The study "Cryopreservation of Platelets for Prophylactic Use in Onco-Hematology: an in vitro Study", coordinated by Dr. Lucia Merolle at the Department of Transfusion Medicine of the Azienda USL-IRCCS of Reggio Emilia, aims to investigate the impact of cryopreservation methods on platelet functionality and metabolism. The primary objective of this project is to determine the effects of cryogenic storage on platelet function. The study design involves the recruitment of donors, preparation of platelet concentrates, and their cryopreservation. After thawing, the products undergo a series of biochemical and functional evaluations, including platelet count, pH measurement, pro-inflammatory cytokine levels, morphological assessment, and activity testing. Data will then be analyzed to determine the minimal effective concentration required for safe and effective use. Platelet apheresis samples are collected and analyzed. Participation is proposed to platelet donors at the Department of Transfusion Medicine, and inclusion requires the signing of written informed consent, while refusal to provide consent will exclude individuals from the study.

Platelets are essential blood components with pivotal roles in both hemostasis and inflammation. Thrombocytopenia, defined as a reduction in circulating platelet count, may result from impaired bone marrow production, increased consumption, splenic sequestration, or bleeding episodes, and is commonly managed through platelet transfusions aimed at restoring adequate counts and, when necessary, controlling hemorrhage. In clinical practice, the majority of platelet transfusions are administered to thrombocytopenic cancer patients with a prophylactic intent, where the primary requirement is sustained platelet survival to minimize the number of transfusions. Conversely, in actively bleeding patients, platelet transfusions are expected to provide rapid and efficient hemostasis, while their circulation time is of lesser relevance. Currently, platelet concentrates are stored at room temperature (22-24 °C) under continuous agitation for up to five days, after which they are discarded if unused. This storage method carries the risk of bacterial contamination and induces metabolic and structural changes that compromise platelet function, a phenomenon known as platelet storage lesion (PSL). Furthermore, the short shelf life limits the availability of platelets in emergency settings, as observed during the SARS-CoV-2 pandemic, when the demand for prophylactic platelet transfusions remained constant while supply was severely strained. In response, the Transfusion Medicine Unit in Reggio Emilia introduced cryopreservation of platelet concentrates to prevent future shortages. Cryopreservation allows storage of platelets for up to two years, offering an attractive alternative to conventional methods, not only extending storage duration but also reducing the risl of bacterial contamination. Although recent studies have demonstrated the safety and efficacy of cryopreserved platelets in bleeding patients, limited evidence exists for their use in prophylaxis among onco-hematologic patients. Major concerns include the potential for exacerbating thrombotic risk in patients with chronic hypercoagulability and the release of bioactive molecules such as growth factors and pro-inflammatory cytokines during thawing, which could have pro-tumorigenic effects. Platelet-tumor interactions are known to be complex, exerting both pro- and anti-tumoral influences, with the balance often favoring tumor progression. The present study therefore aims to evaluate the impact of cryopreservation on platelet functionality, structure and morphology, as well as to assess potential pro-tumoral effects after thawing. Using a comparative approach, cryopreserved platelets obtained by apheresis were analyzed alongside standard room-temperature stored platelets, currently considered the gold standard for prophylactic transfusion. The protocol involves donor recruitment at the Transfusion Medicine Unit of the AUSL-IRCCS of Reggio Emilia, with platelet concentrates prepared and cryopreserved using 6% dimethyl sulfoxide (DMSO) as well as alternative approaches employing intracellular and extracellular cryoprotectants, either individually or in combination, with lower cytotoxicity reported in other transfusion and cryobiology settings. Platelet samples were evaluated before freezing and after thawing at multiple time points (1, 3, and 6 hours post-reconstitution). Analyses included recovery rate, pH changes, release of bioactive factors, activation markers, hemostatic potential (via thromboelastography), and morphological alterations assessed by flow cytometry, microscopy, and synchrotron-based Fourier transform infrared (FTIR) microspectroscopy. Furthermore, the potential pro-tumoral activity of cryopreserved versus fresh platelets was investigated in vitro using co-culture systems with breast cancer and leukemia cell lines, focusing on proliferation, and platelet-cell interaction tests. The primary objective of this project is to determine the effects of cryogenic storage on platelet function. The secondary objective is to identify the cryopreserved platelet product with the most appropriate safety profile for prophylactic transfusion in onco-hematologic patients. The study is conducted by the BioMetLab at the Transfusion Medicine Unit of AUSL-IRCCS of Reggio Emilia in compliance with the Declaration of Helsinki, following informed consent from all donors.

Inclusion Criteria:

• Written informed consent

Exclusion Criteria:

• refusal or inability to provide written informed consent

Platelet donors