Erythrocytes: Member of the immune system that should not be ignored.

Erythrocytes are the most abundant type of cells in the blood and have a relatively simple structure when mature; they have a long life-span in the circulatory system. The primary function of erythrocytes is as oxygen carriers; however, they also play an important role in the immune system. Erythrocytes recognize and adhere to antigens and promote phagocytosis. The abnormal morphology and function of erythrocytes are also involved in the pathological processes of some diseases. Owing to the large number and immune properties of erythrocytes, their immune functions should not be ignored. Currently, research on immunity is focused on immune cells other than erythrocytes. However, research on the immune function of erythrocytes and the development of erythrocyte-mediated applications is of great significance. Therefore, we aimed to review the relevant literature and summarize the immune functions of erythrocytes.

Incidence of acute haemolysis in cats receiving canine packed red blood cells (xenotransfusions).

OBJECTIVES: The aim of this study was to report the incidence of transfusion reactions in cats, including acute haemolysis (AH), occurring within 24 h of receiving a xenotransfusion. An additional aim was to determine whether cases with AH could be classified as having an acute haemolytic transfusion reaction (AHTR) as per the definition provided by the Association of Veterinary Haematology and Transfusion Medicine's Transfusion Reaction Small Animal Consensus Statement. METHODS: Medical records of cats that received canine packed red blood cells (PRBCs) between July 2018 and September 2020 at a veterinary hospital were reviewed. The incidence of AH, AHTRs, febrile non-haemolytic transfusion reactions (FNHTRs), transfusion-associated circulatory overload and septic transfusion reactions were recorded. RESULTS: The medical records of 53 cats were retrospectively evaluated. Twenty-three (43%) cats had transfusion reactions. Thirteen (25%) cats had AH; however, only four (8%) met the definition of an AHTR. Ten (19%) cats were determined to have FNHTRs. Survival to discharge of cats affected by AH was 50% (25% for cases that met the definition of an AHTR). Survival to discharge of cats not suffering from AHTR was 40%. CONCLUSIONS AND RELEVANCE: This report indicates that a higher proportion of cats undergo AH (25%) when administered canine PRBCs than previously reported, although many could not be classed as having an AHTR due to an apparently adequate packed cell volume rise. Challenges with sourcing feline blood in emergency situations occasionally necessitates the use of xenotransfusion in transfusion medicine. Clinicians should be aware that haemolysis after xenotransfusion can occur within 24 h and that a repeat feline transfusion may be required sooner than anticipated in some cases.

[COVID-19 development during the treatment of paroxysmal nocturnal hemoglobinuria].

Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder in which an activated complement causes intravascular hemolysis of erythrocytes that do not have complement regulators. It is critical to monitor the rapid progression of hemolysis caused by infection and thrombosis. As far as we can tell, this is the first report of 5 COVID-19 patients with PNH in Japan. Three patients were being treated with ravulizumab, one with eculizumab, and one with crovalimab. All five cases had received two or more COVID-19 vaccinations. COVID-19 was classified as mild in four cases and moderate in one. None of the cases required the use of oxygen, and none became severe. All of them experienced breakthrough hemolysis, and two required red blood cell transfusions. In any case, no thrombotic complications were observed.

Evaluation of the Hematological Patterns from Up to 985 Days of Long COVID: A Cross-Sectional Study.

Long COVID affects many individuals following acute coronavirus disease 2019 (COVID-19), and hematological changes can persist after the acute COVID-19 phase. This study aimed to evaluate these hematological laboratory markers, linking them to clinical findings and long-term outcomes in patients with long COVID. This cross-sectional study selected participants from a 'long COVID' clinical care program in the Amazon region. Clinical data and baseline demographics were obtained, and blood samples were collected to quantify erythrogram-, leukogram-, and plateletgram-related markers. Long COVID was reported for up to 985 days. Patients hospitalized in the acute phase had higher mean red/white blood cell, platelet, and plateletcrit levels and red blood cell distribution width. Furthermore, hematimetric parameters were higher in shorter periods of long COVID than in longer periods. Patients with more than six concomitant long COVID symptoms had a higher white blood cell count, a shorter prothrombin time (PT), and increased PT activity. Our results indicate there may be a compensatory mechanism for erythrogram-related markers within 985 days of long COVID. Increased levels of leukogram-related markers and coagulation activity were observed in the worst long COVID groups, indicating an exacerbated response after the acute disturbance, which is uncertain and requires further investigation.

Erythrocytes Functionality in SARS-CoV-2 Infection: Potential Link with Alzheimer's Disease.

Coronavirus disease 2019 (COVID-19) is a rapidly spreading acute respiratory infection caused by SARS-CoV-2. The pathogenesis of the disease remains unclear. Recently, several hypotheses have emerged to explain the mechanism of interaction between SARS-CoV-2 and erythrocytes, and its negative effect on the oxygen-transport function that depends on erythrocyte metabolism, which is responsible for hemoglobin-oxygen affinity (Hb-O2 affinity). In clinical settings, the modulators of the Hb-O2 affinity are not currently measured to assess tissue oxygenation, thereby providing inadequate evaluation of erythrocyte dysfunction in the integrated oxygen-transport system. To discover more about hypoxemia/hypoxia in COVID-19 patients, this review highlights the need for further investigation of the relationship between biochemical aberrations in erythrocytes and oxygen-transport efficiency. Furthermore, patients with severe COVID-19 experience symptoms similar to Alzheimer's, suggesting that their brains have been altered in ways that increase the likelihood of Alzheimer's. Mindful of the partly assessed role of structural, metabolic abnormalities that underlie erythrocyte dysfunction in the pathophysiology of Alzheimer's disease (AD), we further summarize the available data showing that COVID-19 neurocognitive impairments most probably share similar patterns with known mechanisms of brain dysfunctions in AD. Identification of parameters responsible for erythrocyte function that vary under SARS-CoV-2 may contribute to the search for additional components of progressive and irreversible failure in the integrated oxygen-transport system leading to tissue hypoperfusion. This is particularly relevant for the older generation who experience age-related disorders of erythrocyte metabolism and are prone to AD, and provide an opportunity for new personalized therapies to control this deadly infection.

Erythrocyte-Derived microRNAs: Emerging Players in Cardiovascular and Metabolic Disease.

Recent studies have demonstrated a novel function of red blood cells (RBCs) beyond their classical role as gas transporters, that is, RBCs undergo functional alterations in cardiovascular and metabolic disease, and RBC dysfunction is associated with hypertension and the development of cardiovascular injury in type 2 diabetes, heart failure, preeclampsia, familial hypercholesterolemia/dyslipidemia, and COVID-19. The underlying mechanisms include decreased nitric oxide bioavailability, increased arginase activity, and reactive oxygen species formation. Of interest, RBCs contain diverse and abundant micro (mi)RNAs. miRNA expression pattern in RBCs reflects the expression in the whole blood, serum, and plasma. miRNA levels in RBCs have been found to be altered in various cardiovascular and metabolic diseases, which contributes to the development of cardiovascular complications. Evidence has shown that RBC-derived miRNAs interact with the cardiovascular system via extracellular vesicles and argonaute RISC catalytic component 2 as carriers. Alteration of RBC-to-vascular communication via miRNAs may serve as potential disease mechanism for vascular complications. The present review summarizes RBCs and their released miRNAs as potential mediators of cardiovascular injury. We further focus on the possible mechanisms by which RBC-derived miRNAs regulate cardiovascular function. A better understanding of the function of RBC-derived miRNAs will increase insights into the disease mechanism and potential targets for the treatment of cardiovascular complications.

Exploring SARS-CoV-2 and Plasmodium falciparum coinfection in human erythrocytes.

The co-occurrence and the similarities between malaria and COVID-19 diseases raise the question of whether SARS-CoV-2 is capable of infecting red blood cells and, if so, whether these cells represent a competent niche for the virus. In this study, we first tested whether CD147 functions as an alternative receptor of SARS-CoV-2 to infect host cells. Our results show that transient expression of ACE2 but not CD147 in HEK293T allows SARS-CoV-2 pseudoviruses entry and infection. Secondly, using a SARS-CoV-2 wild type virus isolate we tested whether the new coronavirus could bind and enter erythrocytes. Here, we report that 10,94% of red blood cells had SARS-CoV-2 bound to the membrane or inside the cell. Finally, we hypothesized that the presence of the malaria parasite, Plasmodium falciparum, could make erythrocytes more vulnerable to SARS-CoV-2 infection due to red blood cell membrane remodelling. However, we found a low coinfection rate (9,13%), suggesting that P. falciparum would not facilitate the entry of SARS-CoV-2 virus into malaria-infected erythrocytes. Besides, the presence of SARS-CoV-2 in a P. falciparum blood culture did not affect the survival or growth rate of the malaria parasite. Our results are significant because they do not support the role of CD147 in SARS-CoV-2 infection, and indicate, that mature erythrocytes would not be an important reservoir for the virus in our body, although they can be transiently infected.

Bat Red Blood Cells Express Nucleic Acid-Sensing Receptors and Bind RNA and DNA.

RBCs demonstrate immunomodulatory capabilities through the expression of nucleic acid sensors. However, little is known about bat RBCs, and no studies have examined the immune function of bat erythrocytes. In this study, we show that bat RBCs express the nucleic acid-sensing TLRs TLR7 and TLR9 and bind the nucleic acid ligands, ssRNA, and CpG DNA. Collectively, these data suggest that, like human RBCs, bat erythrocytes possess immune function and may be reservoirs for nucleic acids. These findings provide unique insight into bat immunity and may uncover potential mechanisms by which virulent pathogens of humans are concealed in bats.

Evaluating patients with autoimmune hemolytic anemia in the transfusion service and immunohematology reference laboratory: pretransfusion testing challenges and best transfusion-management strategies.

The serologic evaluation of autoimmune hemolytic anemia (AIHA) confirms the clinical diagnosis, helps distinguish the type of AIHA, and identifies whether any underlying alloantibodies are present that might complicate the selection of the safest blood for any needed transfusion. The spectrum of testing is generally dependent on the amount and class (immunoglobulin G or M) of autoantibody as well as the resources and methodologies where testing is performed. The approach may range from routine pretransfusion testing, including the direct antiglobulin test, to advanced techniques such as adsorptions, elution, and red cell genotyping. When transfusion is needed, the selection of the optimal unit of red blood cells is based on urgency and whether time allows for the completion of sophisticated serologic and molecular testing methods. From the start of when AIHA is suspected until the completion of testing, communication among the clinical team and medical laboratory scientists in the transfusion service and immunohematology reference laboratory is critical as testing can take several hours and the need for transfusion may be urgent. The frequent exchange of information including the patient's transfusion history and clinical status, the progress of testing, and any available results is invaluable for timely diagnosis, ongoing management of the patient, and the safety of transfusion if required before testing is complete.

Recent updates of stem cell-based erythropoiesis.

Blood transfusions are now an essential part of modern medicine. Transfusable red blood cells (RBCs) are employed in various therapeutic strategies; however, the processes of blood donation, collection, and administration still involve many limitations. Notably, a lack of donors, the risk of transfusion-transmitted disease, and recent pandemics such as COVID-19 have prompted us to search for alternative therapeutics to replace this resource. Originally, RBC production was attempted via the ex vivo differentiation of stem cells. However, a more approachable and effective cell source is now required for broader applications. As a viable alternative, pluripotent stem cells have been actively used in recent research. In this review, we discuss the basic concepts related to erythropoiesis, as well as early research using hematopoietic stem cells ex vivo, and discuss the current trend of in vitro erythropoiesis using human-induced pluripotent stem cells.

Red Blood Cell Substitutes: Liposome Encapsulated Hemoglobin and Magnetite Nanoparticle Conjugates as Oxygen Carriers.

The established blood donation and transfusion system has contributed a lot to human health and welfare, but for this system to function properly, it requires a sufficient number of healthy donors, which is not always possible. Pakistan was a country hit hardest by COVID-19 which additionally reduced the blood donation rates. In order to address such challenges, the present study focused on the development of RBC substitutes that can be transfused to all blood types. This paper reports the development and characterization of RBC substitutes by combining the strategies of conjugated and encapsulated hemoglobin where magnetite nanoparticles would act as the carrier of hemoglobin, and liposomes would separate internal and external environments. The interactions of hemoglobin variants with bare magnetite nanoparticles were studied through molecular docking studies. Moreover, nanoparticles were synthesized, and hemoglobin was purified from blood. These components were then used to make conjugates, and it was observed that only the hemoglobin HbA1 variant was making protein corona. These conjugates were then encapsulated in liposomes to make negatively charged RBC substitutes with a size range of 1-2 µm. Results suggest that these RBC substitutes work potentially in a similar way as natural RBCs work and can be used in the time of emergency.

The ultimate tradeoff: how red cell adaptations to malaria alter the host response during critical illness.

The human immune system evolved in response to pathogens. Among these pathogens, malaria has proven to be one of the deadliest and has exerted the most potent selective pressures on its target cell, the red blood cell. Red blood cells have recently gained recognition for their immunomodulatory properties, yet how red cell adaptations contribute to the host response during critical illness remains understudied. This review will discuss how adaptations that may have been advantageous for host survival might influence immune responses in modern critical illness. We will highlight the current evidence for divergent host resilience arising from the adaptations to malaria and summarize how understanding evolutionary red cell adaptations to malaria may provide insight into the heterogeneity of the host response to critical illness, perhaps driving future precision medicine approaches to syndromes affecting the critically ill such as sepsis and acute respiratory distress syndrome (ARDS).

Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

De novo natural anti-M alloantibody emergence in severe Coronavirus Disease 2019.

The immune response is a key player in the course of SARS-CoV-2 infection, and is often seriously dysfunctional in severe Coronavirus Disease 2019. The hyperinflammatory status has been described to be accompanied by the appearance of autoantibodies. In a lethal COVID-19 infection, we observed the emergence of a de novo natural alloantibody which targeted the M antigen from the MNS blood group on red blood cells (RBC) without evidence of any cross-reaction with SARS-CoV-2 antigens. This IgM lambda alloantibody was unmutated and unswitched. Here, we describe for the first time the emergence of a bystander de novo natural alloantibody against RBCs in a severe COVID-19 patient, highlighting the extra-follicular humoral response reported in these cases.

Automated Recognition of Plasmodium falciparum Parasites from Portable Blood Levitation Imaging.

In many malaria-endemic regions, current detection tools are inadequate in diagnostic accuracy and accessibility. To meet the need for direct, phenotypic, and automated malaria parasite detection in field settings, a portable platform to process, image, and analyze whole blood to detect Plasmodium falciparum parasites, is developed. The liberated parasites from lysed red blood cells suspended in a magnetic field are accurately detected using this cellphone-interfaced, battery-operated imaging platform. A validation study is conducted at Ugandan clinics, processing 45 malaria-negative and 36 malaria-positive clinical samples without external infrastructure. Texture and morphology features are extracted from the sample images, and a random forest classifier is trained to assess infection status, achieving 100% sensitivity and 91% specificity against gold-standard measurements (microscopy and polymerase chain reaction), and limit of detection of 31 parasites per µL. This rapid and user-friendly platform enables portable parasite detection and can support malaria diagnostics, surveillance, and research in resource-constrained environments.

Simulated RBC antibody identification training panels created using SARS-CoV-2 kodecytes and immune plasma.

BACKGROUND: Training is essential to develop and maintain skills required to be a competent serologist, yet samples required to achieve this are often difficult to obtain. We evaluated the feasibility of SARS-CoV-2 peptide modified RBCs (1144-kodecytes) to develop simulated antibody screening and identification panels of reagent RBCs suitable for practical training, recognition, and grading of serologic reactions. STUDY DESIGN AND METHODS: RBCs from a single donor were modified into kodecytes using Kode Technology function-spacer-lipid constructs bearing a short SARS-CoV-2 peptide. Kodecytes and unmodified cells were then arranged in patterns representative of RBC antibody profiles as simulated antibody screening and identification reagent cell panels (SASID), and then tested against immune donor plasma samples containing SARS-CoV-2 antibodies. Manual tube and two different gel card serologic platforms were evaluated by routine techniques. SASID exemplars were created for antibodies including D, Cw , f (ce), Jka (strong, weak, dosing), mixtures of D + E, Jka + K, Fya + E, high and low frequency antibodies and a warm IgG autoantibody. RESULTS: Kodecytes (positive reactions) and unmodified cells (negative) when arranged and tested in appropriate patterns in SASID panels were able to mimic IgG antibody reactions, and were capable of measuring both accuracy and precision in reaction grading. CONCLUSIONS: Kodecytes can be used to rapidly create in-house simulated yet realistic antibody screening and identification panels suitable for large scale training in the recognition and grading of serologic reactions.

Selection of O-negative induced pluripotent stem cell clones for high-density red blood cell production in a scalable perfusion bioreactor system.

OBJECTIVES: Large-scale generation of universal red blood cells (RBCs) from O-negative (O-ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year-round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high-density culture platform for large-scale generation of RBCs in vitro. MATERIALS AND METHODS: We generated 10 O-ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high-density cultures of erythroblasts in vitro. RESULTS: Based on their tri-lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small-scale generation of high-density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation. CONCLUSIONS: This study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume-controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Immunomodulatory roles of red blood cells.

PURPOSE OF THE REVIEW: To discuss recent advances supporting the role of red blood cells (RBCs) in the host immune response. RECENT FINDINGS: Over the last century, research has demonstrated that red blood cells exhibit functions beyond oxygen transport, including immune function. Recent work indicates that the nucleic acid sensing receptor, toll-like receptor 9 (TLR9), is expressed on the RBC surface and implicated in innate immune activation and red cell clearance during inflammatory states. In addition to this DNA-sensing role of RBCs, there is growing evidence that RBCs may influence immune function by inducing vascular dysfunction. RBC proteomics and metabolomics have provided additional insight into RBC immune function, with several studies indicating changes to RBC membrane structure and metabolism in response to severe acute respiratory syndrome coronavirus 2 infection. These structural RBC changes may even provide insight into the pathophysiology of the 'long-coronavirus disease 2019' phenomenon. Finally, evidence suggests that RBCs may influence host immune responses via complement regulation. Taken together, these recent findings indicate RBCs possess immune function. Further studies will be required to elucidate better how RBC immune function contributes to the heterogeneous host response during inflammatory states. SUMMARY: The appreciation for nongas exchanging, red blood cell immune functions is rapidly growing. A better understanding of these RBC functions may provide insight into the heterogeneity observed in the host immune response to infection and inflammation.