Introduction: The impact of blood storage on red blood cell (RBC) alloimmunization remains controversial, with some studies suggesting enhancement of RBC-induced alloantibody production and others failing to observe any impact of storage on alloantibody formation. Since evaluation of storage on RBC alloimmunization in patients has examined antibody formation against a broad range of alloantigens, it remains possible that different clinical outcomes reflect a variable impact of storage on alloimmunization to specific antigens. Methods: RBCs expressing two distinct model antigens, HEL-OVA-Duffy (HOD) and KEL, separately or together (HOD × KEL), were stored for 0, 8, or 14 days, followed by detection of antigen levels prior to transfusion. Transfused donor RBC survival was assessed within 24 h of transfusion, while IgM and IgG antibody production were assessed 5 and 14 days after transfusion. Results: Stored HOD or KEL RBCs retained similar HEL or KEL antigen levels, respectively, as fresh RBCs, but did exhibit enhanced RBC clearance with increased storage age. Storage enhanced IgG antibody formation against HOD, while the oppositive outcome occurred following transfusion of stored KEL RBCs. The distinct impact of storage on HOD or KEL alloimmunization did not appear to reflect intrinsic differences between HOD or KEL RBCs, as transfusion of stored HOD × KEL RBCs resulted in increased IgG anti-HOD antibody development and reduced IgG anti-KEL antibody formation. Conclusions: These data demonstrate a dichotomous impact of storage on immunization to distinct RBC antigens, offering a possible explanation for inconsistent clinical experience and the need for additional studies on the relationship between RBC storage and alloimmunization.

Background: Red cell transfusions remain a mainstay of therapy for patients with sickle cell disease (SCD), but pose significant clinical challenges. Guidance for specific indications and administration of transfusion, as well as screening, prevention, and management of alloimmunization, delayed hemolytic transfusion reactions (DHTRs), and iron overload may improve outcomes. Objective: Our objective was to develop evidence-based guidelines to support patients, clinicians, and other healthcare professionals in their decisions about transfusion support for SCD and the management of transfusion-related complications. Methods: The American Society of Hematology formed a multidisciplinary panel that was balanced to minimize bias from conflicts of interest and that included a patient representative. The panel prioritized clinical questions and outcomes. The Mayo Clinic Evidence-Based Practice Research Program supported the guideline development process. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to form recommendations, which were subject to public comment. Results: The panel developed 10 recommendations focused on red cell antigen typing and matching, indications, and mode of administration (simple vs red cell exchange), as well as screening, prevention, and management of alloimmunization, DHTRs, and iron overload. Conclusions: The majority of panel recommendations were conditional due to the paucity of direct, highcertainty evidence for outcomes of interest. Research priorities were identified, including prospective studies to understand the role of serologic vs genotypic red cell matching, the mechanism of HTRs resulting from specific alloantigens to inform therapy, the role and timing of regular transfusions during pregnancy for women, and the optimal treatment of transfusional iron overload in SCD.

Background: Red blood cell (RBC) alloantibodies to nonself antigens may develop after transfusion or pregnancy, leading to morbidity and mortality in the form of hemolytic transfusion reactions or hemolytic disease of the newborn. A better understanding of the mechanisms of RBC alloantibody induction, or strategies to mitigate the consequences of such antibodies, may ultimately improve transfusion safety. However, such studies are inherently difficult in humans. Study Design and Methods: We recently generated transgenic mice with RBC-specific expression of the human KEL glycoprotein, specifically the KEL2 or KEL1 antigens. Herein, we investigate recipient alloimmune responses to transfused RBCs in this system. Results: Transfusion of RBCs from KEL2 donors into wild-type recipients (lacking the human KEL protein but expressing the murine KEL ortholog) resulted in dose-dependent anti-KEL glycoprotein immunoglobulin (Ig)M and IgG antibody responses, enhanced by recipient inflammation with poly(I:C). Boostable responses were evident upon repeat transfusion, with morbid-appearing alloimmunized recipients experiencing rapid clearance of transfused KEL2 but not control RBCs. Although KEL1 RBCs were also immunogenic after transfusion into wild-type recipients, transfusion of KEL1 RBCs into KEL2 recipients or vice versa failed to lead to detectable anti-KEL1 or anti-KEL2 responses. Conclusions: This murine model, with reproducible and clinically significant KEL glycoprotein alloantibody responses, provides a platform for future mechanistic studies of RBC alloantibody induction and consequences. Long-term translational goals of these studies include improving transfusion safety for at-risk patients.

Background Breakdown of humoral tolerance to RBC antigens may lead to autoimmune hemolytic anemia, a severe and sometimes fatal disease. The underlying mechanisms behind the breakdown of humoral tolerance to RBC antigens are poorly understood. Design and Methods In order to study the pathogenesis of autoimmune hemolytic anemia, we developed a murine model with RBC-specific expression of a model antigen carrying epitopes from hen egg lysozyme and ovalbumin. Results Humoral tolerance was observed; this was not broken even by strong immunogenic stimulation (lysozyme or ovalbumin with adjuvant). Autoreactive CD4 + T cells were detected by tetramer enrichment assays, but failed to activate or expand despite repeat stimulation, indicating a non-responsive population rather than deletion. Adoptive transfer of autoreactive CD4 + T cells (OT-II mice) led to autoantibody (anti-lysozyme) production by B cells in multiple anatomic compartments, including the bone marrow. Conclusions These data demonstrate that B cells autoreactive to RBC antigens survive in healthy mice with normal immune systems. Furthermore, autoreactive B cells are not centrally tolerized and are receptive to T-cell help. As the autoreactive T cells are present but non-responsive, these data indicate that factors that reverse T-cell non-responsiveness may be central to the pathogenesis of autoimmune hemolytic anemia.

In conclusion, RBC alloantibodies and DHTR are not uncommon in patients with SCD. They are underappreciated and, in our opinion, are the single leading cause of transfusion-associated morbidity and mortality in this vulnerable population of patients. Many of the challenges associated with preventing and treating DTHR can be addressed by developing international and national RBC alloantibody databases, limiting RBC transfusions to situations that are evidence-based, implementing more accurate diagnostic strategies (through routine use of HbA quantification and standard antibody screening), better understanding the pathophysiology, and formally testing additional prophylactic and treatment approaches to prevent and treat these reactions. We urge our colleagues in hematology, transfusion medicine (from donor centers to transfusion services), laboratory information technology, funding agencies, and regulatory agencies to view RBC alloimmunization and DTHR in patients with SCD with a similar urgency as TRALI was viewed in past decades. Such a heightened awareness, and subsequent industry changes, are predicted to directly reduce the significant transfusion-associated complications that contribute to the current morbidity and mortality of patients with SCD.

On April 5–6, 2016, the National Heart, Lung, and Blood Institute (NHLBI); the Department of Health and Human Services/Office of the Assistant Secretary of Health; and the Food and Drug Administration (FDA) sponsored a meeting on Scientific Priorities in Pediatric Transfusion Medicine at NHLBI in Bethesda, Maryland. The goal of this meeting was to identify important research questions that could be answered in the next 5 to 10 years and which would have the potential to transform the clinical practice of pediatric transfusion medicine. By applying basic, translational, and/or clinical research studies focused in six areas the science of/transfusion medicine would be advanced. The six areas were selected based on recognized gaps in knowledge and included neonatology and perinatology, oncology and transplant, chronic transfusion, devices and surgery, intensive care, and trauma and teenage blood donation. The meeting and premeeting teleconferences included 80 participants representing multiple stakeholders, academic medicine, the basic and clinical research community, and the government. The participants had expertise in multiple relevant disciplines including transfusion medicine, pediatrics, neonatology, hematology, surgery, critical care, trauma, internal medicine, epidemiology, and study design. The speakers and participants identified research priorities based on unmet needs in the pediatric transfusion medicine arena and proposed strategies to overcome the knowledge gaps and address unanswered questions.

The American Society of Hematology (ASH) 2020 guidelines for transfusion support for individuals with sickle cell disease (SCD)1 included recommendations on specific indications and administration of transfusion, as well as screening, prevention, and management of alloimmunization, delayed hemolytic transfusion reactions (DHTRs), and iron overload. The ASH Guideline Monitoring Expert Working Group conducted an updated literature search that did not identify new studies that would change the current recommendations.

Background: The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P) is a new iteration of prior National Heart, Lung, and Blood Institute (NHLBI) REDS programs that focus on improving transfusion recipient outcomes across the lifespan as well as the safety and availability of the blood supply. Study Design and Methods: The US program includes blood centers and hospitals (22 including 6 free-standing Children's hospitals) in four geographic regions. The Brazilian program has 5 participating hemocenters. A Center for Transfusion Laboratory Studies (CTLS) and a Data Coordinating Center (DCC) support synergistic studies and activities over the 7-year REDS-IV-P program. Results: The US is building a centralized, vein-to-vein (V2V) database, linking information collected from blood donors, their donations, the resulting manufactured components, and data extracts from hospital electronic medical records of transfused and non-transfused patients. Simultaneously, the Brazilian program is building a donor, donation, and component database. The databases will serve as the backbone for retrospective and prospective observational studies in transfusion epidemiology, transfusion recipient outcomes, blood component quality, and emerging blood safety issues. Special focus will be on preterm infants, patients with sickle cell disease, thalassemia or cancer, and the effect of donor biologic variability and component manufacturing on recipient outcomes. A rapid response capability to emerging safety threats has resulted in timely studies related to Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2). Conclusions: The REDS-IV-P program endeavors to improve donor-recipient-linked research with a focus on children and special populations while also maintaining the flexibility to address emerging blood safety issues.

Background and Objectives Transfusion of allogeneic platelet products can result in antibodies against donor MHC I antigens, leading to a refractory state to subsequent platelet transfusions. However, there is disagreement in the field regarding the molecular mechanisms of humoral alloimmunization. One hypothesis states that donor MHC II is a requirement for alloimmunization. However, other studies have suggested that donor MHC I is alone sufficient and MHC II is not required. Materials and Methods We utilized a mouse model of anti-MHC I alloimmunization to transfused blood, which employed donors with a complete deletion of all MHC II genes. BALB/c (H-2d) recipients were transfused with blood from either C57BL/6 (H-2b) or MHC II null donors on a C57BL/6 background. Anti-MHC I alloimmunization was monitored by indirect immunofluorescence. Results Recipients of either wild type or MHC II null blood produced equivalent humoral responses against donor MHC I antigens. However, there was variation in the relative amounts of IgG subclasses. Conclusion These data reject the hypothesis that donor MHC II expression is required for alloimmunization to MHC I antigens.

Generation of alloantibodies to transfused RBCs can be a serious medical problem for patients who require chronic RBC transfusion therapy. Patients with sickle cell disease have a substantially increased rate of alloimmunization compared to other chronically transfused populations. A recent study has forwarded the hypothesis that a polymorphism in an immunoregulatory gene in close proximity to beta-globin (TRIM21 rs660) plays a role in the increased rates of RBC alloimmunization in sickle cell patients. In particular, it was hypothesized that rs660C/T decreases expression of TRIM21, resulting in loss of a negative feedback pathway in immune responses and increased RBC alloimmunization. To test the effects of TRIM21 expression on alloimmunization, we analyzed antibody responses to alloantigens on RBCs and platelets transfused into wild-type and TRIM21 KO mice. No significant increases were seen in the frequency or magnitude of humoral immunization to alloantigens on transfused RBCs or platelets in adult or juvenile TRIM 21 KO recipients compared to wild-type controls. Moreover, recipient inflammation with poly (I:C) enhanced RBC alloimmunization to similar degrees in both TRIM 21 KO and wild-type control recipients. Together, these data rule out the hypothesis that decreased TRIM 21 expression enhances transfusion induced humoral alloimmunization, in the context of a reductionist murine model.