<p>There has been a renewed interest in cold-stored platelets, supported by a growing body of evidence examining their safety and quality characteristics. A protocol termed delayed cold storage, whereby platelets concentrate (PC) are stored in room temperature until near to the expiry date then transfer them to the 4⁰ C; has been suggested to facilitate the logistics of platelet units’ inventory. By delayed cold platelets storage we can make use of the outdated platelets units that will be discarded after the fourth day of storage if not requested. This study aimed to assess the metabolic and functional in-vitro changes in the delayed cold platelets protocol and compare them with room temperature platelets over 8-day period. PC was stored for 4 days under standard optimal storage conditions, named as “Room temperature stored platelet concentrate” (RT-PC). Then, it was transferred for an additional 4 days in a blood bank refrigerator (4&#xa0;°C) and named “Refrigerated platelet concentrate” (REF-PC). Before transferring the platelets to the refrigerator, samples were drawn from the RT-PC to perform the study procedures, these samples were considered the study control. Other samples were drawn from Extended-storage REF-PC (Day 8) to assess the efficacy of delayed cold platelet storage. The test procedures included in the study were measuring biochemical markers, platelet count, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipHenyl-2&#xa0;H-tetrazolium bromide), which is an assay employed to assess platelets metabolic activity. Finally, scanning electron microscopy (SEM) was used to examine the characteristics of the fibrin clots, mainly the thread thickness. Our study showed that RT-PC had double the platelet metabolic activity of Extended-storage REF-PC (Day 8), as assessed by the MTT assay. In addition, measurements of the fibrin networks using SEM examination showed that the RT-PC group had higher fibrin density, which was less porous than that of REF-PC, whereas REF-PC had thicker fibrin fibers. Biochemical examination of the RT-PC and REF-PC groups showed significant differences, highlighting the dynamic biochemical alterations occurring during delayed cold platelet storage, underscoring the potential implications for platelet function and metabolic activity. In summary, these in-vitro findings document significant biochemical and structural deterioration during delayed cold storage when Extended-storage REF-PC (Day 8) is compared with RT-PC at standard expiry (Day4). Notably, these comparisons were not between equivalent storage durations, introducing time-dependent confounding. No in vivo recovery, survival, or hemostatic efficacy data were generated in this study, and no conclusions regarding clinical benefits for prophylactic or therapeutic transfusion can be drawn from the present in-vitro data alone. The potential logistical advantage of an extended shelf life of up to 8 days could be beneficial for actively bleeding patients, simplify logistics for blood banks, and would reduce wastage.</p>