Red Blood Cell Autoantibodies Research Articles

A Review Article on Etiology, Clinical Presentation, Laboratory Evaluation and Treatment of Autoimmune Hemolytic Anemia

Red blood cell (RBC) autoantibodies are a relatively uncommon cause of anemia. Nevertheless, autoimmune hemolytic anemia (AIHA) should consider in the differential diagnosis of hemolytic anemia whenever patient has an accompanying lymphoproliferative disorder, autoimmune disease, viral or mycoplasma infection. AIHA is further categorized as warm AIHA, cold agglutinin syndrome, paroxysmal cold hemoglobinuria, mixed-type and drug-induced AIHA. It is the characteristic of the autoantibodies which are accountable for various clinical entities. Diagnosis is based on clinical presentation and work up of AIHA. This review discusses epidemiology, causes, clinical presentation, and laboratory workup and treatment options available for AIHA.

Prevalence and specificity of red blood cell alloantibodies and autoantibodies in transfused Iranian β-thalassemia patients: A systematic review and meta-analysis.

BACKGROUND:Repeated allogeneic blood transfusions in thalassemia major patients stimulate the patient's immune system to generate antibodies against foreign erythrocyte antigens. This study was carried out to systematically review the findings of available studies about the prevalence of alloantibodies and autoantibodies, as well as the type of causative antigens among transfusion-dependent thalassemia patients in Iran.METHODS:Electronic search was conducted on Medline, PubMed, Cochrane, EMBASE, ScienceDirect, and Persians databases. All relevant articles published from January 1990 to July 2018 were included. s of conference booklets which that been published in the last 5 years were also included in the meta-analysis. The search language was restricted to English and Persian. The quality of studies was evaluated according to a checklist developed by authors, and Cochrane Risk of Bias Assessment Tool was used to evaluate the risk of bias.RESULTS:Twenty-three relevant articles met all the inclusion criteria. The prevalence of alloimmunization was 13%. Our study showed that anti-D (25%) and anti-K (25%) were most prevalent among Iranian β-thalassemia patients. Data analysis shows the autoantibody prevalence to be 1% among 3787 patients. Meta-regression revealed that the prevalence of alloantibodies increases with each year as the average age of the study population increases.CONCLUSION:The prevalence of red blood cell (RBC) alloantibodies in transfused Iranian β-thalassemia patients was high. Appropriate preventive strategies such as RBC phenotyping for patients before beginning transfusion and using extended RBC donor–recipient matching, specifically for Rh and Kell system, could be implemented to avoid complications in thalassemia patients.

Red Blood Cell Autoantibodies in Patients Treated with Immune Checkpoint Inhibitors

Red Blood Cell Autoantibodies in Patients Treated with Immune Checkpoint Inhibitors

Autoantibodies to red blood cell surface Glycophorin A impact the activation poise of circulating leukocytes.

Both M and N alleles encode antigens on Glycophorin A (GPA), a red blood cell (RBC) surface sialoglycoprotein. Interaction between RBC GPA and leukocyte surface lectins may downregulate their activation. The current study investigates if RBC autoantibodies against GPA, such as auto-anti-M/N, prime an activated phenotype in peripheral blood leukocytes. Leukocyte activation was assessed in whole blood from patients with auto-anti-GPA (anti-M/N) and compared to those with allo-anti-M/N and healthy subjects. Control samples from healthy subjects with no antibodies incubated in vitro with either anti-GPA or anti-Rh were analyzed for neutrophil and monocyte surface activation marker expression, reactive oxygen species (ROS) content, and formation of aggregates with RBCs. Samples incubated with an IgG1 isotype antibody served as controls. Ex vivo, neutrophil CD66b and monocyte CD63 surface expression was increased in patients with auto-anti-M/N compared to those with allo anti-M/N (p=.1757; p=.0698) and to healthy subjects (p=.0186; p=.013). In vitro, neutrophil CD66b and monocyte CD63 surface expression was increased following incubation with anti-GPA compared to anti-Rh (p=.0003; p=.0328) and isotype control (p=.000; p=.0062). Intracellular ROS content increased in both neutrophils and monocytes incubated with anti-GPA compared to anti-Rh (p=.0012; p=.0693) and isotype control (p=.001; p=.0021). Percentage of neutrophil-RBC aggregates was decreased when incubated with anti-GPA compared to isotype control (p < .01). Neutrophils and monocytes in peripheral blood exposed to an antibody directed against GPA on RBC surfaces, such as M or N antigens, may be primed towards an activated phenotype.

Characterization of circulating and cultured Tfh-like cells in sickle cell disease in relation to red blood cell alloimmunization status

BackgroundPeople living with sickle cell disease (SCD) are prone to red blood cell (RBC) alloimmunization. We hypothesized that subjects with alloantibodies (responders) would have differences in circulating T-follicular helper (Tfh)-like cells compared to subjects without alloantibodies (non-responders). Materials and methodsPeripheral blood mononuclear cells were collected from 28 subjects, including those with SCD and controls. Circulating CD4 T-cell subsets were first evaluated at baseline. CD4 T-cell subsets were also evaluated after naïve CD4 T-cells were differentiated into Tfh-like cells following in vitro culture with CD3/CD28 beads, IL-7, IL-12, and Activin A. Transfusion and alloantibody histories were extracted from the electronic medical record. ResultsNon-responders had a lower percentage of CD45RA negative Tmemory cells than responders or controls (p<0.05). Notably, there were no differences in circulating Tfh-like cells between any group. However, naïve CD4 T-cells from subjects with SCD were more likely to express CXCR5 after in vitro culture than cells from controls. After culture, CXCR5 expressing cells from responders were more likely to express PD1 and ICOS (16.43 %, sd. 20.23) compared to non-responders (3.69 %, s.d. 3.09) or controls (2.78 %, s.d. 2.04). DiscussionThe tendency for naïve CD4 T-cells from responders to differentiate into Tfh-like cells after in vitro culture may suggest these cells are prepared to assist B-cells with antibody production regardless of antigen specificity. Further studies are needed, but it is possible that these results may explain why some responders form RBC alloantibodies with multiple specificities, in addition to RBC autoantibodies and HLA alloantibodies.

Red blood cell autoantibodies: The importance of being earnestly autoadsorbed

Red blood cell autoantibodies: The importance of being earnestly autoadsorbed

Predictors of autoimmune hemolytic anemia in beta-thalassemia patients with underlying red blood cells autoantibodies

In beta-thalassemia patients, erythrocyte autoantibodies can remain silent or lead to Autoimmune Hemolytic Anemia (AIHA).The aim of this study was to identify predictors of AIHA in beta-thalassemia patients with positive Direct Antiglobulin Test (DAT), in Tunisia.This longitudinal prognosis study was carried out on beta-thalassemia patients with a positive confirmed DAT. Predictors of AIHA were identified the Kaplan-Meier method. A Cox model analysis was used to identify independent predictors.Among 385 beta thalassemia patients, 87 developed positive DAT (22.6%). Autoimmune hemolytic anemia was occurred in 25 patients. Multivariate analysis showed that AIHA was independently associated with beta-thalassemia intermedia and similar family history of AIHA. Splenectomy in patients with positive DAT was independently associated with an increased risk of AIHA (HR = 6.175, CI: 2.049–18.612, p < 0.001). The risk of developing AIHA was higher during the first 72 transfusions. Autoimmune hemolytic anemia was significantly associated with polyspecific DAT (anti-complement and anti-IgG), blood group AB and prior alloimmunization. Whereas transfusion by phenotypic and leukoreduced blood was a protective factor.In summary, splenectomy after autoimmunization, prior alloimmunization, DAT specificity (IgG with complement), thalassemia intermedia, AB blood group and family history of AIHA were strongly associated with AIHA. Leukoreduced blood transfusion had a proven preventive role.

Red blood cell alloimmunizations in beta-thalassemia patients in Casablanca/Morocco: Prevalence and risk factors.

We aimed to study the prevalence and risk factors of red cell alloimmunization in β-thalassemic patients. Retrospective study during 9 years (2009-2018) was conducted on 160 β-thalassemic patients transfused regularly in pediatric department of children's hospital in Casablanca, Morocco. The main clinical, demographic and transfusional characteristics of alloimmunized and non-alloimmunized patients were compared. Red blood cells units transfused were leukodeplated and phenotypically matched for RH-KELL systems and for other systems after immunization. Screening and antibody identification were performed by gel-filtration method on BIO-RAD caseds using 3 and 11 red blood cells panels. To detect autoantibodies, autocontrol and direct antiglobulin tests were carried out using polyspecific coombs (IgG/C3d) gel cards. The prevalence of alloimmunizations was 8.75% during the study period. Seventeen alloantibodies identified were directed mainly against antigens of KELL and RH systems: KEL 1 (35.29%), RH 3 (23.52%), RH1 (11.76%), Kpa (11.76%), RH2 (5.88%). Red blood cells autoantibodies had been detected in 6 of 14 (42.85%) of alloimmunized patients versus 12 of 146 (11.76%) of non-alloimmunized patients (P<0.01). Presence of autoantibodies, transfusional interval<3weeks and gender were associated with high rate of red cells alloimmunization. This study proves the data of literature. The presence of red cell autoantibodies appears to be a major risk factors for alloimmunization in thalassemic children, and could advocate specific transfusion guidelines.

PB2309 INCIDENCE OF ALLOIMMUNIZATION IN SICKLE CELL DISEASE: EXPERIENCE OF A CENTER IN LISBON

Background:Nowadays, red blood cell (RBC) transfusion is an indispensable option for the treatment and prevention of chronic complications of sickle cell disease (SCD). In SCD patients it has been reported at a rate of 18–76% of alloimmunization after transfusion. It has also been suggested, that transfusion is related to the formation of RBC autoantibodies, especially in patients with hemoglobinopathies. Interestingly, some authors correlate higher susceptibility to allo and autoimmunization in patients without the spleen. That could be one of the explanations why SCD patients have a higher incidence of RBC immunization.Aims:To determinate the rate of alloimmunization among the transfused SCD patients residing in Lisbon. The other objectives were to clarify among the same study population the incidence of positive direct antiglobulin (DAT) test and spleen absence and correlate both with alloimmunization.Methods:The subjects for the study were selected from the adult patients (≥18 years of age) of a tertiary hospital in Lisbon, between 01‐01‐2017 and 20‐02‐2018, that were transfused at least one time and monitored for the presence of irregular antibodies. A total of 61 patients met the inclusion criteria. Most of the routine immunohematological tests, i.e. blood group typing, indirect antiglobulin test (IAT) and DAT, were done using a microtube gel technique. Moreover, clinical assessment of the spleen status (absence or presence) were obtained from imagiological data.Results:In our study, we obtained a rate of alloimmunization of 21,3% (N = 13). 40% of the transfused SCD (N = 22) have in DAT positive and 50% of those/these have alloantibodies. We found in our population a significant association between positive DAT with alloimmunization (p &lt; 0.001). In 58 patients we succeed in obtaining their spleen status and among those without a spleen (N = 36) 25% became alloimmunized.Summary/Conclusion:In our study, the risk of alloimmunization was higher in patients who received more than 20 red blood cell units. Our alloimmunization rate is in agreement with the literature as well as the significant association of DAT with alloimmunization. Interestingly, 50% of those patients with DAT positive also have alloantibodies, supporting the idea that those patients develop autoantibodies due to transfusion therapy. This fact advocates some plausibility in screening for autoantibodies at regular intervals prior to each transfusion. There was no significant correlation between the presence or absence of a spleen and alloimmunization (p = 0.7475). In conclusion, this study is one of the few to approach the rate of alloimmunization in SCD patients. Therefore, it is necessary to have further studies in Portugal in order to study and to understand our heterogeneous SCD population.

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.

Prevalence and clinical significances of red cell alloimmunization and red cell bound immunoglobulin G in polytransfused patients with thalassemias

ABSTRACTThe study was to determine the prevalence and clinical significances of red blood cell (RBC)-bound IgG as detected by flow cytometry in polytransfused patients with thalassemias. Relationship of the presence of RBC-bound IgG with RBC alloimmunization was also evaluated. This study included 59 polytransfused patients with β-thalassemia disease. We studied the frequency of RBC autoantibodies and alloimmunization. Direct Coombs test and flow cytometry were performed to detect the presence of RBC autoantibodies while RBC alloantibodies were detected by antibody screening and identification assays.Eight (13.6%) and 34 (57.6%) patients were found a positive direct Coombs test and flow cytometry, respectively. Twenty (33.9%) patients developed RBC alloantibodies. The four most frequent RBC alloantibodies were anti-E (55%), anti-Mia (40%), anti-Di(a) (25%) and anti-c (15%), respectively. There was no significant difference in the presence of RBC-bound IgG between polytransfused with thalassemia patients who developed RBC alloimmunization (13 of 20; 65%) and those without RBC alloantibodies (21 of 39; 53.8%), p = 0.412. Splenectomy and increased transfusion requirement were significantly associated with the presence of RBC-bound IgG but not with RBC alloantibody formation.The overall frequency of RBC alloantibody formation in polytransfused patients with thalassemias was 33.9%. The most common RBC alloantibody was anti-E. RBC autoantibody formation was more frequently detected by flow cytometry (57.6%) than by direct Coombs test (13.6%). Splenectomy was significantly associated with the development of autoreactive RBC-bound IgG antibodies in the polytransfused patients with thalassemias. The presence of the anti-RBC autoantibodies may cause an increase of transfusion requirement.

Severe Warm Autoimmune Hemolytic Anemia in a 7-Month-Old Infant Associated With a Mycoplasma Pneumoniae Infection.

A 7-month-old female infant had clinical and laboratory evidence of severe warm autoimmune hemolytic anemia. She also had clinical evidence of an upper respiratory tract infection with molecular detection of Mycoplasma pneumoniae. Although reticulocytopenic initially, she responded to red blood cell transfusion, steroids, and intravenous immunoglobulin and remains well today. With the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the rare association described in this report may prove to be more common than previously thought and may provide insight into the pathogenesis and clinical consequences of red blood cell autoantibodies.

The Polymorphism of SMIM1 Gene in Chinese Dividuals.

The SMIM1 gene, which encodes the high-frequency blood group antigen Vel, has not been systematicallyanalyzed at the molecular level in Chinese individuals. To better understand the SMIM1 genetic polymorphism, we assessed mutations among healthy Chinese individuals, patients with red blood cell autoantibodies and hematological disease. A total of 130 patients with hematological disease (case I group), 50 patients with red blood cell autoantibodies (case II group), and 500 healthy controls (control group) were enrolled. Exons 3 and 4 in the SMIM1 gene were sequenced to identify genetic variants or mutations. A polyclonal anti-Vel antibody was used to evaluate the expression of the Vel antigenon red blood cells in patients with novel alleles. The novel alleles of the SMIM1 gene were intron 3 position 193 (TT, CT, CC), 194 (GG, AG), 3' untranslated region positions 81 (CC, CA) and 87 (AA, CA). The single nucleotide polymorphism (SNP) frequencies of intron 3 position 193 TT, CT, CC were 13.1, 39.2, 47.7% in case group I, 6.7, 33.3, 60.0% in case group II and 8.5, 35.6, 56.2% in the control group, respectively. Other minor allele frequencies were all greater than 10% and all SNPs in Chinese showed Vel antigen expression on RBC membranes. The allele at intron 3 position 193 was the most frequent mutant allele found in the Chinese population and Vel antigen deficiency may not cause problems in Chinese patients with hematological diseases and RBC autoantibodies.

Red blood cell alloantibodies and autoantibodies: different presentation, same physiopathology

Red blood cell alloantibodies and autoantibodies: different presentation, same physiopathology

Optimized Antigen-Matched in Sickle Cell Disease Patients: Chances and Challenges in Molecular Times - the Brazilian Way

The development of red blood cell (RBC) alloantibodies and autoantibodies complicates transfusion therapy in sickle cell disease (SCD) patients. In an effort to reduce the risk of alloimmunization, some strategies have been used to provide antigen-matched RBC transfusions to patients with SCD in Brazil, including molecular matching in 3 levels: RH and K matching; extended matching (RH, KEL, FY, JK, MNS, DI), and extended matching including RHD and RHCE variant alleles. Molecular matching has shown clinical benefits to the patients with SCD, contributing significantly to reduce the rates of alloimmunization. Improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hemoglobin levels and reduction in their percentage of hemoglobin S as well as better in vivo RBC survival and diminished frequency of transfusions. However, prevention of RBC alloimmunization still remains a challenge in Brazil due to the difficulty to fulfill all transfusion requests of the patients with antigen-matching units, inaccuracy of RBC phenotyping, RBC transfusions outside the institution where the patient is treated, advanced age of some patients, the RBC antigen discrepancy between donors and recipients, and the presence of RH variants.

Induction of IL-10 cytokine and the suppression of T cell proliferation by specific peptides from red cell band 3 and in vivo effects of these peptides on autoimmune hemolytic anemia in NZB mice

PurposeThe anion channel protein band 3 is the main target of the pathogenic red blood cells (RBC) autoantibodies in New Zealand black (NZB) mice. CD4 T cells from NZB mice with autoimmune hemolytic anemia respond to band 3. Previously, we have shown that IL-10 and peptides containing a dominant T cell epitope from red cell band 3 modulate autoimmune hemolytic anemia in NZB mice. Because of the immunoregulatory role of IL-10 in autoimmune diseases, we aim to identify individual band 3 peptides that induce high IL-10 production and simultaneously suppress CD4 T cell proliferation and to investigate the effect intranasal administration of IL-10 producing band 3 peptides on autoantibody responses of NZB mice.MethodsSplenic CD4 T cells of NZB mice were isolated and stimulated by co-culture of T cells with individual band 3 peptides. IL-10 production was measured by enzyme-linked immunosorbent assay and proliferative response of CD4 T cells was estimated by incorporation of [3H] thymidine assay. NZB mice were given either PBS, or peptides 25 (241–251) and 29 (282–296) or both peptides intranasally on three occasions at 2-day intervals. The mice were bled at 6, 10 and 18 weeks after peptide inhalation, and the number of RBC auto-antibodies was measured by DELAT and hematocrit values were assessed.ResultsPeptides 25 (241–251) and 29 (282–296) induced the highest IL-10 production by CD4 T cells. These peptides also inhibited the peak T cell proliferative response. 6 and 10 weeks after peptide inhalation, the total IgG, IgG1 and IgG2a in mice treated with both peptides 241–251 and 282–296 were significantly higher than control (P < 0.05). However, no significant difference in the mean hematocrit between of the peptide-treated mice and the control group was found.ConclusionsAlthough band 3 peptides 241–251 and 282–296 induced to the highest IL-10 production by CD4 T cells in vitro but fail to reverse the RBC autoantibody response in vivo. Modifications to improve solubility these peptides might help to modulate the immune response toward a T helper-2 profile and decrease the severity of anemia.

Autoimmune hemolytic anemia: transfusion challenges and solutions

Autoimmune hemolytic anemia: transfusion challenges and solutions Melca M O Barros, Dante M Langhi Jr, Jos&eacute; O Bordin Department of Clinical and Experimental Oncology, Universidade Federal de S&atilde;o Paulo, S&atilde;o Paulo, Brazil Abstract: Autoimmune hemolytic anemia (AIHA) is defined as the increased destruction of red blood cells (RBCs) in the presence of anti-RBC autoantibodies and/or complement. Classification of AIHA is based on the optimal auto-RBC antibody reactivity temperatures and includes warm, cold-reactive, mixed AIHA, and drug-induced AIHA subtypes. AIHA is a rare disease, and recommendations for transfusion are based mainly on results from retrospective data and relatively small cohort studies, including heterogeneous patient samples or single case reports. In this article, we will review the challenges and solutions to safely transfuse AIHA patients. We&nbsp;will reflect on the indication for transfusion in AIHA and the difficulty in the accomplishment of immunohematological procedures for the selection of the safest and most compatible RBC units. Keywords: hemolytic anemia, RBC autoantibodies, autoimmunity, hemolysis, direct &shy;antiglobulin test

Association of HLA-DRB1 and HLA-DQB1 alleles with red blood cell auto-immunization in the Chinese population

Red blood cell (RBC) auto-immunization poses a clinical dilemma, because the formation of RBC autoantibodies in certain patients is a significant causative factor for autoimmune hemolytic anemia. To clarify whether specific HLA-DRB1 and HLA-DQB1 alleles are associated with the response to RBC auto-immunization, we conducted a case-controlled retrospective study. The case group included 117 patients with autoantibodies potentially harmful to RBC after transfusion at the Beijing Red Cross Blood Center, and the controls were 1.6 million healthy subjects whose data are stored in the China Marrow Donor Program database. In the case group, the frequencies of HLA-DRB1 and HLA-DQB1 allele were determined by PCR-rSSO Luminex high-resolution technology. HLA-DRB1*13 allele expression was not detected in the 75 autoantibody positive patients with anti-C3d production (adjusted P=0.0383), whereas the presence of HLA-DRB1*14 allele showed a positive correlation with the autoantibody positive group with anti-C3d production (odds ratio = 2.22, 95% confidence interval = 1.293.84, adjusted P=0.0383). The HLA-DQB1*02 allele was more frequent in patients with both RBC auto-antibodies and alloantibodies. There was no significant association between a positive antiglobulin test result and the presence of HLA-DRB1 and HLA-DQB1 alleles. In summary, we found that HLA-DRB1*13 may serve as a protective factor, whereas HLA-DRB1*14 appears to be a risk factor for the production of autoantibodies positive with anti-C3d in RBC auto-immunization. To our knowledge, this is the first study to propose HLA-DRB1*13 as a protective factor in auto-immunization.

Development of red blood cell autoantibodies following treatment with checkpoint inhibitors: a new class of anti-neoplastic, immunotherapeutic agents associated with immune dysregulation

Ipilimumab, nivolumab, and pembrolizumab represent a new class of immunotherapeutic drugs for treating patients with advanced cancer. Known as checkpoint inhibitors, these drugs act to upregulate the cellular and humoral immune response to tumor antigens by inhibiting T-cell autoregulation. As a consequence, they can be associated with immune-related adverse events (irAEs) due to loss of self-tolerance, including rare cases of immune-related cytopenias. We performed a retrospective clinical chart review, including serologic, hematology, and chemistry laboratory results, of two patients who developed red blood cell (RBC) autoantibodies during treatment with a checkpoint inhibitor. Serologic testing of blood samples from these patients during induction therapy with ipilimumab and nivolumab, respectively, showed their RBCs to be positive by the direct antiglobulin test (IgG+, C3+) and their plasma to contain panreactive RBC autoantibodies. Neither patient had evidence of hemolysis. Both patients developed an additional irAE during treatment. A literature review for patients who had developed immune-mediated cytopenia following treatment with a checkpoint inhibitor was performed. Nine other patients were reported with a hematologic irAE, including six with anemia attributable to autoimmune anemia, aplastic anemia, or pure RBC aplasia. Hematologic irAEs tend to occur early during induction therapy, often coincident with irAEs of other organs. In conclusion, checkpoint inhibitors can be associated with the development of autoantibodies, immune-mediated cytopenias, pure RBC aplasia, and aplastic anemia. Immunohematology reference laboratories should be aware of these agents when evaluating patients with advanced cancer and new-onset autoantibodies, anemia, and other cytopenias.

Patients with Autoimmune Anti-Red Blood Cell IgG1 Have Different Total- and Autoantibody -IgG1 Glycosylation Compared to Healthy Controls

IntroductionAutoimmune hemolytic anemia (AIHA) is a potentially severe disease in which red blood cells (RBC) are destroyed by autoantibodies. IgG anti-RBC autoantibodies bind at 37°C and can lead to extravascular hemolysis by binding to phagocytes in the spleen.The composition of the N-linked sugar at position 297 in the IgG-Fc tail influences the binding affinity to IgG Fc receptors (FcγR) on effector cells. By mass spectrometric analysis we recently extensively studied the contribution of this sugar moiety to the clinical effect of RBC and platelet alloantibodies in pregnant women. Compared to the IgG glycoforms detected in plasma of these women, anti-D, anti-K and anti-Human Platelet Antigen 1a were found to be skewed towards IgG1 isoforms with low fucosylation, increasing binding capacity to IgG-Fc receptors type IIIa and IIIb (Kapur et al Blood 2014;123:471-80 & BHJ 2015;166:936-45; Sonneveld et al BJH 2016; 174:310-320 and oral presentation ASH Blood 2015;126:660). This feature has only been described for anti-HIV antibodies, but never for any other immune response. Furthermore, we observed an additional role for the level of bisected GlcNAc (bisection), galactosylation and sialylation of the RBC alloantibody-bound sugar moiety in predicting clinical outcome of HDFN. It is known that in autoimmune diseases the galactosylation and sialylation of plasma IgG1 is lowered, the glycoforms of specific autoantibodies have not yet been studied.AimThe aim of this study was to investigate the type of glycoforms of RBC autoantibodies in relation to the plasma IgG1 glycoforms in these patients and occurrence of hemolysis.Methods109 patients with a positive direct antiglobulin test (DAT) with anti-IgG were included, of whom blood was sent to our laboratory for diagnostic purposes. From 90 patients clinical data were collected. Based on laboratory markers (hemoglobin (Hb), haptoglobin, lactate dehydrogenase, reticulocyte count, bilirubin level), these patients were divided in three groups: 1. with hemolysis (n=36), 2. no hemolysis (n=33) or 3. possible hemolysis (n=21). RBC-bound autoantibodies were purified by acid elution. This RBC-eluted IgG and total IgG purified from paired plasma samples were subjected to tryptic digestion followed by mass spectrometry for glycoprofiling. Total IgG1 glycosylation patterns from healthy donors were used as controls.ResultsIn agreement with the previously described low IgG1-galactosylation and sialylation in patients with autoimmune diseases, we observed that IgG1 purified from plasma of patients with RBC-bound antibodies (a positive DAT) showed decreased galactosylation and sialylation levels compared to healthy controls: average 36% (95% CI, 34-37%) versus 53% (95%CI, 50-56%) (p<0.0001) and 2.4% (95% CI, 1.8-2%) versus 4% (95% CI, 3.4-4.5%), respectively. We now observed that total bisection levels were higher in patients with RBC autoantibodies (19%) versus healthy controls (12%) (p<0.001).Compared to plasma IgG1 glycosylation, the RBC-bound autoantibodies showed a profile with lower galactosylation, higher sialylation and lower bisecting GlcNAc levels (p=0.0029, p=0.0128 and p<0.0001 respectively). Fc-fucosylation was not different between healthy controls and patients for plasma IgG1 and not for RBC-bound IgG1 fractions (p=0.4986 and p=0.29 resp.).Analysis of Fc glycoprofiles in relation to Hb levels showed that higher bisection was associated with higher Hb levels for the total group of patients (p<0.001). This correlation was not found analyzing the three patients groups separately, probably due to low sample number.In conclusion, we found a low total IgG1 Fc-galactosylation in patients with RBC-bound IgG antibodies irrespective of occurrence of hemolysis. This supports the hypothesis that galactosylation is a marker for activation of the immune system in autoimmune diseases. In addition, we found RBC-bound IgG1 to be differently skewed in the composition of the N-linked sugar at position 297 compared to plasma-derived IgG1 in these patients. DisclosuresNo relevant conflicts of interest to declare.