Impact of transfusion of blood components on the recipient immune system

Transfusions of blood provide essential therapeutic measures in a number of pathological conditions. However, when carrying out blood component therapy, it is important to consider probability of post-transfusion complications. Most of them are immune-mediated side effects. The unfavorable consequences of blood transfusions can manifest at long-range time periods, and pathogenesis of these phenomena may be associated not only with the presence of alloantibodies. They may be caused by alloimmunization to HLA antigens, leukocyte factors, including cytokines, products of leukocyte degranulation, as well as storage-related erythrocyte damage («storage lesion»), immunomodulatory properties of extracellular vesicles or microparticles derived from blood components, and other factors. Despite significant number of publications on this issue, a lot of unresolved issues still remain, concerning transfusion-related effects of blood components on the immune system of recipients. The review article provides the results of current studies in this area. We present and discuss the results of current studies and the features of transfusion-mediated immunomodulation (TRIM) revealed over recent years, when transfusing different blood components. The role of plasma factors, microparticles, platelets and erythrocytes, HLA sensitization and microchimerism in the development of TRIM is highlighted, the data on occurrence and clinical features of TRIM in perioperative period are presented. A separate section of the review provides information about recent clinical studies, devoted to the issues of TRIM in different clinical cohorts, including newborns, patients with malignant neoplasms, immunocompromised patients after heart and vascular surgery. The data on TRIM incidence in the patients with exhausted immune system due to previous disease or treatment, severe comorbidity, extensive surgical thoracic/abdominal intervention and artificial circulation are also in scope. As based on the studies performed, the role of distinct measures, e.g., washing of erythrocyte concentrates, leukodepletion, and gamma irradiation are discussed in view of potential TRIM prevention. The results of published research do not allow us to draw definite conclusions about the effects of blood component transfusion on the immune system of recipients with respect to differences between the studied groups of patients, characteristics of the studied disorders and clinical situations, diversity of hemocomponents, as well as varying standards of transfusion therapy adopted in different countries. However, the systematic literature review may provide some guidance in transfusion-mediated immune modulation.

Degradation of Red Blood Cell Deformability during Cold Storage in Blood Bags

RBC transfusions are a life-saving procedure, aiding both chronic and acute patients in restoring tissue oxygenation. The ability to store collected RBC units for prolonged periods has been one of the most transformative advances in medicine, significantly improving the reliability and the speed of access to blood. However, RBCs undergo a number of metabolic, structural, and biochemical changes during storage, collectively known as the storage lesion, that is detrimental to the quality of the RBC. A major challenge is the ability to evaluate the extent of the storage lesion, and thus the quality of the stored RBC unit directly prior to transfusion. The storage lesion can directly or indirectly reduce the ability of the RBC to deform through the small openings in the microvasculature. Rigid RBCs pose a risk of sequestration in capillaries, impeding blood flow and reducing tissue oxygenation, and are more likely to be cleared out by endothelial macrophages. Studies have shown that there is a loss in RBC deformability during storage and that the rate of RBC deformability loss is donor-dependent. Thus, RBC deformability can be a valuable and reliable biophysical marker of RBC unit quality. Currently, there is a need for a reliable measurement technique that is repeatable and sensitive enough to observe individual differences in RBC deformability in healthy donors, to enable quality control testing of RBC units. We have developed the microfluidic ratchet device, which sorts RBCs based on their deformability, allowing the measurement of both rigid and deformable sup-populations of RBCs within the sample, and generating a unique deformability curve. Here, we use this assay to predict the quality of stored RBC units. We assessed the deformability of 14 healthy donor RBC units through 8 weeks of cold storage at 4°C, which is 2 weeks beyond the Canadian Blood Services approved 6-week standard in Canada. We measured RBC deformability, standard hematological parameters (MCV, MCHC, MCH, and RDW), and hemolysis levels at the time of RBC unit manufacture (week 0), followed by weeks 2, 4, 6, and 8. The microfluidic ratchet device operates by forcing RBCs to deform and travel through rows of tapered constrictions. Constriction size changes from 7.5 to 1.5 µm and is reflective of the microvasculature and vessel opening sizes encountered by RBCs in circulation. RBCs are sorted into 12 distinct outlets based on their deformability. Distribution of RBCs in outlets 1-12 can be quantified and used to calculate the cumulative distribution curve. The cumulative distribution curve provides a distinct deformability signature of each individual RBC sample, which can be defined as rigidity score (RS). RS provides an easy metric to compare the changes in RBC deformability throughout storage (ΔRS) in a single donor as well as across multiple donors. We show that there are both donor- and sex-specific differences in the RBC deformability signatures of stored RBC units. We observed significant inter-donor variability in RBC deformability measured on the day of the RBC unit manufacture, where male donors showed a more stable RBC deformability range (n=8, RS=3.00±0.18) compared to female donors (n=6, RS= 3.29±0.48). The average RS scores were stable between weeks 0-2 (ΔRS 0.07) and showed a reduction in deformability between weeks 1-6 (ΔRS 0.35), with the greatest loss seen between weeks 6-8 (ΔRS 0.42) of cold storage. Interestingly, the response to cold storage is variable, with ΔRS 0.22 to 0.90, suggesting that some donors are more susceptible to storage related changes in RBC deformability than others. Notably, the change in RS over time was donor-specific and did not correlate with RBC deformability at week 0. The majority of RBCs from male donors (ΔRS 0.485, p<0.05), but none of the female donors (ΔRS 0.172) showed changes in deformability during cold storage, suggesting that RBCs from female donors degrade at a slower rate compared to RBCs from male donors. The ability to profile RBC deformability at the individual blood bag level may help identify more stable RBCs for use in chronic and sensitive patients, or RBC units that can be safely stored beyond the 6-week storage window. Disclosures No relevant conflicts of interest to declare.

Role of 12-LOX in the Platelet Storage Lesion

Background: 12-lipoxygenase (12-LOX) is an enzyme abundant in platelets which can contribute to the platelet storage lesion by oxidizing polyunsaturated fatty acids (PUFAs) released from phospholipid membranes. We and others have shown that the PUFA arachidonic acid (AA) and its lipid oxidation products, such as 12-hydroxyeicosatetraenoic acid (12-HETE), accumulate during storage and have inhibitory effects on platelet recovery, survival, and function. However, several PUFAs are substrates for 12-LOX, and their resulting oxylipins may have different effects. We used targeted metabolomics to quantify PUFAs and oxylipins and platelet function assays to characterize function of fresh and stored wild-type (WT) and 12-LOX -/- platelets. Methods: Blood from WT and 12-LOX -/- mice was collected by retro-orbital bleeding. Platelet-rich plasma (PRP) was generated from whole blood. After fresh samples were aliquoted, the remaining PRP was separated in two groups. One group was stored at room temperature with agitation (RT) for 24 hours, and the other for 48 hours. Metabolites were extracted from samples and quantified by targeted metabolomics as described previously. We assessed platelet function by αIIbβ3 integrin activation by flow cytometry. In vivo recovery of function was measured by transfusing stored platelets into UBiC-GFP mice and stimulating platelets with agonists, followed by gating for transfused (GFP-negative) platelets by flow cytometry. For recovery and survival, we traced biotinylated fresh, 24h, or 48h-stored platelets after transfusion in vivo. Results: We quantified metabolites present in platelets by targeted metabolomics to monitor their changes in concentration over storage time. Among the 10 PUFAs and 28 related oxylipins we analyzed, 15 of 38 analytes showed a significant difference in PRP from WT and 12-LOX-/- mouse samples. The major metabolites of 12-LOX include 12-HETE, 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA), from AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). 12-HETE, 12-HEPE, and 14-HDHA were only detected at <8 nmol/L levels in fresh PRP from 12-LOX -/- mice compared to 668 ± 409nM, 149 ± 85nM, and 295 ± 154nM from WT mice, respectively. After 24 hours of storage at RT, 12-HETE, 12-HEPE, and 14-HDHA dramatically increased to 29.0±4.2µM, 3.7±1.1µM, and 6.3±0.8µM in PRP from WT mice, respectively. As expected, these same metabolites remained at low nmol/L levels in 12-LOX-/- samples during storage accompanied by a significant increase of their precursors AA, EPA, and DHA due to lack of 12-LOX activity. Interestingly, there was also a significant reduction in 15-HETE, 17-HDHA, and 13-hydroxyoctadecadienoic acid (13-HODE) in the 12-LOX -/- mice compared to the WT mice, which are primarily produced by the 15-LOX enzyme. Additionally, we observed a significant decrease of metabolites mediated via the cyclooxygenase (COX) pathway in PRP from 12-LOX-/- mice, including prostaglandin E2 (PGE2), PGD2, thromboxane B2, and 12-hydroxyheptadecatrienoic acid (12-HHTrE). Function-wise, fresh 12-LOX -/- platelets were less responsive to agonists compared to WT platelets. Surprisingly, after transfusion of fresh 12-LOX -/- platelets, we found comparable αIIbβ3-integrin activation results after 1, 4, and 24h of circulation time. In contrast, 24h and 48h of storage of 12-LOX -/- platelets led to significantly lower pre-activation at baseline and a significantly lower activation response than WT platelets after 1h and 4h of circulation time. No significant differences were observed after 24h of circulation time. We observed a clear trend for longer survival after 24 and 48h of storage. Conclusions: We found many metabolic changes between 12-LOX -/- and WT mice during storage. While the 12-LOX -/- mouse model highlights the primary metabolic differences that occur without 12-LOX activity, other changes, such as differences in COX or additional LOX isoform activity, may attenuate oxylipin production. Functionally, we observed less pre-activation and better survival in functional studies, but this may be due to a combined effect of each of these individual metabolites. Future studies will have to determine the roles of individual oxylipins. Disclosures Stolla: Cerus: Research Funding.

Transfusion-Associated Hyperkalemic Cardiac Arrest in Neonatal, Infant, and Pediatric Patients

Red blood cell (RBC) transfusions are a life-saving intervention, with nearly 14 million RBC units transfused in the United States each year. However, the safety and efficacy of this procedure can be influenced by variations in the collection, processing, and administration of RBCs. Procedures or manipulations that increase potassium (K+) levels in stored blood products can also predispose patients to hyperkalemia and transfusion-associated hyperkalemic cardiac arrest (TAHCA). In this mini review, we aimed to provide a brief overview of blood storage, the red cell storage lesion, and variables that increase extracellular [K+]. We also summarize cases of TAHCA and identify potential mitigation strategies. Hyperkalemia and cardiac arrhythmias can occur in pediatric patients when RBCs are transfused quickly, delivered directly to the heart without time for electrolyte equilibration, or accumulate extracellular K+ due to storage time or irradiation. Advances in blood banking have improved the availability and quality of RBCs, yet, some patient populations are sensitive to transfusion-associated hyperkalemia. Future research studies should further investigate potential mitigation strategies to reduce the risk of TAHCA, which may include using fresh RBCs, reducing storage time after irradiation, transfusing at slower rates, implementing manipulations that wash or remove excess extracellular K+, and implementing restrictive transfusion strategies.

Innate and adaptive immunity to transfused allogenic red blood cells in mice requires MyD88

Red blood cell (RBC) transfusion therapy is essential for the survival of patients with hematological disorders such as sickle cell anemia. A potentially fatal complication of transfusion is development of non-ABO alloantibodies to polymorphic RBC antigens, yet mechanisms of alloantibody formation remain unclear. Human and mouse RBCs acquire a “storage lesion” prior to transfusion, which in mice contributes to immunogenicity. We previously reported that mouse splenic dendritic cells (DCs) are required for RBC alloimmunization and are activated by sterile and leukoreduced mouse RBCs after storage. Yet how syngeneic RBCs activate innate immune pathways to induce DC activation is unknown. We now show that DC activation to transfused RBCs occurs regardless of alloantigen presence, suggesting that RBC damage induced during storage triggers innate immune receptors. We discovered an unexpected dependence of RBC alloimmunization on the Toll-like receptor (TLR) signaling adaptor molecule MyD88. TLRs are a class of pattern recognition receptors (PRRs) that regulate DC activation and signal through two adaptor molecules, MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation, and the subsequent alloantibody response to transfused syngeneic RBCs require MyD88 but not TRIF, suggesting a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.

Degradation of Red Blood Cell Deformability during Cold Storage in Blood Bags

Red blood cells (RBCs) stored in blood bags develop a storage lesion that include structural, metabolic, and morphologic transformations resulting in a progressive loss of RBC deformability. The speed of RBC deformability loss is donor-dependent, which if properly characterized, could be used as a biomarker to select high-quality RBC units for sensitive recipients or to provide customized storage timelines depending on the donor. We used the microfluidic ratchet device to measure the deformability of red blood cells stored in blood bags every 14 days over a span of 56 days. We observed that storage in blood bags generally prevented RBC deformability loss over the current standard 42-day storage window. However, between 42 and 56 days, the deformability loss profile varied dramatically between donors. In particular, we observed accelerated RBC deformability loss for a majority of male donors, but for none of the female donors. Together, our results suggest that RBC deformability loss could be used to screen for donors who can provide stable RBCs for sensitive transfusion recipients or to identify donors capable of providing RBCs that could be stored for longer than the current 42-day expiration window.

Effect of microaggregate filter passage on feline whole blood stored for 35 days

Objectives The aim of this study was to compare the characteristics of fresh and stored feline red blood cells (RBCs) after passage through an 18 μm microaggregate filter. Methods Nine cats were recruited for a single blood donation using an open collection system. A simulated transfusion using a syringe driver and microaggregate filter was performed over 2 h with half the blood on the day of donation and the other half after 35 days of storage. Differences in haematological parameters, haemolysis percentage and osmotic fragility (OF) were compared on the day of donation pre-filter passage (D0–) vs day of donation post-filter (D0+) or day 35 storage pre-filter (D35–) and post-filter (D35+). Blood was cultured at D0+ and D35+. Results There were no statistically significant differences in the D0– vs D0+ comparisons. There were statistically significant ( P <0.05) increases in haemolysis percentage, red cell distribution width (RDW) percentage and mean OF, and decreases in packed cell volume (PCV), RBC count, haemoglobin and haematocrit for D0– vs D35–. The same was found for D0– vs D35+ with the addition of a significant increase in mean cell haemoglobin (MCH). For D35– vs D35+ only MCH significantly increased. At day 35, 6/9 units had haemolysis percentages that exceeded 1%. This increased to 8/9 of stored units post-filter passage. All blood units cultured negative. Conclusions and relevance Fresh RBCs exhibited no in vitro evidence of injury following passage through an 18 μm microaggregate filter. Increased MCH was observed in the stored blood and may represent haemolysis induced by the filter. All other changes can be explained by storage lesion rather than filter passage. The findings highlight the importance of blood banking quality controls and the need for further research to assess the effects of transfusion technique, specifically filter passage, on storage lesion-affected feline blood.

The effect of N-acetyl-cysteine (NAC) on RBC oxidative damage and RBC metabolism during storage of red blood cell product in blood bank condition

Background: Oxidative damage is one of the main causes of Red blood cell (RBC) storage lesion which can reduce RBC survival during RBC storage in blood bank condition. In this study, we evaluate the effect of N-acetyl cysteine (NAC) as an anti-oxidant compound on RBC oxidative damage and RBC metabolism during storage of this product. Methods: In this experimental study, 10 bag of packed RBC were provided to the Iranian Blood Transfusion Organization's Innovation Center were randomly selected and effect of NAC was investigated on metabolism, oxidative status and hematologic variables of RBC during RBC storage. The results of this study were compared between two groups of NAC treated RBC and untreated RBC (without NAC). All of the data were analyzed with SPSS statistical program (version 22). Results: In this study, the concentration of lactate and LDH enzyme activity in the NAC treated RBC were lower than the control group (without NAC). The concentration of malondialdehyde (MDA) as a lipid peroxidation marker in the NAC treated RBC was lower increase than the untreated RBC. Also antioxidant capacity was so higher in the NAC - RBC than in the control group, especially in the 28th day of RBC storage (Pday28 <0.05). Conclusion: The results of this study indicated that the use of NAC as an additive solution could decrease oxidative damage via maintaining of the RBC oxidant capacity during RBC storage. In the future NAC may be used as an additive for maintaining of the RBC survival and RBC quality during storage of RBC in blood bank condition.

From bench to bar side: Evaluating the red wine storage lesion

Vitally essential red fluids like packed cells and red wine are seriously influenced in quality when stored over prolonged periods. In the case of red cell concentrates, the resulting storage lesion has particular significance in perioperative medicine. We hypothesized that, in contrast, aging rather improves the properties of red wine in several ways. A translational approach, including (I) in vitro experiments, (II) a randomized, blinded crossover trial of acute clinical effects, and (III) a standardized red wine blind tasting was used. Three monovarietal wines (Cabernet Sauvignon, Chianti, Shiraz) in three different vintages (range 2004–2016), each 5 years different, were assessed. Assessments were performed at a German university hospital (I, II) and on a garden terrace during a mild summer evening (III). Young wines induced cell stress and damage while significantly reducing cytoprotective proteins in HepG2 hepatoma cells. Sympathetic activity and multitasking skills were altered depending on wines’ ages. Hangovers tended to be aggravated by young red wine. Aged variants performed better in terms of aroma and overall quality but worse in optical appearance. We found no evidence for a red wine storage lesion. However, we plead for consensus-based guidelines for proper storage, as it is common in clinical medicine.