INTRODUCTION:The immunomodulatory consequences of blood transfusion, known as transfusion-related immune modulation (TRIM), are often not captured by hemovigilance systems. Changes to blood product manufacturing processes may impact patient outcomes. DESIGN AND METHODS:We conducted a retrospective study of hospitalized adults (≥18 years) in Hamilton, ON, who received ≥1 red blood cell (RBC) transfusion(s) between 2010 and 2014. Primary outcome was in hospital mortality; TRIM outcomes included respiratory failure, organ dysfunction, and sepsis. We evaluated outcomes before and after the change made by Canadian Blood Services (2012) to consolidate manufacturing of blood products in Ontario. Exclusions included autologous, washed, or deglycerolized RBC transfusions, RBCs manufactured outside select regional sites, or patients who received both pre-/post-consolidation RBCs. Data was obtained from the TRUST database. Logistic regression adjusted for key covariates. RESULTS:A total of 9871 pre- and 7871 post-consolidation patients met inclusion criteria. Multivariate analysis demonstrated no change in in-hospital mortality (odds ratio [OR] 1.00, 95% confidence interval [CI] 0.89-1.14, p = 0.95), respiratory failure (OR 0.83, CI 0.65-1.06, p = 0.14) or organ dysfunction (OR 0.95, 95% CI 0.84-1.08, p = 0.42) comparing post to pre-consolidation. However, hospital-onset sepsis was lower post-consolidation (OR 0.59, 95% CI 0.48-0.73, p < 0.001). CONCLUSIONS:Consolidation of blood production in Ontario was not associated with changes in rates of in-hospital mortality, respiratory failure, or organ dysfunction among transfusion recipients, but may be associated with a lower risk of sepsis. TRIM and the clinical impacts of changes to blood processing require further study.

Red blood cell (RBC) transfusion is a critical medical therapy but is limited by a decline in cell quality over 6 weeks of standard cold storage. Supercooled storage in tailored additive solutions has recently emerged as a means of attenuating RBC damage, with improved retention of 2,3 DPG and ATP during storage. However, prior studies of supercooled storage have been constrained to small experimental volumes, relying on oil-phase surface sealing to prevent ice formation at the liquid-air interface, limiting practical clinical translation. Here, we evaluated RBC storage in ESOL-5 additive solution at -4 °C across scales: 1 mL samples in tubes with and without oil-phase surface sealing versus large volume, 15-100 mL, bags without oil sealing. Smaller volume tests were conducted across 4 experiments of 2 type-matched, pooled donors, while 100 mL storage was evaluated via 3 independent experiments of a single donor. RBCs stored at -4 °C in large volume bags experienced no freezing events and demonstrated lower hemolysis than 1-mL equivalents while retaining lower lactate production, glucose consumption, and acidification relative to cold-stored samples. The presented scale-up approach for supercooled RBC storage addresses critical technical and regulatory barriers, enabling its eventual clinical translation.

Adverse reactions from donor-recipient sex-mismatched transfusions have been reported in the field of transfusion medicine. Immature CD71+ red blood cells (CD71+ RBCs) may influence these reactions through an immunomodulatory mechanism potentially linked to enriched intracellular reactive oxygen species (ROS). This study was conducted to investigate the effect of CD71+ RBCs on erythrophagocytosis, a common in vitro test for hemolysis risk in incompatible blood transfusions. Enriched CD71+ RBCs and CD71- RBCs were exposed to allogenic peripheral blood mononuclear cells (PBMCs). CD71+ RBCs were also incubated with anti-D sensitised CD71- RBCs and PBMCs, in varying ratios, to assess any dose-dependent behaviour. An ROS scavenger, apocynin, was applied to understand how CD71+ RBCs impact erythrophagocytosis. The phagocytosis index (RBC PI or CD71+ RBC PI) was measured using image flow cytometry. Supernatant haemoglobin was examined spectrophotometrically. The enriched CD71+ RBC group showed a significantly higher RBC PI and a reduction in the monocyte number compared to the CD71- RBC group. Compared to the non-treated group, the reduction of monocyte number and the increase of the phagocytosis index of CD71+ RBCs was partially reversed in the apocynin-treated group. There were positive correlations between the dose of CD71+ RBC and CD71+ RBC PI and supernatant haemoglobin. Donated CD71+ RBCs enhance in vitro erythrophagocytosis of CD71+ RBCs in a dose-dependent manner and reduce monocyte number. This work contributes to our understanding of immature CD71+ RBCs' role in post-transfusion immunobiology.

Hypothermic storage is constrained by the progressive depletion of energy reserves, curtailing the shelf-life of organs, cell therapies, and blood products. High sub-zero 'supercooling' helps preserve energy homeostasis by slowing catabolic processes; however, the resulting injury in this setting is not primarily driven by energy depletion. Here, we investigated whether low-dose ethanol could prevent forms of injury that arise independently of disrupted energy homeostasis and remain unaddressed in supercooled storage. Human red blood cells treated with 4% (v/v) ethanol were stored 4 °C, -4 °C, or -8 °C and subject to a series of functional assessments and integrated metabolomic/lipidomic profiling after 21 and 42 days of storage. Metabolomics data showed that energy homeostasis was better preserved at lower temperatures, yet these supercooled conditions simultaneously intensified hemolysis and caused a marked depletion of lysophospholipid species that did not occur at 4 °C. Ethanol blunted these effects, cutting hemolysis by ∼50 % at -4 °C, by ∼85 % at -8 °C, and attenuating lysophospholipid depletion. These results uncover a previously unrecognized, lipid-centric injury that arises during supercooled storage and establish low-dose ethanol as a simple, readily deployable countermeasure that could help extend storage intervals of diverse biological systems. SUMMARY:Cellular preservation has traditionally focused on maintaining energy metabolism during hypothermic storage, but whether this is sufficient at high sub-zero temperatures remains unclear. Human red blood cells were stored for up to 42 days at 4°C, -4°C, or -8°C with or without 4% ethanol and evaluated using functional assays and integrated metabolomic and lipidomic profiling. Although supercooling preserved energy homeostasis, it increased hemolysis and caused pronounced lysophospholipid depletion. Ethanol reduced hemolysis by approximately 50% at -4°C and 85% at -8°C, attenuated lysophospholipid loss, and produced comparatively modest changes in cellular metabolism. These findings identify membrane integrity as a critical determinant of preservation outcome and support strategies that protect membrane stability alongside metabolic homeostasis.

BACKGROUND:Many blood centers now accept donors with diabetes, provided their condition is "well-controlled". However, glycemic control is not routinely assessed at donation, and the impact of donor diabetes on blood product quality remains unclear. STUDY DESIGN AND METHODS:To screen glycemic control, glycated hemoglobin A1c (HbA1c) was measured in whole blood from 256 unique donors with either type 1 diabetes (T1D), type 2 diabetes (T2D), or without known diabetes. In parallel, routine quality control (QC) data were linked with donor metadata to assess the impact of donor diabetes on red cell concentrate (RCC) and apheresis platelet concentrate (PC) quality at the time of their expiry. RESULTS:Nearly half of donors with diabetes had suboptimal glycemic control (HbA1c >7 %) at donation, including 57.1 % of donors with T1D and 43.4 % with T2D. RCC units from donors with T2D (n = 1,329) had higher hemolysis (p <.001) and a greater proportion exceeding the clinical threshold of 0.8 % hemolysis (p <.001). These differences were not observed with T1D (n = 46). Apheresis PC units from donors with T2D (n = 164) had lower expiry pH (p = .014), with a trend toward more units below the clinical cutoff of pH 6.4 (p = .059). Differences in both products were most pronounced in donors with T2D using both metformin and insulin. CONCLUSION:Many donors with diabetes have suboptimal glycemic control, and RCC and PC from donors with T2D have reduced quality. The observed effects are modest, likely not driven by glycemic control alone, and do not support additional deferral criteria for donors with diabetes.

BACKGROUND:Cryopreserved red cell concentrates (RCCs) glycerolized using a high glycerol (40%) method can be stored below -65°C for up to 30 years; however, units may be inadvertently warmed above -65°C due to freezer failures, human errors, or routine inventory management. RCCs known to have experienced transient warming events (TWEs) or storage temperatures ≥-65°C are frequently discarded. STUDY DESIGN:Six groups of ABO/Rh matched RCCs were pooled-and-split (n = 9), glycerolized using the ACP 215 (Haemonetics), and frozen. Three types of TWEs were performed: (1) "Fast" TWEs (warming at room temperature [RT] to -25°C), (2) "Slow" TWEs (exposure to -25°C for 2 h), and (3) "Thaw" TWEs (single warming to RT). Units (n = 6) were exposed to 0 (control), 1, 10, or 30 "Slow" or "Fast" TWEs. RCCs were deglycerolized using the ACP 215, stored at 1-6°C, and tested at 0, 1, 7, and 14 days post-deglycerolization for red blood cell (RBC) quality. RESULTS:No significant differences among RCCs over hypothermic storage were found when exposed to "Fast" or "Slow" single or "Thaw" TWEs, although a significant decrease in the quality of RCCs was observed when units were subjected to 10 or more "Fast" or "Slow" TWEs. DISCUSSION:Our data suggests the single TWEs examined in this study do not significantly impact the quality of RCCs post-deglycerolization. Based on these observations, TWE exposure considerations should be made on a unit-by-unit basis, with additional policy support for unit retention.

Effective cryopreservation of biological materials is essential for enabling advances in food storage, biomedical research and clinical applications worldwide. Isochoric pressure-assisted supercooling (Isochoric-PAS) is a promising cryopreservation technique that can preserve biomaterials in a supercooled state without ice damage by leveraging high hydrostatic pressure. However, exposure to the elevated pressure leads to significant cellular injury. This study investigates trehalose as a protective agent against high-pressure stress during storage of cells with Isochoric-PAS. We hypothesized that although trehalose is generally membrane-impermeant under ambient conditions, high hydrostatic pressure can facilitate its intracellular uptake, enabling stabilization of biomembranes and proteins on both sides of the cell membrane and thereby enhancing cell survival. The experiment was conducted in which Jurkat cells were preserved with trehalose compared with various other cryoprotective agents (CPAs) and subjected to Isochoric-PAS at -2.5 degrees C (31 MPa) and -10 degrees C (111 MPa). Viability and cell size were assessed post-thaw and after a 1-hour recovery. Results show that trehalose at 800 mM yielded superior post-thaw viability (55.0%+1.0% at -10 degrees C) and significantly better recovery after incubation compared with other tested impermeable CPAs. Furthermore, milder freezing conditions (-2.5 degrees C) with lower pressure further improved outcomes. Intracellular trehalose uptake was confirmed and was enhanced under high pressure. This study demonstrates that trehalose is highly effective at mitigating pressure-induced damage, underscoring its potential as a key CPA for optimizing isochoric cryopreservation protocols.

Red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation during storage. Preserving RBCs for clinical transfusion remains a challenge due to storage-induced damage and limitations of traditional freezing methods. This study investigates isochoric freezing-a constant-volume, high-pressure cryopreservation technique that suppresses ice formation-as an alternative approach for RBC cryopreservation. RBC samples were preserved under isochoric freezing conditions at -2.5°C to -15°C with corresponding pressures of 31-156 MPa and a comparator supercooled control group. Hemolysis, cell count, morphology, and membrane integrity were assessed using hematological analyses, imaging flow cytometry, and dextran permeability assays. It is reported that hemolysis and morphological deterioration increased with decreasing temperature and rising pressure, with higher membrane damage compared to supercooling. Lower-temperature isochoric freezing resulted in loss of membrane integrity that was irreversible. While isochoric freezing minimized ice formation, elevated pressures adversely affected RBC viability. These findings highlight critical pressure-temperature thresholds necessary for optimizing isochoric freezing protocols for RBC preservation and inform future development of safer, long-term blood storage strategies.

The global infrastructure supporting nearly 100 million transfusions annually relies on the ability to store red cell concentrates (RCCs) for up to 42 days at hypothermic temperatures or indefinitely at low sub-zero temperatures. While these methods are generally effective, there is both an opportunity and, in specific settings, a need to refine storage techniques that have remained largely unchanged since the 1980s. Recent research has identified ways to address limitations that were not fully understood when these methods were first implemented in blood banks, with much of it focusing on modifying conventional storage strategies, while some studies explore alternative approaches. In this review, we explore the current state of RBC preservation and the future prospects for advancing both short- and long-term storage strategies.

Red blood cell (RBC) deformability-the ability of RBCs to change shape-is crucial for their passage through microvessels and effective oxygen delivery. Deformability depends on the cell's surface area-to-volume ratio, internal viscosity, and membrane elasticity. Deformability is assessed using single-cell methods like micropipette aspiration and bulk flow techniques such as ektacytometry. RBC deformability decreases during cold storage due to biochemical and structural changes known as storage lesions. Additional processes like cryopreservation and irradiation further impair the deformability of stored RBCs. Donor factors, including age, sex, lifestyle, and health, also influence RBC mechanical properties, affecting transfusion outcomes. Clinically, transfusing RBCs with reduced deformability is linked to impaired microvascular flow, decreased oxygen delivery, and faster clearance from the circulation, especially in patients with chronic or inflammatory conditions. Despite its importance, deformability testing is not yet standard in transfusion practice. New technologies offer potential for routine deformability assessment which could improve transfusion outcomes by optimizing RBC quality and matching units to patient needs.

Extreme temperature fluctuations during routine handling and shipping of cryopreserved cell products significantly compromise product quality in ways that extend beyond the duration and peak temperature of the fluctuation. The type of cryoprotectant used and the initial ice nucleation temperature influence ice crystal growth during rewarming events, in turn impacting cell survival. Using a cryomicroscope together with temperature profiles recorded in cord-blood units, ice crystal growth was tracked through five transient-warming events (TWEs) that peaked at -30 °C, -20 °C, or -10 °C. Initial freezing conditions were modified either by adding 6 % (w/v) hydroxyethyl starch (HES) or by lowering the ice-nucleation temperature by 10 °C. Across five TWEs, ice-crystal area saw the greatest increase when the peak rewarming temperature was -10 °C. Although adding HES further accelerated this recrystallization, it still protected Jurkat cells after a single TWE. Lowering the nucleation temperature also improved viability in samples warmed to -20 °C, regardless of HES supplementation. These findings show that ice crystal growth is not the sole cause of injury during transient rewarming; other temperature-dependent stresses also play a role. Importantly, careful optimisation of cryoprotectant composition and nucleation temperature can bolster cellular resilience to temperature excursions, potentially reducing quality losses during the storage and transport of cryopreserved therapeutics.

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare but often fatal complication of allogeneic transfusion, caused by the activation and expansion of donor T lymphocytes in susceptible recipients. Prevention focuses on reducing these immune cells through leukoreduction and irradiation. While leukoreduction of blood components decreases white blood cell content and improves overall transfusion safety, it does not fully prevent TA-GVHD, as viable T cells may persist. Irradiation using gamma or X-ray methods remain the most effective strategy, inactivating donor T cells by inducing DNA damage and suppressing proliferation. However, it also compromises red blood cell quality by increasing hemolysis, oxidative injury, membrane damage, extracellular potassium, and reducing storage duration. As an alternative, hypothermic storage of leukoreduced red blood cells is gaining attention. Evidence suggests that extending storage beyond 21 days significantly reduces T cell viability and proliferation without compromising red blood cell integrity. Further research is needed to directly compare the efficacy of hypothermic storage to irradiation.

[This corrects the article DOI: 10.3389/fphys.2023.1165330.].

The quality of stored red cell concentrates (RCCs) has been linked to the biological age distribution of red blood cell (RBC) subpopulations. Teenage male RCCs contain higher proportions of biologically old RBCs, with poorer quality. This study sought to assess the contribution of donor sex and age on the deformability characteristics of RBC subpopulations in stored RCCs. On days 5, 14, 28, and 42 of hypothermic storage, RCCs from healthy teenage male (n = 15), senior male (n = 15), teenage female (n = 15), and senior female (n = 15) donors were biologically age profiled. The deformability of the resulting young RBCs and old RBCs (O-RBCs) was assessed using ektacytometry. Over storage, donor age was the biggest factor influencing the rheology of RBC subpopulations. Teenage male RCCs had the largest reduction in Ohyper (osmolality in the hypertonic region corresponding to 50% of the maximum RBC elongation [EImax]). The strongest correlations between Ohyper and mean corpuscular hemoglobin content (R2> 0.5) were witnessed with O-RBCs from senior donors, and to a lesser extent with teenage males. Teen O-RBCs, particularly from males, had higher elongation indices, both under isotonic conditions and in the presence of an increasing osmotic gradient. Teen RBCs, regardless of biological age, were discovered to be more rigid (higher shear stress required to reach half the EImax). Donor variation in the age distribution of RBC subpopulations and its downstream effect on deformability serves as further evidence that factors beyond storage could potentially affect RCC quality and transfusion outcomes.

BACKGROUND:Historically, red cell concentrates (RCCs) have been manually glycerolized and deglycerolized using an open system (COBE 2991, Terumo). Implementation of a closed system cell processor (ACP-215, Haemonetics) for glycerolization and deglycerolization of RCCs creates a challenge for management of the historic cryopreserved RCC inventory. A study was undertaken to determine whether manually glycerolized frozen RCCs could be deglycerolized using the closed system processor, as the open system processors are being discontinued. STUDY DESIGN AND METHODS:Thirteen ABO/Rh matched RCCs were pooled and split to produce six large (approximately 354 mL) and six small (approximately 244 mL) RCCs. All units were stored for 14 days post-collection, manually glycerolized and frozen at ≤ -65°C for ≥72 h. Half of the units of each size were deglycerolized using the COBE 2991 and resuspended in 0.9% saline, and the remaining units were centrifuged, deglycerolized on the ACP-215, and resuspended AS-3. RBC quality was tested at 24 ± 2 h post-deglycerolization. RESULTS:All units deglycerolized on the ACP-215 had significantly lower hemolysis (p < .001) levels than those processed on the COBE2991. Large ACP-215 deglycerolized units had lower hematocrits (p < .05), hemoglobin (p < .01), and recovery (p = .001) than did large units deglycerolized on the COBE 2991. All ACP-215 units met the regulatory standards for hemolysis, hematocrit, hemoglobin, and recovery. DISCUSSION:The closed-system ACP-215 processor significantly reduced post-deglycerolization hemolysis in all units, and hemoglobin content in large units. The ACP-215, in combination with a centrifugation step, is suitable for processing cryopreserved RCCs that have been manually glycerolized.

Assessment of the morphology of red blood cells (RBCs) can improve clinical benefits following blood transfusion. Deep machine learning surpasses traditional microscopy-based classification methods, offering more accurate and consistent results while reducing time and labor intensity. RBCs from teenage males, teenage females, senior males, and senior females were biologically age-profiled or density-separated into dense/old RBCs (O-RBCs) and less-dense/young (Y-RBCs) throughout hypothermic storage and assessed using image flow cytometry with deep machine learning analysis. Regardless of biological age, morphology index decreased with hypothermic storage. Significant differences in RBC morphology index were not seen when comparing unseparated RBCs (U-RBCs), O-RBCs, and Y-RBCs, although the proportions of morphology subclasses revealed differences between RBCs groups from different donor groups and in samples with different biological age. Cold storage remains the most significant influence on morphology, although teenage male donors demonstrated slightly more susceptibility to storage lesions compared with senior males and females. Our work highlights that hypothermic storage most significantly impacts RBC morphology over biological age and donor characteristics, emphasizing the importance of storage effects on transfusion quality and safety.