives: to evaluate the performance of the new Applied Biosystems™ SeqStudio™ Genetic Analyzer by validating the PowerPlex® 16 HS System kit (Promega Italia Srl, Milan, Italy), at the Transfusion Medicine Laboratory of the “SS. Antonio e Biagio and Cesare Arrigo” Hospital of Alessandria. Materials and Methods: three different studies were conducted: sensitivity, mixture, reproducibility, using DNA from hematology patients undergoing allogeneic hematopoietic stem cell transplantation and DNA from blood donors. Results: the sensitivity studies using the AB SeqStudio genetic analyzer proved to be more sensitive than those used previously (AB 3130), while the values obtained in the mixture studies overlapped with the expected values and the results obtained in the reproducibility studies were replicable and the same correlation exists in the tests performed on both instruments. Conclusions: the AB SeqStudio genetic analyzer through the PowerPlex® 16 HS System kit demonstrates excellent performance in accuracy and reproducibility of results and is a better performing instrument in sensitivity than the previous analyzer.
Objectives: The purpose of this work was to validate the procedure of extracorporeal photopheresis (ECP) offline after the addition of 8- MOP and UV-A (PUVA) irradiation by carrying out functional tests as recommended by the GITMO/SIdEM guidelines. There is currently no general agreement on the protocol to be adopted for the biological validation of the ECP; in fact, it is required that the test needs to be reproducible, reliable and standardisable. So, the protocol needs to be replicated in every processing laboratory.Methods: in absence of guidelines and evidence to support ECP, we have chosen to monitor cellular apoptosis induced by 8-MPO and UV-A (PUVA) irradiation, after 1 h, 48 h, and 96 h, using double marking with FITC-Annexine V and 7-AAD and consequent cytofluorimetric evaluation.Results: the functional tests utilized reproduce in vitro the 8-MOP activation effects. Our data suggest that the use of double staining with FITC-Annexine V and 7-AAD and subsequent cytofluorimetric evaluation may represent a reliable and reproducible method for ECP biological validation.Conclusions: the work allowed us to review our ECP validation procedure. We will adopt as functional test the double marking FITC- Annex V and 7-AAD and reading in cytofluorimetry at +48h post PUVA. The test needs to be performed on each enlisted patient, during the first two procedures, with each modification of the set used and at each 8-MOP batch change. We do not know what is the impact of the ECP procedure in terms of clinical efficacy or if reinfused cells are biologically inactivated or not active in the patient with GvHD. Clinical follow-up must remain the key parameter for evaluating the response to ECP treatment because even qualified therapy becomes clinically irrelevant in a patient not responding to treatment.
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