Processing methods and storage duration impact extracellular vesicle counts in red blood cell units.

Extracellular vesicles (EVs) are active components of red blood cell (RBC) concentrates and may be associated with beneficial and adverse effects of transfusion. Elucidating controllable factors associated with EV release in RBC products is thus important to better manage the quality and properties of RBC units. Erythrocyte-derived EVs (EEVs) and platelet-derived EVs (PEVs) were counted in 1226 RBC units (administered to 280 patients) using a standardized cytometry-based method. EV size and CD47 and annexin V expression were also measured. The effects of donor characteristics, processing methods, and storage duration on EV counts were analyzed by using standard comparison tests, and analysis of covariance was used to determine factors independently associated with EV counts. PEV as well as EEV counts were higher in whole-blood-filtered RBC units compared with RBC-filtered units; PEV counts were associated with filter type (higher with filters associated with higher residual platelets), and CD47 expression was higher on EEVs in RBC units stored longer. Multivariate analysis showed that EEV counts were strongly associated with filter type (P < .0001), preparation, and storage time (+25.4 EEV/µL per day [P = .01] and +42.4 EEV/µL per day [P < .0001], respectively). The only independent factor associated with PEV counts was the residual platelet count in the unit (+67.1 PEV/µL; P < .0001). Overall, processing methods have an impact on EV counts and characteristics, leading to large variations in EV quantities transfused into patients. RBC unit processing methods might be standardized to control the EV content of RBC units if any impacts on patient outcomes can be confirmed. The IMIB (Impact of Microparticles in Blood) study is ancillary to the French ABLE (Age of Transfused Blood in Critically Ill Adults) trial (ISRCTN44878718).

How to quantify microparticles in RBCs? A validated flow cytometry method allows the detection of an increase in microparticles during storage.

BACKGROUND: The procoagulant and proinflammatory microparticles (MPs) released during storage of packed red blood cells (pRBCs) can potentially modify transfusion benefits. A robust method to quantify MPs in pRBCs is needed to evaluate their impact in clinical trials. STUDY DESIGN AND METHODS: The objective was to validate the preanalytic conditions required to prepare pRBC supernatant as well as a method to quantify and evaluate MP variations over 42 days of pRBC storage.A flow cytometry method with size-calibrated beads was developed and fully validated. Quantification of MPs in pRBCs (n = 109) was assessed during short-term (7 days) and long-term (42 days) storage at 4°C, during short-term storage (8 hours) at room temperature, and after 2 years frozen. RESULTS: Repeatability, reproducibility, and linearity of the quantification method were validated, and variations during conservation are presented. There was high variability in RBC (erythrocyte) MP (ERMP) and platelet MP (PMP) levels between RBC units, depending on the filter used for leukocyte reduction. During the 42 days of storage at 4°C, significant increases in ERMPs and PMPs occurred (from 58 to 138 ERMPs/µL from Day 2 to Day 42; p = 0.0002; and from 326 to 771 PMPs/µL from Day 2 to Day 42; p = 0.00026). CONCLUSION: We use a robust method to confirm that ERMPs and PMPs are present to various degrees in pRBCs and that storage for 42 days significantly increases their generation. This method is robust enough to allow MP quantification in pRBCs and is adapted to evaluate the clinical impact of transfused MPs in prospective clinical trials.

Isolation of viable human hepatic progenitors from adult livers is possible even after 48 hours of cold ischemia.

Liver transplantation, utilized routinely for end-stage liver disease, has been constrained by the paucity of organ donors, and is being complemented by alternative strategies such as liver cell transplantation. One of the most promising forms of liver cell transplantation is hepatic stem cell therapies, as the number of human hepatic stem cells (hHpSCs) and other early hepatic progenitor cells (HPCs) are sufficient to provide treatment for multiple patients from a single liver source. In the present study, human adult livers were exposed to cold ischemia and then processed after <24 or 48 h. Cells positive for epithelial cell adhesion molecule (EpCAM), a marker on early lineage stage HPCs, were immunoselected and counted. Approximately 100,000 EpCAM(+) cells/gram of tissue was obtained from surgical resection of livers subjected to cold ischemia up to 24 h and comparable numbers, albeit somewhat lower, were obtained from those exposed to 48 h of cold ischemia. The yields are similar to those reported from livers with minimal exposure to ischemia. When cultured on plastic dishes and in Kubota's Medium, a serum-free medium designed for early lineage stage HPCs, colonies of rapidly expanding cells formed. They were confirmed to be probable hHpSCs by their ability to survive and expand on plastic and in Kubota's Medium for months, by co-expression of EpCAM and neural cell adhesion molecule, minimal if any albumin expression, with EpCAM found throughout the cells, and no expression of alpha-fetoprotein. The yields of viable EpCAM(+) cells were surprisingly large, and the numbers from a single donor liver are sufficient to treat approximately 50-100 patients given the numbers of EpCAM(+) cells currently used in hepatic stem cell therapies. Thus, cold ischemic livers for up to 48 h are a new source of cells that might be used for liver cell therapies.