Cryopreserved platelets: from in vitro thrombin generation potential to in vivo safety

Background: Cryopreservation of platelets (PLTs) at -80°C with dimethyl sulfoxide (DMSO) can extend their shelf life up to 2 years. Cryopreserved PLTs (CRY-PLTs) are reported to have a greater in vivo hemostatic effect than liquid-stored PLTs. Aims of this study were: i. to evaluate the thrombin generation potential of buffy coat derived cryopreserved PLTs (CRY- BC PLT) in comparison with fresh buffy coat derived platelets concentrates; ii. to determine the efficacy and safety of CRY-PLTs transfusion in hematological patients with severe thrombocytopenia. Materials and methods: BC PLTs were obtained from 5 buffy coats and pooled. The final PLTs concentrates were leukoreduced by filtration and transferred to a 650 mL patented cryopreservation kit (Promedical ®) which allowed mixing with DMSO 25% in a closed system and following removal of supernatant without further manipulations. BC-PLTs were washed prior freezing, suspended in homologous plasma from 1 of the 5 donors to a final concentration of 200 mL and frozen at - 80°. CRY- BC PLTs were preserved at -80°C with 6% DMSO. A system of 3 accessory bags directly connected to the mother bag was adopted for the in vitro study, to avoid repeated freezing/thawing of samples. In vitro assays were performed before freezing and at 3,6 and 9 months after thawing. Before assay, CRY-BC PLT were thawed at 37°C and diluted in plasma to adjust to 300× 109/L PLTs. Fresh BC PLTs underwent the same dilution to adjust to 300 ×109/L PLTs. Thrombin generation (TGA) was tested in CRY BC-PLTs and compared to TG potential of fresh BC PLTs. TGA was triggered by the addition of 0.5 pmol/L of recombinant human tissue factor. Endogenous thrombin potential (ETP) and peak height (PH) were determined. Flow Cytometry assays for PLTs activation markers and thromboelastography were also determined on each sample. CRY-BC PLTs, separately prepared according to the above described method for in vivo study, were infused in five hematological patients with acute leukemia (AL) and severe thrombocytopenia (PLTs Results Fourty nine BC-PLTs from 245 healthy volunteer donors (145 males and 100 females, mean age: 48.16.±18.91) were prepared, cryopreserved and analyzed up to 9 months after storage. Cryopreserved PLTs show a good thrombin generation potential that is stably maintained up to 9 months after cryopreservation [ETP (nM min): 529.25±98.64 at T0, 558.82±114.67 at T3, at 548.57±93.38 T6 and 533.04±103.15 at T9 months, respectively; PH(nM): 132.77±44.9 at T0, 103.4±44.9 at T3, 108.0±36.7 at T6 and 132.0±44.6 at T9 months, respectively]. At TGA, fresh BC-PLTs (n=35) had a mean ETP of 760.13±130.11, PH was 138.9±40.2. Thrombin generation of CRY-BC PLTs is comparable to fresh BC-PLTs, even if slightly decreased. Infusion of CRY-BC PLT (1U) was effective in controlling mucosal bleeding (epistaxis) in two patients with AL and severe thrombocytopenia. CRY-PLT were also effective when administered for prophylaxis in 3 patients with very low platelets count secondary to chemotherapy. In vivo, thrombin generation is stably maintained up to 24 hours after infusion of 1 Unit of CRY-BC PLTs, without any adverse effect (mean ETP pre-treatment was: 414.13±160.60, 24 hours after transfusion: 326.95±152.54). CRY-BC PLTs were safe and they did not determine any thrombotic event. At flow-cytometry, CRY-BC PLTs expressed higher activation markers (CD62P,CD63) than fresh BC PLTs. CRY-BC PLTs are able to significantly decrease the time to clot formation and clot strength, as measured also by thromboelastography. CRY-BCPLTs activation/deterioration is accompanied by an effective hemostatic in vivo function. Conclusions: Cryopreserved PLTs have an enhanced hemostatic activity and a good thrombin generation potential. They are effective and safe in preventing and controlling bleeding, being available in emergency/urgency settings also for patients with acute leukemia and severe thrombocytopenia. Disclosures Reina: Promedical: Consultancy.