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Stored red blood cells (RBCs) incur biochemical and morphological changes, collectively termed the storage lesion. Functionally, the storage lesion manifests as slower oxygen unloading from RBCs, which may compromise the efficacy of transfusions where the clinical imperative is to rapidly boost oxygen delivery to tissues. Recent analysis of large real-world data linked longer storage with increased recipient mortality. Biochemical rejuvenation with a formulation of adenosine, inosine, and pyruvate can restore gas-handling properties, but its implementation is impractical for most clinical scenarios. We tested whether storage under hypoxia, previously shown to slow biochemical degradation, also preserves gas-handling properties of RBCs. A microfluidic chamber, designed to rapidly switch between oxygenated and anoxic superfusates, was used for single-cell oxygen saturation imaging on samples stored for up to 49 days. Aliquots were also analyzed flow cytometrically for side-scatter (a proposed proxy of O2 unloading kinetics), metabolomics, lipidomics, and redox proteomics. For benchmarking, units were biochemically rejuvenated at 4 weeks of standard storage. Hypoxic storage hastened O2 unloading in units stored to 35 days, an effect that correlated with side-scatter but was not linked to posttranslational modifications of hemoglobin. Although hypoxic storage and rejuvenation produced distinct biochemical changes, a subset of metabolites including pyruvate, sedoheptulose 1-phosphate, and 2/3 phospho-d-glycerate, was a common signature that correlated with changes in O2 unloading. Correlations between gas handling and lipidomic changes were modest. Thus, hypoxic storage of RBCs preserves key metabolic pathways and O2 exchange properties, thereby improving the functional quality of blood products and potentially influencing transfusion outcomes., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: The COVID19 pandemic highlights the need for contingency planning in the event of blood shortages. To increase platelet supply, we assessed the operational impact and effect on platelet quality of splitting units prior to storage., Study Design and Methods: Using production figures, we modeled the impact on unit numbers, platelet counts, and volumes of splitting only apheresis double donations into three units (yielding ⅔ doses), or all standard dose units in half. To assess quality, eight pools of three ABO/Rh-matched apheresis (Trima Accel) double donations in plasma were split to ⅔ and ½ volumes in both Terumo and Fresenius storage bags. These were irradiated and subject to maximal permitted periods of nonagitation (3 × 8 h) before comparing platelet quality markers (including pH, CD62P expression) to Day 9 of storage., Results: Splitting all double donations into three predicted inventory expansion of 23% overall whereas halving all standard dose units clearly doubles stock. In our study, ⅔ and ½ doses contained 153 ± 15 × 10 9 (~138 ml) and 113 ± 11 × 10 9 (~102 ml) platelets respectively. Following storage, higher pH was observed in ⅔ than in ½ doses and in Terumo compared to Fresenius bags. The higher pH was reflected in better quality markers, including lower CD62P expression. Despite the differences, on Day 8 (of pH monitoring at expiry) all ⅔ doses and most ½ doses were ≥pH 6.4., Conclusion: A strategy to split apheresis platelets in plasma to lower doses is feasible, maintains acceptable platelet quality, and should be considered by blood services in response to extreme shortages., (© 2021 Crown copyright. Transfusion © 2021 AABB. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.)

Background: Rejuvenation of stored red blood cells (RBCs) increases levels of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) to those of fresh cells. This study aimed to optimize and validate the US-approved process to a UK setting for manufacture and issue of rejuvenated RBCs for a multicenter randomized controlled clinical trial in cardiac surgery., Study Design and Methods: Rejuvenation of leukoreduced RBC units involved adding a solution containing pyruvate, inosine, phosphate, and adenine (Rejuvesol, Zimmer Biomet), warming at 37°C for 60 minutes, then "manual" washing with saline adenine glucose mannitol solution. A laboratory study was conducted on six pools of ABO/D-matched units made the day after donation. On Days 7, 21, and 28 of 4 ± 2°C storage, one unit per pool was rejuvenated and measured over 96 hours for volume, hematocrit, hemolysis, ATP, 2,3-DPG, supernatant potassium, lactate, and purines added (inosine) or produced (hypoxanthine) by rejuvenation. Subsequently, an operational validation (two phases of 32 units each) was undertaken, with results from the first informing a trial component specification applied to the second. Rejuvenation effects were also tested on crossmatch reactivity and RBC antigen profiles., Results: Rejuvenation raised 2,3-DPG to, and ATP above, levels of fresh cells. The final component had potassium and hemolysis values below those of standard storage Days 7 and 21, respectively, containing 1.2% exogenous inosine and 500 to 1900 μmoles/unit of hypoxanthine. The second operational validation met compliance to the trial component specification. Rejuvenation did not adversely affect crossmatch reactivity or RBC antigen profiles., Conclusion: The validated rejuvenation process operates within defined quality limits, preserving RBC immunophenotypes, enabling manufacture for clinical trials., (© 2019 Crown copyright. Transfusion © 2019 AABB.)

Mutations in NBEAL2 , the gene encoding the scaffolding protein Nbeal2, are causal of gray platelet syndrome (GPS), a rare recessive bleeding disorder characterized by platelets lacking α-granules and progressive marrow fibrosis. We present here the interactome of Nbeal2 with additional validation by reverse immunoprecipitation of Dock7, Sec16a, and Vac14 as interactors of Nbeal2. We show that GPS-causing mutations in its BEACH domain have profound and possible effects on the interaction with Dock7 and Vac14, respectively. Proximity ligation assays show that these 2 proteins are physically proximal to Nbeal2 in human megakaryocytes. In addition, we demonstrate that Nbeal2 is primarily localized in the cytoplasm and Dock7 on the membrane of or in α-granules. Interestingly, platelets from GPS cases and Nbeal2 -/- mice are almost devoid of Dock7, resulting in a profound dysregulation of its signaling pathway, leading to defective actin polymerization, platelet activation, and shape change. This study shows for the first time proteins interacting with Nbeal2 and points to the dysregulation of the canonical signaling pathway of Dock7 as a possible cause of the aberrant formation of platelets in GPS cases and Nbeal2- deficient mice., (© 2018 by The American Society of Hematology.)

Allogeneic platelet transfusions are widely used for the prevention and treatment of bleeding in thrombocytopenia. Recent evidence suggests platelet transfusions have limited efficacy and are associated with uncertain immunomodulatory risks and concerns about viral or bacterial transmission. Alternatives to transfusion are a well-recognised tenet of Patient Blood Management, but there has been less focus on different strategies to reduce bleeding risk by comparison to platelet transfusion. Direct alternatives to platelet transfusion include agents to stimulate endogenous platelet production (thrombopoietin mimetics), optimising platelet adhesion to endothelium by treating anaemia or increasing von Willebrand factor levels (desmopressin), increasing formation of cross-linked fibrinogen (activated recombinant factor VII, fibrinogen concentrate or recombinant factor XIII), decreasing fibrinolysis (tranexamic acid or epsilon aminocaproic acid) or using artificial or modified platelets (cryopreserved platelets, lyophilised platelets, haemostatic particles, liposomes, engineered nanoparticles or infusible platelet membranes). The evidence base to support the use of these alternatives is variable, but an area of active research. Much of the current randomised controlled trial focus is on evaluation of the use of thrombopoietin mimetics and anti-fibrinolytics. It is also recognised that one alternative strategy to platelet transfusion is choosing not to transfuse at all., (© 2016 John Wiley & Sons Ltd.)

A goal of platelet storage is to maintain the quality of platelets from the point of donation to the point of transfusion - to suspend the aging process. This effort is judged by clinical and laboratory measures with varying degrees of success. Recent work gives encouragement that platelets can be maintained ex vivo beyond the current 5 -7 day shelf life whilst maintaining their quality, as measured by posttransfusion recovery and survival. However, additional measures are needed to validate the development of technologies that may further reduce the aging of stored platelets, or enhance their hemostatic properties.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD., (© 2016 by The American Society of Hematology.)