Red Blood Cell Alloimmunization In Patients Research Articles

Identifying Inflammatory Pathways That Promote RBC Alloimmunization in Patients with Sickle Cell Disease

Introduction: During red blood cell (RBC) transfusion, exposure to unmatched non-ABO antigens can lead to production of alloantibodies that promote potentially fatal hemolytic events. Patients with Sickle Cell Disease (SCD) have the highest incidence of RBC alloimmunization. Prior studies indicate that an altered immuno-regulatory and/or pro-inflammatory state may determine the risk of RBC alloimmunization. For example, one study reported that patients with SCD have inflammatory cytokine gene polymorphisms that could be associated with diverse disease presentations and alloimmunization. We previously reported that inflammatory type 1 interferons (IFNα/β) produced by innate immune cells are required for RBC alloimmunization in mice, while others have suggested that NFkB-induced cytokines play a role in murine transfusion models. However, the inflammatory factors that regulate RBC alloimmunization in patients with SCD are poorly understood. Alloimmunized patients with SCD also have elevated levels of anti-RBC autoantibodies, which facilitate erythrophagocytosis. Given the role of erythrophagocytosis by spleen macrophages in processing RBC antigens, we hypothesized that erythrophagocytosis of SCD RBCs may promote inflammatory pathways that regulate alloimmune responses. Methods: RBCs from control subjects (AA), and reticulocytes and mature RBCs from patients with SCD, were opsonized or not with anti-RBC antibodies (anti-RhD) and co-cultured for 3 hrs with human macrophages (hMac) differentiated from monocytes of healthy subjects. Phagocytosis of labeled RBCs was analyzed by flow cytometry. Bulk mRNA sequencing of co-cultured macrophages was performed to identify differential gene expression. Ingenuity Pathway Analysis (IPA) software was used to identify relevant pathways and upstream regulators of differentially expressed genes. To validate sequencing results, cytokine levels from co-culture supernatants were measured by multiplex cytokine assays. Results: Flow cytometric analysis revealed that opsonization was required for erythrophagocytosis in an FcR dependent manner. RNA-seq Differential Expression analysis of macrophages co-cultured with AA opsonized RBCs vs. non-opsonized RBCs identified 166 significantly upregulated and 24 downregulated genes (p<0.0001). IPA analysis revealed that the top 12 canonical pathways were related to inflammation, including TNF, IL-17, NF-kappa-B and NOD-like receptor signaling pathways. TNF signaling was the top predicted upstream regulator pathway with a fold enrichment score of 10.3 (16 genes) (p<0.0001). Co-cultures of mature RBCs from patients with SCD showed comparable results. When comparing macrophages from co-cultures of opsonized reticulocytes vs. non-opsonized reticulocytes from patients with SCD, 174 genes were upregulated and 31 were downregulated (p<0.0001). Pathway analysis also revealed unique signatures for opsonized reticulocytes from patients with SCD. The top 12 canonical pathways included TNF, NF-kappa-B and NOD-like receptor signaling pathways (Figure 1). The top predicted upstream regulators were TNF and the NF-kappa B signaling pathway with a fold enrichment score of 12.3 (22 genes) (p<0.0001) and 10.9 (18 genes), respectively (Figure 1). Finally, to validate the sequencing results, cytokines levels from supernatants of hMac co-cultures with the RBCs from the patients with SCD and control subjects were measured. Erythrophagocytosis of opsonized reticulocytes and AA RBCs, compared to non-opsonized co-cultures, led to production of NFκB-mediated cytokines, including TNF. Our results show that erythrophagocytosis of SCD RBCs promote NFκB-mediated TNF production. These results warrant further investigation of the role of NFκB-mediated inflammation in RBC alloimmunization in patients with SCD.

Storage differentially impacts alloimmunization to distinct red cell antigens following transfusion in mice.

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. 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. 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. 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.

Type 1 Interferon Gene Signature Promotes RBC Alloimmunization in a Lupus Mouse Model.

Red blood cell (RBC) transfusion exposes recipients to hundreds of unmatched minor RBC antigens. This exposure can lead to production of alloantibodies that promote clinically significant hemolytic events. Multiple studies have reported an increased frequency of RBC alloimmunization in patients with autoimmunity. However, cellular and molecular mechanisms that underlie autoimmunity-induced alloimmunization have not been reported. Patients with systemic lupus erythematosus (SLE) have a high frequency of alloimmunization and express a type 1 interferon (IFNα/β) gene signature. Thus, we utilized the pristane-induced lupus mouse model to test the hypothesis that inflammation in lupus promotes RBC alloimmunization, and to examine the potential role of IFNα/β. Intraperitoneal injection of pristane, a hydrocarbon oil, led to autoantibody production, glomerulonephritis, and pulmonary hemorrhage in wild type (WT) mice. Pristane treatment significantly induced serum IFNα and expression of multiple interferon-stimulated genes (ISGs) in peripheral blood and peritoneal fluid cells, including inflammatory macrophages. Following transfusion with allogeneic RBCs expressing the KEL glycoprotein, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG, compared to untreated mice. Pristane induced comparable levels of inflammatory cells and cytokines in mice lacking the IFNα/β receptor (IFNAR1–/–) or the IFNα/β-inducing transcriptions factors (IRF3/7–/–), compared to WT mice. However, pristane-treated IFNAR1–/– and IRF3/7–/– mice failed to produce ISGs and produced significantly lower levels of transfusion-induced anti-KEL IgG, compared to WT mice. Thus, pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. To our knowledge, this is the first examination of molecular mechanisms contributing to RBC alloimmunization in a model of autoimmunity. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens and the contribution of the IFNα/β gene signature to the elevated frequency of alloimmunization in patients with SLE.

Red Blood Cell Alloimmunization in Transfused Patients With Sickle Cell Disease in Sub-Saharan Africa; a Systematic Review and Meta-Analysis.

Sickle cell disease (SCD) is the most common monogenic disorder in sub-Saharan Africa (SSA). Blood transfusion to increase the oxygen carrying capacity of blood is vital in the management of many patients with SCD. However, red blood cell (RBC) alloimmunization is a major challenge to transfusions in these patients. Commonly in SSA, pretransfusion tests only involve ABO D grouping and compatibility without RBC antibody testing. Data on the frequency of RBC alloimmunization in patients with SCD in SSA are limited. We performed a systematic review and meta-analysis on available data on alloimmunization in transfused patients with SCD to determine the published prevalence of RBC alloimmunization in SCD patients in SSA. Six databases were systematically searched to identify relevant studies, without year or language restrictions. In all, 249 articles were identified and 15 met our selection criteria. The overall proportion of alloimmunization was 7.4 (95% confidence interval: 5.1-10.0) per 100 transfused patients. Antibodies against E, D, C, and K antigens accounted for almost half of antibody specificities, and antibodies to low- and high-frequency antigens were also common and represented almost 30% (20% to low-frequency antigens and 9% to high-frequency antigens) of specificities. Heterogeneity between studies was moderate, and meta-analysis found region of Africa as the major contributor to the heterogeneity. We also observed inconsistencies across studies in reporting of factors that may influence alloimmunization. This review provides an overview of the extent of the alloimmunization problem in SSA and provides a baseline against which to compare the effect of any interventions to reduce the alloimmunization risk.

Red blood cell alloimmunization and delayed hemolytic transfusion reactions in patients with sickle cell disease

Red blood cell (RBC) alloimmunization is more common in patients with sickle cell disease (SCD) than in any other studied patient population. The high prevalence of RBC alloimmunization is multi-factorial, likely involving the chronic hemolysis and inflammatory status of SCD itself, the transfusion burden of patients, and the RH genetic diversity of patients and blood donors, among other reasons. Antibody evanescence, or the decrease of RBC alloantibodies below levels detectable by blood bank testing, occurs frequently with fewer than 30% of alloantibodies estimated to be detected by current screening practices. Evanescence increases the likelihood that a patient with SCD will have a delayed hemolytic transfusion reaction upon future RBC exposure, with previously undetected alloantibodies coming roaring back in an anamnestic manner after exposure to the cognate RBC antigen. A subset of patients having delayed hemolytic transfusion reactions go on to experience hyperhemolysis; some but not all cases of hyperhemolysis are associated with previously evanescent RBC alloantibodies. There is an increasing appreciation of the association between RBC alloantibodies and RBC autoantibodies, as well as involvement of the alternative complement pathway in some instances of hyperhemolysis. A case report in this manuscript describes a highly alloimmunized patient with SCD who experiences a delayed hemolytic transfusion reaction with bystander hemolysis due to a previously evanescent, complement binding anti-M RBC alloantibody. Additional studies, including those involving multiple centers and countries, are needed to further understand RBC alloimmunization in patients with SCD and to develop strategies to prevent or mitigate potentially life-threatening hemolytic transfusion reactions.

FCGR2B B2.4 Haplotype Predicts Increased Risk of Red Blood Cell Alloimmunization in Sickle Cell Disease Patients

Introduction: Red blood cell (RBC) alloimmunization is an important transfusion complication which is very prevalent among patients with sickle cell disease (SCD). Only part of patients is able to develop alloantibodies following antigen-mismatched transfusions, named the ‘immune responders’, who should be prospectively transfused with antigen-matched RBC units, comprising the most immunogenic erythrocyte antigens. Auto-immune diseases are a known risk factor for RBC alloimmunization, suggesting that auto-immunity and post-transfusion alloantibody development happen through similar physiopathological pathways. In this context, polymorphisms in FCGR2B gene have already been associated with several auto-immune disorders and, hypothetically, could be associated with RBC alloimmunization and help to identify the ‘immune responders’ in transfusion practice.Methods: In this case-control study, we enrolled 277 transfused SCD patients with retrospective data on alloimmunization status and transfusion exposure from two Brazilian centers. DNA samples (collected from March, 2017 to June, 2018) were extracted from whole blood samples using commercial kits (PureLink™ Genomic DNA®, Invitrogen, CA), and used for PCR amplification of promoter region of FCGR2B gene containing both -386G/C and -120T/A single nucleotide polymorphisms (SNPs) using gene-specific primer. SNPs of the FCGR2B were genotyped by direct Sanger sequencing (ABI3730XL, Applied Biosystems, CA). Cases had a positive history of alloimmunization with a clinically significant alloantibody and a minimum transfusion exposure of 1 RBC unit. Controls had a negative history of alloimmunization and having received ≥ 2 RBC units. Patients with autoimmune diseases or poor quality of DNA were excluded. Unadjusted and adjusted analyses were conducted by using Logistic Regression models to determine the association of covariates with binary outcome. Since the assumption of linearity in the logit was not supported, continuous covariates were categorized based on optimum cut-off point derived from ROC analysis. All statistical analyses were performed using SAS University Edition. A p-value of <0.05 was considered to be statistically significant.Results: A total of 237 patients met the eligibility criteria, 121 cases (alloimmunized) and 117 controls (non-alloimmunized). Majority of patients were female in cases (61%) and male in controls (86%), with mean age of 27.9 years (SD 15.3). Table 1 list the characteristics between the groups. RBC alloimmunization was associated with female sex (p<0.001), lifetime number of RBC units transfused (p=0.002) and FCGR2B 120T/A SNP (p=0.031). No statistically differences in the allele frequency of c.386C were observed between groups (p=0.602). FCGR2B -386G/C and -120T/A SNPs were in intense linkage disequilibrium. Three haplotypes of promoter region were identified as 2B.1 (-386G-120T), 2B.2 (-386C-120T) and 2B.4 (-386C-120A) with statistical significance (p=0.045). Logistic regression model was performed to assess the effects of age, gender, lifetime number of RBC units transfused and FCGR2B 2B.4 haplotype on the likelihood of RBC alloimmunization in patients with SCD. The analysis identified that female sex (OR 10.03, CI95% 5.16-19.49; p<0.001) and FCGR2B 2B.4 haplotype (OR 4.55, CI95% 1.11-18.65; p=0.035) predict increased risk of RBC alloimmunization in SCD patients as show in Table 2.Conclusions: SCD patients with the FCGR2B 2B.4 haplotype had over a 4-fold higher risk for RBC alloimmunization in comparison with patients without this haplotype and probably may be helpful to identify the ‘immune responders’. The presence of FCGR2B 2B.4 haplotype have been associated with increased expression of this inhibitory Fc gamma receptor in immune system cells, that was strongly associated with capacity of immune tolerance, regulating the development of antibodies. Future studies are needed to understand more about the functional and immune mechanism behind alloimmunization in SCD patients and ultimately improve our current preventive strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The severity of illness and inflammatory markers cannot predict red blood cell alloimmunization in cancer patients

Red Blood Cell (RBC) alloimmunization remains a major complication for transfusion-dependent patients but factors governing risk for alloimmunization are little known. Antibody production is a serious complication in patients on long-term transfusion therapy; it may cause potentially life-threatening delayed hemolytic transfusion reactions (DHTRs), autoantibody formation as well as logistic problems, for example, to obtain timely and properly matched transfusion blood for patients in which new alloantibodies are detected. Even non-chronically transfused patients who become alloimmunized are 20 times more likely to form additional antibodies after one or more repeat transfusion events(1-3). This is not an ideal scenario for a patient population in which the benefit is proportional to the longevity of transfused RBCs and this may result in an extensive laboratory workup that consumes significant time and resources. In an effort to reduce alloimmunization, some programs have been designed and implemented to provide Rh and K antigen-matched RBC transfusions to patients who are in need of chronic transfusion support but some patients still become alloimmunized despite this antigen matching(4,5). In contrast to prophylactic antigen matching, some institutions argue that the data supporting a reduction in DHTRs are insufficient to offset the cost of labor and resources required to perform extended matching for polytransfused patients. Characterization of patients and clinical conditions with high alloimmunization risk could help in the selection of patients who need to be extensively matched. By identifying such risks, it may be possible to predict responders and non-responders, thereby avoiding the use of costly antigen-matched units for non-responders, and only selecting phenotyped/genotyped-matched units for responders to reduce alloimmunization-associated morbidity and mortality. Several factors are predicted to influence the reaction of the recipient's immune system to alloantigens including the dose and the immunogenicity of the antigen as well as genetic, and acquired patient-related factors and clinical conditions(6,7). It has also been shown that the number of transfusions plays an important role in RBC alloimmunization and that immune-compromised patients have a lower risk to develop red cell antibodies. However, other patient-related risks are little known. Although unexpected risk factors were found, such as solid tumors because of their chronic inflammatory state, there are few and limited studies showing that the inflammatory status of human transfusion recipients may regulate the immunogenicity of transfused RBCs(8). In this issue of the Revista Brasileira de Hematologia e Hemoterapia, there is an important retrospective case-control study on a small group of alloimmunized and nonalloimmunized patients with solid cancer, regarding the risk of RBC alloimmunization(9). The authors evaluated clinical factors related to the severity of the disease (ECOG performance scale, Karnofsky scale, presence of metastasis and body mass index) and inflammatory background (C-reactive protein) and concluded that these factors cannot be used to predict the risk of RBC alloimmunization in such patients. Although the relatively small number of patients is a limitation of the study, this paper represents an additional contribution in the field of RBC alloimmunization in patients with solid tumors. The results of the studies of Dinardo et al. do not ratify the thesis that the inflammatory status regulates the immunogenicity of transfused RBCs(9). This finding supports the concept that there are combinations of factors influencing the immune response to RBC antigens. We will have to look to future studies by this group and others to better understand the risk factors of RBC alloimmunization in cancer patients in order to provide or not antigen-matched RBC transfusions.