Biotin labeling allows for post-transfusion functional assessment of stored human platelets in mice.

BACKGROUND: Platelet radiolabeling with radioisotopes is currently used for human platelet recovery and survival studies. Biotinylation enables ex vivo post-transfusion platelet function testing. Whether platelet biotinylation itself affects platelet function is controversial. STUDY DESIGN AND METHODS: Platelet concentrates from healthy humans were stored for 6 days. Samples were obtained at 1 or 2 and 6 days, and platelets were labeled following a radiolabeling protocol using saline instead of radioactive indium-111 (sham radiolabeling [sham-RL]). Alternatively, a newly developed biotinylation protocol, a washing protocol, or an unmanipulated control sample were used. Platelet function was assessed by flow cytometry after stimulation with platelet agonists and labeling of platelets with platelet activation markers. To test whether platelets can be activated after transfusion, labeled platelets were transfused into nonobese diabetic/severe combined immunodeficiency mice, and samples were obtained 1 h after transfusion. RESULTS: The activation profile of biotinylated platelets was comparable to sham-RL platelets before transfusion except for significantly less α-degranulation and more phosphatidyl serine exposure on storage day 1/2. There was no significant difference between sham-RL and biotinylated platelets on storage day 6. Sham-RL and biotinylated platelets were significantly less activatable than washed and unmanipulated control platelets. After transfusion, the activation profile of biotinylated platelets was largely indistinguishable from unmanipulated ones. DISCUSSION: The decrease in activation level in biotinylated platelets we and others observed appears mainly due to the physical manipulation during the labeling process. In conclusion, biotinylated platelets allow for post-transfusion function assessment, a major advantage over radiolabeling.

Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

Quality assessment of red blood cell concentrates from blood donors at the extremes of the age spectrum: The BEST collaborative study.

BACKGROUND: Blood donors at the extremes of the age spectrum (16-19 years vs. ≥75 years) are characterized by increased risks of iron deficiency and anemia, and are often underrepresented in studies evaluating the effects of donor characteristics on red blood cells (RBC) transfusion effectiveness. The aim of this study was to conduct quality assessments of RBC concentrates from these unique age groups. STUDY DESIGN: We characterized 150 leukocyte-reduced (LR)-RBCs units from 75 teenage donors, who were matched by sex, and ethnicity with 75 older donors. LR-RBC units were manufactured at three large blood collection centers in the USA and Canada. Quality assessments included storage hemolysis, osmotic hemolysis, oxidative hemolysis, osmotic gradient ektacytometry, hematological indices, and RBC bioactivity. RESULTS: RBC concentrates from teenage donors had smaller (9%) mean corpuscular volume and higher (5%) RBC concentration compared with older donors counterparts. Stored RBCs from teenage donors exhibited increased susceptibility to oxidative hemolysis (>2-fold) compared with RBCs from older donors. This was observed at all testing centers independent of sex, storage duration, or the type of additive solution. RBCs from teenage male donors had increased cytoplasmatic viscosity and lower hydration compared with older donor RBCs. Evaluations of RBC supernatant bioactivity suggested that donor age was not associated with altered expression of inflammatory markers (CD31, CD54, and IL-6) on endothelial cells. CONCLUSIONS: The reported findings are likely intrinsic to RBCs and reflect age-specific changes in RBC antioxidant capacity and physical characteristics that may impact RBC survival during cold storage and after transfusion.

An assessment of the safety, hemostatic efficacy, and clinical impact of low-titer group O whole blood in children and adolescents.

BACKGROUND: Low-titer group O whole blood (LTOWB) use has been associated with improved survival and less blood transfusions in adult trauma patients. Its use in pediatric trauma has been shown to be safe when using leukoreduced, LTOWB with anti-A, anti-B antibody titers of <1:50. We set out to evaluate the safety, hemostatic potential, and impact on pediatric outcomes at a center using non-leukoreduced, LTOWB with anti-A, anti-B antibody titers of <1:200. METHODS: Patients younger than 18 years, who received emergency-release, uncrossed matched blood, and presented to our trauma center from November 2017 to April 2021 were included. Patients were divided into those receiving any LTOWB and those receiving only RBC and or plasma (COMP). Primary outcome was 30-day survival. RESULTS: One hundred sixty-four patients received emergency release blood products. Of these, 73 received at least one unit of LTOWB. The LTOWB group were younger (14 years vs. 13 years), more likely to be male (87% vs. 49%), and to have sustained penetrating trauma (44% vs. 23%); all p < 0.05. Low-titer group O whole blood patients received more blood than their COMP counterparts prior to arrival. Serial hemolysis panels (K+, bilirubin, LDH, haptoglobin) obtained at 24 hours, 48 hours, and 72 hours were similar between groups; all p > 0.05. There was no difference in survival by univariate analysis but after adjusting for inverse probability of treatment weights there was an observed association between WB administration and improved survival, with an odds ratio of 2.48 (1.15-5.47). CONCLUSION: Non-leukoreduced, LTOWB in anti-A/anti-B antibody titers of <1:200 appear safe in children and adolescents. While patients receiving LTOWB had more evidence of shock, higher torso injury severity, and received more prehospital blood products, there may be a mortality benefit with whole blood. Larger, multicenter studies are needed. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

Assessment of a multisite standardized biospecimen collection protocol for immune phenotyping in neurodevelopmental disorders.

Multisite collection and preservation of peripheral blood mononuclear cells (PBMCs) for centralized analysis is an indispensable strategy for large cohort immune phenotyping studies. However, the absence of cross-site standardized protocols introduces unnecessary sample variance. Here we describe the protocol implemented by the Province of Ontario Neurodevelopmental Disorders (POND) Network's immune platform for the multisite collection, processing, and cryopreservation of PBMCs. We outline quality control standards and evaluate the performance of our PBMC processing and storage protocol. We also describe the Child Immune History Questionnaire results, an assessment tool evaluating pre-existing immune conditions in children with neurodevelopmental disorders (NDDs). Cell viability was assessed in samples from 178 participants based on strict quality control criteria. Overall, 83.1% of samples passed quality control standards. Samples collected and processed at the same site had higher quality control pass rates than samples that were collected and subsequently shipped to another site for processing. We investigated if freezer time impacted sample viability and found no difference in mean freezer time between samples that passed and failed quality control. The Child Immune History Questionnaire had a response rate of 87.1%. The described protocol produces viable samples that may be used in future immune phenotyping experiments.

Assessment of the soluble proteins HMGB1, CD40L and CD62P during various platelet preparation processes and the storage of platelet concentrates: The BEST collaborative study.

BACKGROUND: Structural and biochemical changes in stored platelets are influenced by collection and processing methods. This international study investigates the effects of platelet (PLT) processing and storage conditions on HMGB1, sCD40L, and sCD62P protein levels in platelet concentrate supernatants (PCs). STUDY DESIGN/METHODS: PC supernatants (n = 3748) were collected by each international centre using identical centrifugation methods (n = 9) and tested centrally using the ELISA/Luminex platform. Apheresis versus the buffy coat (BC-PC) method, plasma storage versus PAS and RT storage versus cold (4°C) were investigated. We focused on PC preparation collecting samples during early (RT: day 1-3; cold: day 1-5) and late (RT: day 4-7; cold: day 7-10) storage time points. RESULTS: HMGB1, sCD40L, and sCD62P concentrations were similar during early storage periods, regardless of storage solution (BC-PC plasma and BC-PC PAS-E) or temperature. During storage and without PAS, sCD40L and CD62P in BC-PC supernatants increased significantly (+33% and +41%, respectively) depending on storage temperature (22 vs. 4°C). However, without PAS-E, levels decreased significantly (-31% and -20%, respectively), depending on storage temperature (22 vs. 4°C). Contrastingly, the processing method appeared to have greater impact on HMGB1 release versus storage duration. These data highlight increases in these parameters during storage and differences between preparation methods and storage temperatures. CONCLUSIONS: The HMGB1 release mechanism/intracellular pathways appear to differ from sCD62P and sCD40L. The extent to which these differences affect patient outcomes, particularly post-transfusion platelet increment and adverse events, warrants further investigation in clinical trials with various therapeutic indications.

Long-term whole blood DNA preservation by cost-efficient cryosilicification.

Deoxyribonucleic acid (DNA) is the blueprint of life, and cost-effective methods for its long-term storage could have many potential benefits to society. Here we present the method of in situ cryosilicification of whole blood cells, which allows long-term preservation of DNA. Importantly, our straightforward approach is inexpensive, reliable, and yields cryosilicified samples that fulfill the essential criteria for safe, long-term DNA preservation, namely robustness against external stressors, such as radical oxygen species or ultraviolet radiation, and long-term stability in humid conditions at elevated temperatures. Our approach could enable the room temperature storage of genomic information in book-size format for more than one thousand years (thermally equivalent), costing only 0.5 $/person. Additionally, our demonstration of 3D-printed DNA banking artefacts, could potentially allow 'artificial fossilization'.

Assessment of bacterial growth in leukoreduced cold-stored whole blood supports overnight hold at room temperature prior to filtration: A pilot study.

BACKGROUND AND OBJECTIVES: Whole blood (WB) transfusion has regained attention to treat trauma patients. We reported no significant changes in in vitro quality through 21 days of cold storage for leukoreduced WB (LCWB) when time to filtration was extended from 8 to 24 h from collection. This study evaluated the impact of extended WB-hold at room temperature (RT) prior to leukoreduction on proliferation of transfusion-relevant bacteria. MATERIALS AND METHODS: WB units were spiked with suspensions of Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Listeria monocytogenes prepared in saline solution (SS) or trypticase soy broth (TSB) to a concentration of ~0.2 CFU/ml (N = 6). Spiked units were held at RT for 18-24 h before leukoreduction and cold-stored for 21 days. Bacterial growth was determined on days 2, 7, 14 and 21. In vitro quality of WB inoculated with unspiked diluents was assessed. RESULTS: K. pneumoniae and S. pyogenes proliferated in WB prior to leukoreduction reaching concentrations ≤102 CFU/ml. These bacteria, however, did not proliferate during the subsequent cold storage. S. aureus did not survive in WB while L. monocytogenes reached a concentration of ~102 CFU/ml by day 21. LCWB in vitro quality was not affected by SS or TSB. CONCLUSION: Extended WB-hold prior to leukoreduction allowed proliferation of bacteria able to resist immune clearance, although they did not grow to clinically significant levels. While L. monocytogenes proliferated in LCWB, clinically relevant concentrations were not reached by day 21. These data suggest that transfusing LCWB may not pose a significant bacterial contamination safety risk to transfusion patients.

Assessment of apheresis platelets during 5 days of storage: A National Cancer Institute, Cairo University experience.

BACKGROUND: Platelet transfusion therapy is widely used to prevent hemorrhage in patients with thrombocytopenia and platelet disorders. The platelet concentrate (PC) quality is affected by increased storage time, as reflected in the decreased number of platelets, morphological changes, and impaired functions. This study aimed to analyze the impact of 5 days storage on platelets count and the expression of CD63, and Annexin V as activation markers during PC storage. METHODS: Fifty PCs collected from single donors were tested for platelet count on days 0, 3, and 5 using a Sysmex blood counter. CD61, CD63, and Annexin V expression was analyzed by a multicolor Navios flow cytometer. RESULTS: There was a significant decrease in platelet count during 5 days of storage. There was a direct relationship between storage time and degree of platelet activation. CD63 had almost double increased expression on day 5 than day 3. Annexin V showed significantly increased expression on day 3 with minor differences between days 3 and 5. CONCLUSION: According to standard blood bank conditions, PC stored for 5 days showed a degree of in vitro activation as evidenced by CD63 and Annexin V expression, may lead to reduced therapeutic efficacy. Flow cytometry monitoring platelet activation in PC offers a better understanding of the changes during PC storage and may help improve platelet products.

Quantitative assessment of infusion pump-mediated haemolysis in feline packed red blood cell transfusions.

OBJECTIVES: Haemolysis caused by the use of peristaltic infusion pumps (PIPs) has been described in human and canine packed red blood cells (pRBCs). The aim of this study was to evaluate the effects of two different linear PIPs on the haemolysis of feline pRBC units stored for a long time. METHODS: Feline pRBC units stored with adenine, dextrose, mannitol and sodium chloride (SAGM) were manufactured. After 35-42 days of storage at 2-4°C, a line administration system with a 180 µm filter was attached to every pRBC bag, the system was drained by gravity alone (8 drops/min) and a 1.3 ml sample was collected (G). A NIKI V4 pump was then used at a flow rate of 25 ml/h, the flow was stopped when the infusion system was filled with blood coming from the infusion pump and another 1.3 ml sample was collected (NK). Finally, an Infusomat FmS pump was evaluated, collecting another 1.3 ml sample (IM). Packed cell volume (PCV) was measured in all samples by microhaematocrit centrifugation, total haemoglobin (HGB) was measured using a specific haemoglobin analyser and, after centrifugation, free HGB was determined by spectrophotometry. The percentage of haemolysis was calculated. Friedman's test was used to compare the samples. RESULTS: Fifteen feline pRBC units were evaluated. The average degree of haemolysis for sample G (gravity-assisted) was 1.12%. Comparison of the degree of gravity-assisted haemolysis with haemolysis in PIP NK (1.13%) and IM (1.14%) samples revealed no significant differences, with differences of only 0.01% and 0.02%, respectively. CONCLUSIONS AND RELEVANCE: The results of this study demonstrate that the use of two common PIPs in veterinary hospitals does not produce levels of haemolysis that are significantly different than that caused by gravity alone during transfusion of feline pRBCs at a rate of 25 ml/h.

Cord blood beyond transplantation: can we use the experience to advance all cell therapies?

Unrelated cord blood (CB) units, already manufactured, fully tested and stored, are high-quality products for haematopoietic stem cell transplantation and cell therapies, as well as an optimal starting material for cell expansion, cell engineering or cell re-programming technologies. CB banks have been pioneers in the development and implementation of Current Good Manufacturing Practices for cell-therapy products. Sharing their technological and regulatory experience will help advance all cell therapies, CB-derived or not, particularly as they transition from autologous, individually manufactured products to stored, 'off-the shelf' treatments. Such strategies will allow broader patient access and wide product utilisation.

Maturity Assessment model for Patient Blood Management to assist hospitals in improving patients' safety and outcomes. The MAPBM project.

BACKGROUND: Patient blood management (PBM) is an evidence-based care bundle with proven ability to improve patients' outcomes by managing and preserving the patient's own blood. Since 2010, the World Health Organisation has urged member states to implement PBM. However, there has been limited progress in developing PBM programmes in hospitals due to the implicit challenges of implementing them. To address these challenges, we developed a Maturity Assessment Model (MAPBM) to assist healthcare organisations to measure, benchmark, assess in PBM, and communicate the results of their PBM programmes. We describe the MAPBM model, its benchmarking programme, and the feasibility of implementing it nationwide in Spain. MATERIALS AND METHODS: The MAPBM considers the three dimensions of a transformation effort (structure, process and outcomes) and grades these within a maturity scale matrix. Each dimension includes the various drivers of a PBM programme, and their corresponding measures and key performance indicators. The structure measures are qualitative, and obtained using a survey and structured self-assessment checklist. The key performance indicators for process and outcomes are quantitative, and based on clinical data from the hospitals' electronic medical records. Key performance indicators for process address major clinical recommendations in each PBM pillar, and are applied to six common procedures characterised by significant blood loss. RESULTS: In its first 5 years, the MAPBM was deployed in 59 hospitals and used to analyse 181,826 hospital episodes, which proves the feasibility of implementing a sustainable model to measure and compare PBM clinical practice and outcomes across hospitals in Spain. CONCLUSION: The MAPBM initiative aims to become a useful tool for healthcare organisations to implement PBM programmes and improve patients' safety and outcomes.

The current state of the platelet supply in the US and proposed options to decrease the risk of critical shortages.

Due to circumstances such as increased demand and an aging donor pool, the likelihood of critical platelet shortages is increasing. The platelet supply could be improved through the expansion of the donor pool, the identification and sustained utilization of high-quality donors, and changes in component processing and storage that result in a longer platelet shelf-life. Refrigerated platelets, stored at 1° to 6°C, have the potential to improve patient safety by decreasing the risk of bacterial contamination while concurrently allowing for a longer storage period (eg, 14 days) and improved hemostatic effectiveness in actively bleeding patients. An approach utilizing remuneration of apheresis platelet donors combined with pathogen reduction of the platelet components could be used as a means to increase the donor pool and identify and sustain safe, reliable, high-quality donors. Remuneration might provide an incentive for underutilized populations (eg, individuals <30 years old) to enter the apheresis platelet donor population resulting in a significant expansion of the platelet donor pool. Over time, approaches such as the use of refrigerated platelets, platelet donor remuneration, and the application of pathogen reduction technology, might serve to attract a large, reliable, and safe donor base that provides platelet collections with high yields, longer shelf-lives and, excellent hemostatic function.

Quality assessment of red blood cell suspensions derived from pathogen-reduced whole blood.

BACKGROUND: We used laboratory indicators to evaluate the quality of pathogen-reduced red blood cell suspension (RBCS) compared with gamma-irradiated RBCS. MATERIALS AND METHODS: To determine biochemical and metabolic parameters of RBCS, we obtained 50 whole blood units from healthy volunteers and randomized them into 2 groups: 25 were pathogen-reduced, and then, RBCS prepared from them. RBCS from the other 25 was gamma-irradiated. Sampling was carried out on day zero before and after treatment and at 7, 14, 21 and 28 days. To determine lymphocyte inactivation, we collected another 35 whole blood units. Each was sampled to form 3 study groups: untreated, gamma-irradiated and pathogen-reduced. Daily sampling was carried out during 3 days of storage. RESULTS: The quality of RBCS from both groups was largely the same, except for haemolysis and red blood cell fragility, which were more pronounced in the pathogen-reduced group. This finding limited the shelf life of pathogen-reduced RBCS to 14 days. Lymphocyte viability was significantly reduced after both treatments. Proliferation of lymphocytes after pathogen reduction was reduced to the detection limit, while low-level proliferation was observed in gamma-irradiated samples. CONCLUSION: Pathogen-reduced red blood cells have acceptable quality and can be used for transfusion within 14 days. Results of inactivation of lymphocytes demonstrate that pathogen reduction technology, applied on WB, can serve as an alternative to irradiation.

Longitudinal assessment and stability of long non-coding RNA gene expression profiles measured in human peripheral whole blood collected into PAXgene blood RNA tubes.

OBJECTIVE: Long non-coding RNAs (lncRNAs) are emerging as novel biomarkers for a variety of chronic conditions including autoimmune disease. PAXgene Blood RNA tubes are routinely used in clinical research and molecular diagnostic development to capture RNA profiles in peripheral whole blood. While the stability of mRNA expression profiles captured using PAXgene tubes has been documented previously, no previous work has determined the stability of lncRNA expression profiles observed in PAXgene tubes stored at - 80 °C. Here we sought to determine the effects on lncRNA expression profiles following - 80 °C storage of total RNA templates, cDNA synthesized using fresh or frozen total RNA template, and the impact of freeze-thaw cycles on both total RNA and cDNA obtained from PAXgene tubes. RESULTS: We find that storage of whole blood in PAXgene tubes, total RNA and cDNA for up to 1 year at - 80 °C or up to ten total RNA or cDNA freeze-thaw cycles do not significantly alter lncRNA expression profiles compared to baseline. As monthly expression profiles were determined, some month to month lncRNA expression variability was observed. However, all monthly observations fell within the 95% confidence interval calculated at baseline.

Stochastic Inventory Model for Minimizing Blood Shortage and Outdating in a Blood Supply Chain under Supply and Demand Uncertainty.

Purpose: Blood, like fresh produce, is a perishable element, with platelets having a limited lifetime of five days and red blood cells lasting 42 days. To manage the blood supply chain more effectively under demand and supply uncertainty, it is of considerable importance to developing a practical blood supply chain model. This paper proposed an essential blood supply chain model under demand and supply uncertainty. Methods: This study focused on how to manage the blood supply chain under demand and supply uncertainty effectively. A stochastic mixed-integer linear programming (MILP) model for the blood supply chain is proposed. Furthermore, this study conducted a sensitivity analysis to examine the impacts of the coefficient of demand and supply variation and the cost parameters on the average total cost and the performance measures (units of shortage, outdated units, inventory holding units, and purchased units) for both the blood center and hospitals. Results: Based on the results, the hospitals and the blood center can choose the optimal ordering policy that works best for them. From the results, we observed that when the coefficient of demand and supply variation is increased, the expected supply chain cost increased with more outdating units, shortages units, and holding units due to the impacts of supply and demand fluctuation. Variation in the inventory holding and expiration costs has an insignificant effect on the total cost. Conclusions: The model developed in this paper can assist managers and pathologists at the blood donation centers and hospitals to determine the most efficient inventory policy with a minimum cost based on the uncertainty of blood supply and demand. The model also performs as a decision support system to help health care professionals manage and control blood inventory more effectively under blood supply and demand uncertainty, thus reducing shortage of blood and expired wastage of blood.

Pathogen reduction of double-dose platelet concentrates from pools of eight buffy coats: Product quality, safety, and economic aspects.

BACKGROUND: Pathogen reduction (PR) of platelet concentrates (PCs) contributes to the safety of platelet (PLT) transfusion by reducing the risk of transfusion-transmitted infections and transfusion-associated graft-versus-host disease. In vitro quality of pathogen-reduced double-dose PC (PR-PC) made of eight whole blood (WB)-derived buffy coats (BCs) were evaluated. METHODS: Eight small-volume WB BCs from donors with at least 200 × 109 PLT/L were pooled with an additive solution to produce double-dose PCs (DD-PCs), which were treated with amotosalen/ultraviolet A light in a dual storage processing set, yielding 2 units of PR-PC. Quality controls were undertaken as per European Directive for the Quality of Medicines (EDQM) guidelines. PLT recovery rates were measured. Production costs and savings were compared over the 3 years before and after PR implementation. RESULTS: In the pre-PR period, 19 666 PCs were produced, compared to 17 307 PCs in the PR period. Single BC in the PR period had 41 ± 2 mL, hematocrit 0.39 ± 0.04 and 1.06 ± 0.18 × 1011 PLTs, and showed a recovery of 91% ± 8%. After pooling, separation, PR treatment of DD-PC, and splitting, each single PC had 189 ± 6 mL with 2.52 ± 0.34 × 1011 PLTs, compared to 2.48 ± 0.40 in the pre-PR period. The PLT recovery rate after PR was 87% ± 14%. EDQM requirements were met. An increase of about €12 (+7.5%) per PC from the pre-PR to the PR period was identified. CONCLUSION: A new production method resulting in two PR-PCs made from pools of 8 BCs with use of one PR set was successfully introduced, and our experience of nearly 3 years demonstrated the high efficacy and in vitro quality of the PR-PCs obtained.

Effects of the COVID-19 pandemic on supply and use of blood for transfusion.

The COVID-19 pandemic has major implications for blood transfusion. There are uncertain patterns of demand, and transfusion institutions need to plan for reductions in donations and loss of crucial staff because of sickness and public health restrictions. We systematically searched for relevant studies addressing the transfusion chain-from donor, through collection and processing, to patients-to provide a synthesis of the published literature and guidance during times of potential or actual shortage. A reduction in donor numbers has largely been matched by reductions in demand for transfusion. Contingency planning includes prioritisation policies for patients in the event of predicted shortage. A range of strategies maintain ongoing equitable access to blood for transfusion during the pandemic, in addition to providing new therapies such as convalescent plasma. Sharing experience and developing expert consensus on the basis of evolving publications will help transfusion services and hospitals in countries at different stages in the pandemic.