Anti-red Blood Cell Antibodies Research Articles

Impact of human leucocyte antigen class II polymorphism on anti-red blood cell antibody development: Correlations and indications.

Blood transfusion therapy is vital for many patient groups. They can cause many complications, and the development of anti-red blood cell (RBC) antibodies is of significant importance. Molecules of class II human leucocyte antigens (HLA) are one of the several factors that influence antibody development in patients. In this study, we investigated 108 patients who developed antibodies against different erythrocyte antigens and 115 patients on multiple transfusion therapies who did not develop anti-RBC antibodies. The HLA loci HLA-DRB1 and HLA-DQB1 were typed using commercial molecular assays routinely used in HLA laboratories. An increased frequency of the HLA-DRB1*04 allele group was observed in patients who developed antibodies. Additionally, HLA-DRB1*09 was also significant for anti-E development and in patients with multi-specific alloimmunization. It was found that the HLA-DRB1*07 allele group is associated with antibodies to antigents of the Rh and MNS systems but also lacks an association with anti-K development. The HLA-DRB1*11 and -DRB1*01 allele groups displayed a protective mechanism for anti-E development, similar to that of HLA-DQB1*02 for anti-K. There is an association between various HLA class II alleles and anti-RBC development.

PREVALENCE OF IRREGULAR RED CELLANTIBODIES IN HEALTHY BLOOD DONORS ATTENDING A TERTIARY CARE HOSPITAL IN SOUTH INDIA

Introduction: Red cell antibodies that are found normally in human serum are considered naturally occurring and those are anti A and anti B. All other antibodies directed against RBC antigens are considered “unexpected or irregular". Aim: This study is aimed to evaluate the prevalence of the anti-red blood cell antibodies among healthy blood donors. Material and Methods: Antibody screening and identification was done using commercially available 3 cell and 11 cell reagent cells (0.8% Surgiscreen, Ortho Clinical Diagnostics Limited, USA and Low ionic Strength Saline Ortho Bliss with AHG Cassettes) in antihuman globulin phase. Results: A total of 36,684 donors were screened for the presence of irregular erythrocyte antibodies. Among these donors, twenty donors showed presence of alloantibodies in their serum (0.054%). Most frequent alloantibodies identified were from Lewis blood group system. The results showed statistically a higher prevalence of RBC alloantibodies in males than in females. Conclusion: Screening for presence of alloantibodies in donor blood is important to provide compatible blood products and to avoid transfusion reactions.

Use of a whole-cell ELISA to detect additional antibodies in setting of suspected heparin-induced thrombocytopenia.

Type II heparin-induced thrombocytopenia (HIT) is mediated by formation of antibodies to platelet factor 4 (PF4)-heparin complexes. We evaluated anti-PF4-heparin-negative samples for the presence of additional anti-platelet and anti-red blood cell (RBC) antibodies using whole-cell platelet/ RBC ELISAs we developed. Seventy-three samples tested for anti-PF4-heparin by ELISA were included: 62 tested negative, 9 tested positive, and 2 had equivocal results. Plasma specimens from healthy donors were used as controls. 100% (9/9) anti-PF4-positive samples had anti-platelet antibodies detected by whole-cell platelet ELISA. 42.2% (27/64) anti-PF4-heparin-negative samples were negative for anti-platelet and anti-RBC antibodies. 32.8% (21/64) negative samples showed reactivity to both platelets and RBC; 12.5% (8/64) negative samples were each reactive with either platelet or RBC ELISA, respectively. Additionally, two samples that tested equivocal by anti-PF4-heparin ELISA had antibodies to both platelets and RBC by whole-cell ELISA. Our study suggests that patients with thrombocytopenia testing negative for anti-PF4-heparin may still harbor antibodies to platelets. However, additional research is needed to determine the significance of these antibodies. Nevertheless, these findings may encourage clinicians to further investigate patients with possible immune-mediated etiologies of thrombocytopenia and anemia.

PS1596 AUTOIMMUNIZATION AND ALLOIMMUNIZATION IN TRANSFUSION DEPENDANT THALASSEMIA MAJOR PATIENTS:STUDY ON 300 PATIENTS

Background:Thalassemia is the most common genetic disorder worldwide and one of the major public health problems. Thalassemia patients are dependent on blood transfusions for their survival but they may be associated with many complications. Development of anti red blood cell antibodies(bot alloantibodies and autoantibodies) is one of them. There is a paucity of data on the incidence of alloimmunization and autoimmunization in thalassemic patients in Pakistan,as pre transfusion antibody screening is not routinely performed. Autoantibodies can result in clinical hemolysis and difficulty in cross matching blood. We studied the frequency of Red cell alloimmunization and autoimmunization among Thalassemia patients who received regular transfusions at Sundas Foundation and also to determine types of these antibodies among Thalassemia patients.Aims:To study the frequency of Red Cell alloimmunization and autoimmunization among multiple transfused Thalassemia patients and to determine the types of these antibodies.Methods:A total of 300 multiple transfused Thalassemia patients were included in this study. Clinical and serological records of all the patients were examined. Screening and identification of antibodies was performed on fresh serum of all patients using 3 cell and 11 cell panel respectively. Detection of autoantibodies was done using using polyspecific Coombs(IgG+C3d) gel cards. Approval of this study was taken from the institutional review board. Informed consent was provided.Results:We identified alloantibodies in 17 patients,the most common were Anti E followed by Anti D,Anti C,Anti K,Anti Fyb,Anti‐M.4 patients developed autoantibodies.Summary/Conclusion:Alloimmunization is an immune respone stimulated by the transfusion of blood products. Overall rate of alloimmunization was 5.66% in our study while it is 30%/190 in Kuwait,4.97%/161 in India,5%/1435 in Italy,3.7%/1200 in Greece,5%/711 in Iran,9.2%/97 and 22.7%/75 in Pakistan.

Prevention of hemolytic disease of the fetus and newborn: what have we learned from animal models?

This review aims to highlight recent advances in our understanding of how anti-red blood cell (RBC) antibodies prevent erythrocyte immunization with an emphasis on new murine models. New murine models with clinically relevant human erythrocyte antigens have been used to understand the alloimmunization process and its inhibition. The search to elucidate the mechanism of action of IgG-mediated inhibition of erythrocyte alloimmunization has provided new evidence in support of a potential role for epitope masking, immune deviation and/or antigen modulation in this process. In addition, recent evidence suggests that blends of monoclonal antibodies targeting nonoverlapping epitopes on the RBC surface can improve the efficacy of monoclonal antibodies approaching that of polyclonal IgG. Animal models with defined alloantigens have helped to identify important mechanistic components that lead to alloimmunization and its inhibition by IgG. A better understanding of the underlying mechanisms leading to hemolytic disease of the fetus and newborn is required to develop the most effective prevention strategies for future patients.

Anti-H Antibody Showing Agglutination in the Anti-Human Globulin Phase in a Korean Patient

Dear Editor, Anti-H antibody is a type of anti-red blood cell (RBC) antibody that agglutinates with H antigen, which is universally present on human RBCs. The most widely known problem related to the anti-H antibody is the potentially lethal hemolytic transfusion reaction seen in individuals with the Bombay O blood type [1,2,3]. Other anti-H antibodies are mostly found in A1 or A1B blood type individuals and are usually cold reactive and clinically benign [3]. We report a Korean patient with an anti-H antibody exhibiting a wide thermal range. A 79-yr-old woman diagnosed with pneumonia was due to receive a transfusion of RBCs in the Guro hospital of Korea University, Seoul, Korea. Her blood type was A, RhD+. However, the antibody-screening test was positive; an antibody identification test revealed 11 positive panels with no reaction to autologous RBCs by using the Ortho BioVue Innova system (Ortho-Clinical Diagnostics, Raritan, NJ, USA). The manufacturer's test RBCs used in the antibody screening and identification tests were O cells. ABO serotyping of the patient's serum with O cells showed strong agglutination. We suspected anti-IH or anti-H antibodies with wide thermal amplitude and conducted further evaluation [4]. An ABO genotyping was performed for accurate genotype identification. Various types of RBCs from random donors were used, including autologous A1, adult O, RhD- O, A1, and enzyme-treated O cells. A1 cells were tested with the patient's serum using both the column agglutination test (CAT) and tube method [5,6]. Cord blood A1, B, O, and A1B cells were tested to rule out the possibility of anti-IH antibodies [7]. Additionally, dithiothreitol (DTT)-treated serum was tested with O cells to specify the antibody's immunoglobulin type. The Ortho BioVue Innova system was used for the CAT; tests were carried out at room temperature and Coombs' phase where appropriate. All tests were conducted according to the procedures indicated in the AABB Technical Manual and with methods described by the relevant manufacturers [3]. The patient was identified as having an A102/A102 genotype through sequence analysis. The antibody identification test showed 4+ in all panels through saline, 30 min cold incubation, albumin, and Coombs' phase; no agglutination with autologous RBCs was observed as noted above. The CAT of the patient's serum with adult A1 cells showed no agglutination, including autologous RBCs. Tests with adult O cells revealed agglutination of 3+ or more in all phases. These results suggested the presence of anti-IH or anti-H antibodies, as did the strong reaction with H antigen-abundant O cells and weak or absent reactions with A1 cells that lacked H antigens. Enzyme treatment of RBCs did not cause any significant changes in reactivity to O cells, while papain-treated A1 cells showed agglutination of 2+ or more. The specific effect of enzyme treatment on A1 cells in reaction with anti-H or anti-IH antibodies was unclear; results of this test did not favor any specific type of antibody (Table 1). Table 1 Column agglutination test with various RBCs Cord blood A1 cells showed weak reactions only in the tube method performed at room temperature. Neonatal RBCs have incomplete development of ABO antigens and have fewer H antigens on their surface compared with adult RBCs [3]. Thus, these results suggested that the antibody reacted with a small amount of H antigen left on RBCs with an incomplete A1 phenotype. Cord blood O cells showed 3+ reactions only at room temperature. The lack of agglutination in Coombs' phase was interpreted as a weakened reaction due to fewer H antigens on RBC surfaces. DTT-treated serum showed no agglutination with adult O cells in contrast to phosphate-buffered saline (PBS)-mixed control samples, as the treatment inactivated IgM, which were identified as cold antibodies with a sufficiently high titer to react in the Coombs' phase (Table 2). Table 2 Adult O cell with dithiothreitol-treated serum Anti-IH antibody reacts weakly or not at all with I-RBCs, and reacts strongly only in the presence of both I and H antigens [7,8]. Cord blood RBCs lack I antigens; therefore, 3+ agglutination of cord blood O cells at room temperature signified the presence of anti-H antibodies. Adult Oi cells could have further confirmed this diagnosis; however, blood of that type was unavailable. The patient received a transfusion shortly after the identification test, and further evaluations at 4℃ could not be performed owing to insufficient sample amount. In conclusion, we identified a case of anti-H antibody with a high titer, which was not reported in previous studies of the frequencies of unexpected antibodies in the Korean population [9,10]. This report contributes to identification of anti-H antibodies in cases of unusual ABO discrepancies.

Alloimmunization and autoimmunization in transfusion dependent thalassemia major patients: Study on 319 patients.

Background:The development of anti-red blood cell antibodies (both allo-and autoantibodies) remains a major problem in thalassemia major patients. We studied the frequency of red blood cell (RBC) alloimmunization and autoimmunization among thalassemia patients who received regular transfusions at our center and analyzed the factors, which may be responsible for development of these antibodies.Materials and Methods:The study was carried out on 319 multiply transfused patients with β-thalassemia major registered with thalassemia clinic at our institute. Clinical and transfusion records of all the patients were examined for age of patients, age at initiation of transfusion therapy, total number of blood units transfused, transfusion interval, status of splenectomy or other interventions. Alloantibody screening and identification was done using three cell and 11 cell panel (Diapanel, Bio-rad, Switzerland) respectively. To detect autoantibodies, autocontrol was carried out using polyspecific coombs (IgG + C3d) gel cards.Results:Eighteen patients out of total 319 patients (5.64%) developed alloantibodies and 90 (28.2%) developed autoantibodies. Nine out of 18 patients with alloantibodies also had autoantibodies. Age at first transfusion was significantly higher in alloimmunized than non-immunized patients (P = 0.042). Out of 23 alloantibodies, 52.17% belonged to Rh blood group system (Anti-E = 17%, Anti D = 13%, Anti-C = 13%, Anti-Cw = 9%), 35% belonged to Kell blood group system, 9% of Kidd and 4% of Xg blood group system.Conclusion:Alloimmunization was detected in 5.64% of multitransfused thalassemia patients. Rh and Kell blood group system antibodies accounted for more than 80% of alloantibodies. This study re-emphasizes the need for RBC antigen typing before first transfusion and issue of antigen matched blood (at least for Rh and Kell antigen). Early institution of transfusion therapy after diagnosis is another means of decreasing alloimmunization.

Combinatorial Effect of Pharmacological Inhibitors of Lsd-1 and DNMT on γ-Globin Synthesis in Cultured Baboon Bone Marrow Erythroid Progenitors

Abstract 249Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). New therapies to increase HbF levels in patients are urgently needed. Previous studies from our laboratory have shown that decitabine, an inhibitor of DNA methyltransferase (DNMT), increases HbF to therapeutic levels in non-human experimental primates and SCD patients, thus demonstrating that drugs targeting repressive epigenetic modifications can be effective HbF-inducing agents. Both DNMT1 and Lysine Specific Demethylase-1, (LSD-1), a mono- and dimethyl- histone H3 K4 demethylating enzyme, are components of the DRED multiprotein complex, a repressor of γ-globin expression (Cui et al Mol Cell Biol 31:3298–3311, 2011). Experiments were performed to test the effect of the pharmacological LSD-1 inhibitor, tranylcypromine (TC), alone and in combination with the DNA methyltransferase inhibitor decitabine (DAC), on γ-globin expression. Baboon bone marrow (BM) CD34+ erythroid progenitors were isolated from BM aspirates of normal baboons and grown in a co-culture system with the AFT024 murine fetal liver cell line in Iscove’s media containing 30% fetal bovine serum, 200ng/ml SCF, 2U/ml epo, and 1μM dexamethasone. TC (0.5 to 5.0μM) was added to cultures on d7 and d10, while decitabine (0.1μM) was added on d7. On d14 erythroid cells were purified by immunomagnetic column chromatograpy using a baboon-specific anti-red blood cell antibody (BD Bioscience). Effects on globin expression were measured by biosynthetic radiolabelling of nascent globin chain synthesis in the presence of [3H] leucine followed by HPLC separation of individual globin chains. DNA was isolated and effect of the drugs on DNA methylation of the γ-globin promoter determined by bisulfite sequence analysis. Effects on erythroid differentiation were investigated by examination of Wright’s stained cytospin preparations. In three independent cultures TC induced dose-dependent increases in γ-globin chain synthesis (γ/γ+β). In addition TC and DAC in combination increased γ-globin chain synthesis in an additive manner (see Table).CD34+ cell sourceTC (μM)DAC (μM)γ/γ+βPA 7680000.0600.10.325.000.305.00.10.62PA 7482000.0900.10.352.500.165.000.302.50.10.545.00.10.68Bisulfite sequence analysis showed that increased γ-globin chain synthesis in TC-treated cultures was not associated with increased DNA hypomethylaton of the γ-globin promoter. Examination of Wright’s stained cytospin preparations of d14 cultures showed increased numbers of basophilic and polychromatic erythroblasts in cultures exposed to TC compared to untreated controls suggesting that TC reduced the rate of erythroid differentiation. In contrast, numbers of basophilic and polychromatic cells were reduced and orthochromatic cells were increased in cultures treated with TC and DAC compared to TC alone. We conclude that TC increased γ-globin chain synthesis in a dose-dependent manner in baboon erythroid progenitor cell cultures while TC and DAC, in combination, produced additive effects on γ-globin chain synthesis with minimal effects on erythroid differentiation. These results suggest that combinatorial use of epigenetic modifiers may be superior to single agents and support further studies to determine whether TC and other LSD-1 pharmacological inhibitors can effectively be used in combination with DNMT inhibition to increase HbF levels in vivo. Disclosures:No relevant conflicts of interest to declare.

Critical role of Th17 cells in development of autoimmune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is defined as the increased destruction of red blood cells (RBCs) in the presence of anti-RBC autoantibodies with or without complement activation. However, the underlying mechanism for the development of AIHA remains largely unclear. In this study, we carefully evaluated the potential role of Th17 cells in the development of AIHA. We found an elevated frequency of Th17 cells in patients with AIHA, which were closely correlated with their disease activity, including the level of anti-RBC IgG antibodies, hemoglobin, serum C3, and lactate dehydrogenase activity. Furthermore, we observed that interleukin (IL)-17 was also closely correlated with the disease activity in AIHA patients. To further elucidate the potential role of Th17 cells in induction of AIHA, we used the Marshall-Clarke and Playfair model of murine AIHA. Notably, we found that Th17 cells affected development of AIHA by enhancing the adaptive humoral responses. Specifically, we found that adoptive transfer of Th17 cells heightened the initial anti-rat RBC antibody responses and concomitantly increased the onset of AIHA. In addition, invivo neutralization of IL-17 abrogated the development of AIHA, while initiation of anti-rat RBC IgG responses and induction of AIHA in IL-17(-/-) mice were impaired. Our findings suggest that Th17 cells contribute to the development of AIHA, which could facilitate our better understanding of AIHA pathogenesis and provide clues to developing novel forms of immunotherapy against AIHA.

Screening of blood donors for erythrocyte alloantibodies

ObjectiveTo assess the prevalence of the anti-red blood cell antibodies in the donor population of Delhi.MethodsThis prospective study was conducted in Regional Blood Transfusion Centre (RBTC), Lady Hardinge Medical College (LHMC) and associated hospitals from March 2010 to March 2011. Antibody screening of all donor serum/plasma was performed as routine immunohaematological procedure. Any positive sera were further investigated to identify the specificity of irregular erythrocyte antibody by commercially available red cell panel (ID-Dia Panel, Diamed-ID Microtyping System). The titres and thermal amplitude of the identified antibodies were evaluated.ResultsA total of 7756 donors were screened, of which 7648 donors were males (98.6%) and 108 were females (1.4%). The maximum number of donors belonged to age group of 26–30 years. A total of four donors showed presence of alloantibodies in their serum (0.05%). On antibody identification, two of them were anti-C, one was anti-Lewisa antibody and one was autoantibody.DiscussionThis study was conducted to highlight the significance of detecting irregular erythrocyte antibodies in healthy donors.

Histocompatibility Assessment in the Chicken Colonies of the RIR-Y8/NU, YL, WL-G, and BL-E Closed for 28-48 Years

Histocompatibility was assessed in the RIR-Y8/NU, BL-E, YL, and WL-G chicken closed colonies by hemagglutination test using anti-red blood cell (RBC) antibodies (HT), skin transplantation test (STT), and formation of isohemagglutinins (FIHs) during STT. The YL individuals all showed the survival of skingrafts for more than 17 days with no FIHs in STT and no RBC antigenic variations in HT, indicating a histocompatible nature together with high homogeneity at serological loci. The BL-E as well as WL-G closed colonies were also found to be histocompatible in the STT with no FIHs, although the HT showed heterogeneities at serological locus/loci other than the B and C blood group loci which have significant effects on histocompatibility or FIHs in chicken. In the RIR-Y8/NU closed colonies, one individual in 6 reciprocal combinations of the STT showed early skingraft rejection with positive FIHs caused by different B locus alleles, and the HT suggested relatively high heterogeneities at the other serological loci too. The closed colonies of YL, BL-E, and WL-G will be useful avian materials for transplantation or related experiments, but RIR-Y8/NU needs further pedigree selection for serological homogeneity.

Anti‐Erythrocyte Antibodies and Disease Associations in Anemic and Nonanemic Dogs

Flow cytometry has been used to detect anti-red blood cell (RBC) antibodies in dogs with immune-mediated hemolytic anemia (IMHA), but the prevalence of anti-RBC antibodies in anemic and nonanemic dogs with a variety of different diseases has not been assessed previously. We hypothesized that anti-RBC antibodies would be more common in anemic dogs and in dogs with immune-mediated disorders and cancer. Blood samples from 292 dogs were analyzed prospectively by flow cytometry for anti-RBC antibodies. Blood samples from 147 anemic and 145 nonanemic dogs were evaluated by flow cytometry to detect surface-bound immunoglobulin (Ig) G and IgM antibodies on RBC. Disease associations with RBC antibodies were determined, as was the correlation between disease status and the percentage of Ig(+) RBC. The specificity and sensitivity of flow cytometry and clinical variables for the diagnosis of IMHA were compared by Bayesian analysis. Anemic dogs were significantly more likely to be positive for anti-RBC antibodies (IgG, IgM, or both) than nonanemic dogs. Anemic dogs also had significantly higher percentages of Ig(+) RBC than nonanemic dogs, whereas dogs with IMHA had significantly higher percentages of Ig(+) RBC than dogs with all other diseases. Dogs with IMHA, infectious diseases, and immune-mediated thrombocytopenia were significantly more likely to have anti-RBC antibodies than dogs with other medical or surgical diseases. Anemic dogs with immune-mediated diseases and infectious diseases were at the highest risk for the development of anti-RBC antibodies, and flow cytometry for the detection of IgG on RBC was highly sensitive and specific for the diagnosis of IMHA.

Emergence of Anti-Red Blood Cell Antibodies Triggers Red Cell Phagocytosis by Activated Macrophages in a Rabbit Model of Epstein-Barr Virus-Associated Hemophagocytic Syndrome

Hemophagocytic syndrome (HPS) is a fatal complication frequently associated with viral infections. In childhood HPS, Epstein-Barr virus (EBV) is the major causative agent, and red blood cells (RBCs) are predominantly phagocytosed by macrophages. To investigate the mechanism of RBC phagocytosis triggered by EBV infection, we adopted a rabbit model of EBV-associated HPS previously established by using Herpesvirus papio (HVP). The kinetics of virus-host interaction was studied. Using flow cytometry, we detected the emergence of antibody-coated RBCs, as well as anti-platelet antibodies, at peak virus load period at weeks 3 to 4 after HVP injection, and the titers increased thereafter. The presence of anti-RBCs preceded RBC phagocytosis in tissues and predicted the full-blown development of HPS. The anti-RBC antibodies showed cross-reactivity with Paul-Bunnell heterophile antibodies. Preabsorption of the HVP-infected serum with control RBCs removed the majority of anti-RBC activities and remarkably reduced RBC phagocytosis. The RBC phagocytosis was specifically mediated via an Fc fragment of antibodies in the presence of macrophage activation. Therefore, the emergence of anti-RBC antibodies and the presence of macrophage activation are both essential in the development of HPS. Our observations in this animal model provide a potential mechanism for hemophagocytosis in EBV infection.

Inhibition of IL-1β and TNFα Gene Expression by IVIg or Anti-Red Blood Cell Antibodies in a Mouse Model of ITP.

Plasma-derived human IgG (IVIg) have been used for more than 2 decades for the treatment of several autoimmune diseases such as ITP, even if their mechanism of action is still not fully understood. Recently, several studies with IVIg-treated patients have shown that IVIg can modulate the serum level of both pro-inflammatory and anti-inflammatory cytokines. In ITP, it is known that the destruction of autoantibody-coated platelets occurs mainly in the spleen. We thus used a mouse model of ITP to evaluate the modulatory effects of IVIg on the expression of several cytokine genes in the spleen of the animals by quantitative RT-PCR. The results showed that the induction of platelet destruction following injection of monoclonal anti-platelet antibodies (MWReg30) in BALB/c mice did not change the expression level of IL-1ra and IL-10 (anti-inflammatory cytokines) and IL-6, IFNγ and MCP-1 (pro-inflammatory cytokines). In contrast, MWReg30-treated mice showed an increase in IL-1β and TNFα mRNA expression by up to 4.2 and 2.1 fold respectively compared to control mice. Treatment of thrombocytopenic mice with IVIg (2g/Kg) prevented the increase of IL-1β mRNA but not TNFα mRNA. In parallel, the effect of anti-red blood cell (anti-RBC) antibodies, used to mimic anti-D treatment of ITP patients, was analysed. The results were similar to those observed with IVIg for IL-1β mRNA, but in contrast to IVIg, anti-RBC treatment also prevented the increase in TNFα gene expression, suggesting that the mechanisms of action of IVIg and anti-D in ITP treatment are different, in agreement with previous studies. In summary, we showed that IVIg and anti-RBC treatments attenuate the expression of important pro-inflammatory cytokines. IL-1β and TNFα are known to increase the phagocytic activity of macrophages and also to enhance the production of antibodies by B cells. Therefore, IVIg and anti-D could contribute to the prevention of platelet destruction in ITP by limiting the activation of phagocytes and the production of autoantibodies. This study was assisted by a grant from the Bayer/CBS/HQ partnership Fund to RB and RL.

Multiple loci are linked with anti-red blood cell antibody production in NZB mice -- comparison with other phenotypes implies complex modes of action.

The New Zealand Black (NZB) mouse strain is a model of autoimmune haemolytic anaemia (AHA) and systemic lupus erythematosus (SLE), characterized by the production of anti-red blood cell (RBC) antibodies and anti-nuclear antibodies (ANA), respectively. A linkage analysis was carried out in an (NZB x BALB/c) F(2) cross in order to identify loci involved in the production of both anti-RBC IgM and IgG antibodies. These regions of linkage were compared with linkage data to ANA from the same cohort and other linkage analyses involving New Zealand mice. Four previously described NZB loci linked to anti-RBC antibodies were confirmed, and eight novel loci linked to this trait were also mapped: five of which were of NZB origin, and three derived from the non-autoimmune BALB/c background. A comparison between loci linked with anti-RBC antibodies and ANA demonstrated many that co-localize, suggesting the presence of genes that result in the general breaking of tolerance to self-antigen. Furthermore, the observation that some loci were associated only with the anti-RBC response suggests an antigen specific mechanism in addition to a general breaking of tolerance. A locus linked with anti-RBC antibodies and ANA on distal chromosome 7 in this cohort is orthologous to one on the q arm of human chromosome 11, a region linked to AHA and ANA in human SLE.

Predicting the severity of fetal anemia using time-domain measurement of volume flow in the fetal aorta.

To assess the value of fetal aortic time-domain measurement of volume flow (using color velocity imaging quantification (CVI-Q)) in predicting the severity of fetal anemia. This was a prospective observational study, in which 24 pregnant women with suspected fetal anemia due to rising anti-red blood cell antibody titers underwent cordocentesis. The fetal aortic time-domain volume flow was measured before fetal blood sampling for fetal hemoglobin investigation. We examined the correlation between increased fetal aortic time-domain volume flow (>2 SD for gestational age) and fetal anemia (hemoglobin level <2 SD for gestational age). Seventeen fetuses had anemia, and seven had normal hemoglobin. There was a strong correlation between the increase in fetal aortic time-domain volume flow and the drop in hemoglobin value (r = 0.81; P < 0.01). The sensitivity of this technique to predict fetal anemia was 81.3% and the specificity was 71.4%. The mean increase over time in aortic CVI-Q in anemic fetuses was 323.2 mL/min (95% CI, 200.1 to 446.4) compared with 86.9 mL/min (95% CI, -17.7 to 191.5) in the non-anemic group (P = 0.004). Fetal aortic time-domain measurement of volume flow is significantly increased in cases of fetal anemia due to red-cell alloimmunization. These findings can be used to improve the sensitivity, specificity and positive predictive value of the non-invasive techniques used to predict fetal anemia, and may help in the selection of pregnancies that require cordocentesis and transfusion.

In vitro quantification of anti-red blood cell antibody production in idiopathic autoimmune haemolytic anaemia: effect of mitogen and cytokine stimulation.

The immunopathogenic mechanisms underlying idiopathic autoimmune haemolytic anaemia (AIHA) are still unknown, although regulatory cytokines are thought to play an important role. We investigated cytokine production by mitogen-stimulated whole blood cultures from 21 patients with AIHA and from 22 age- and sex-matched controls. In parallel experiments, we studied the effect of mitogen and cytokine stimulation on anti-red blood cell (RBC) IgG antibody production, assessed as both binding on autologous RBCs and secretion in culture supernatants. To quantify anti-RBC antibody, we set up a sensitive and quantitative solid phase competitive immunoassay. The results showed that in AIHA patients production of interleukin (IL)-4, IL-6 and IL-13 was significantly increased, whereas that of interferon (IFN)-gamma was reduced. Multivariate analysis showed that IFN-gamma was the only independent factor significantly associated with the reduced T-helper-1-like cytokine profile. Patients with active haemolysis showed further reduction of IFN-gamma and IL-2 production and increased secretion of transforming growth factor (TGF)-beta. In AIHA patients, mitogen stimulation, as well as IL-6, significantly increased autologous anti-RBC-binding relative to unstimulated cultures. Mitogen stimulation and addition of IL-4, IL-6, IL-10, IL-13 and TGF-beta significantly increased both autologous anti-RBC binding and antibody secretion in AIHA patients compared with controls. The results suggest that a reduced T-helper-1- and a predominant T-helper-2-like profile and elevated TGF-beta levels might play a role in the immunopathogenesis of AIHA. Furthermore, our competitive anti-RBC antibody was able to detect anti-RBC antibody production in some direct antiglobulin test (DAT)-negative AIHA patients.

Sperm, nuclear, phospholipid, and red blood cell antibodies and isotype RF in infertile couples and patients with autoimmune rheumatic diseases.

To determine if measuring of nonorgan-specific autoantibodies is useful for better understanding and management of unexplained infertility. Sera were obtained from 70 infertile couples, 57 rheumatic patients, and 76 fertile donors. Sperm antibodies (SA) were detected by the tests of Kibrick and Friberg, anti-histones, anti-cardiolipin antibodies, and RF isotypes by ELISA, antinuclear antibodies by indirect immunofluorescence, and anti-red blood cell antibodies by Capture-R. Multiple autoimmune reactivity (both partners positive and/or more than one type of autoantibody involved), higher than naturally occurring in fertile individuals, was found in 55% of the idiopathically infertile couples. IgA-RF was the dominant autoimmune marker. SA revealed similar rates in patients with rheumatic diseases and in infertiles with or without other autoantibodies. Although no single autoimmunity marker could predict occurrence of SA, the coincidence of enhanced polyclonal autoimmunity in both partners of infertile couples might potentiate their negative effect on reproduction.

Immunoglobulin G-mediated inflammatory responses develop normally in complement-deficient mice.

The role of complement in immunoglobulin G-triggered inflammation was studied in mice genetically deficient in complement components C3 and C4. Using the reverse passive Arthus reaction and experimental models of immune hemolytic anemia and immune thrombocytopenia, we show that these mice have types II and III inflammatory responses that are indistinguishable from those of wild-type animals. Complement-deficient and wild-type animals exhibit comparable levels of erythrophagocytosis and platelet clearance in response to cytotoxic anti-red blood cell and antiplatelet antibodies. Furthermore, in the reverse passive Arthus reaction, soluble immune complexes induce equivalent levels of hemmorhage, edema, and neutrophillic infiltration in complement-deficient and wild-type animals. In contrast, mice that are genetically deficient in the expression of Fc receptors exhibit grossly diminished reactions by both cytotoxic antibodies and soluble immune complexes. These studies provide strong evidence that the activation of cell-based Fc gamma R receptors, but not complement, are required for antibody-triggered murine inflammatory responses.

Production of anti-erythrocyte antibodies by leukemic and nonleukemic B cells in chronic lymphocytic leukemia patients.

We have assessed the specificity of antibodies from the leukemic B cells of five patients with both chronic lymphocytic leukemia and autoimmune hemolytic anemia (CLL-AHA). Leukemic cells from one patient displayed surface immunoglobulin with heavy and light chain isotypes identical to that of the patient's anti-red blood cell (RBC) antibodies, and the leukemic cells secreted antibodies in vitro with anti-RBC activity. However, in the remaining patients, the leukemic cells displayed surface immunoglobulin with light chain isotypes different from that of the patient's anti-RBC antibodies and secreted antibodies in vitro with no detectable anti-RBC activity. Thus, there are two distinct classes of CLL-AHA patients, differentiated by the presence or absence of an anti-RBC antibody-producing leukemic B cell clone. The apparent heterogeneity in the source of pathogenic anti-RBC antibodies may impact the treatment response of the two classes of CLL-AHA patients.