Clinical profile and serological correlation with haemolysis in DAT-positive autoimmune haemolytic anaemia patients in Bangladesh.

Discerning the type of autoimmune haemolytic anaemia (AIHA) is crucial for transfusion support and initiation of treatment. This study aimed to establish the clinical profile and serological character of red cell autoantibodies and to investigate the relationship with haemolysis in AIHA patients who were direct antiglobulin test (DAT)-positive. A total of 59 DAT-positive AIHA patients were included in this study. Clinical, laboratory and serological findings were evaluated to find the gradation of haemolysis and to investigate its correlation with age, sex, type of autoantibody and level of autoantibody. Study findings revealed that most patients (89.8%) had haemolysis, wherein moderate haemolysis (67.8%) was predominant. Weakness, palpitations, fever, pallor, tachycardia and splenomegaly were common among patients with severe and moderate haemolysis. The majority (66.1%) had an associated disorder. Warm autoantibody was the most common, followed by cold and mixed cases. The severity of haemolysis correlated strongly with the strength of the DAT reaction (Cramer V 0.636, p<0.001). These findings may be useful to clinicians while determining a treatment plan. The direct relationship between severity of haemolysis and strength of DAT needs further exploration in a large population to establish whether it can be used as a tool to formulate a treatment plan when assessing AIHA patients in low resourced countries.

Sutimlimab in patients with cold agglutinin disease: results of the randomized placebo-controlled phase 3 CADENZA trial.

Sutimlimab, a first-in-class humanized immunoglobulin G4 (IgG4) monoclonal antibody that selectively inhibits the classical complement pathway at C1s, rapidly halted hemolysis in the single-arm CARDINAL study in recently transfused patients with cold agglutinin disease (CAD). CADENZA was a 26-week randomized, placebo-controlled phase 3 study to assess safety and efficacy of sutimlimab in patients with CAD without recent (within 6 months prior to enrollment) transfusion history. Forty-two patients with screening hemoglobin ≤10 g/dL, elevated bilirubin, and ≥1 CAD symptom received sutimlimab (n = 22) or placebo (n = 20) on days 0 and 7 and then biweekly. Composite primary endpoint criteria (hemoglobin increase ≥1.5 g/dL at treatment assessment timepoint [mean of weeks 23, 25, 26], avoidance of transfusion, and study-prohibited CAD therapy [weeks 5-26]) were met by 16 patients (73%) on sutimlimab, and 3 patients (15%) on placebo (odds ratio, 15.9 [95% confidence interval, 2.9, 88.0; P < .001]). Sutimlimab, but not placebo, significantly increased mean hemoglobin and FACIT-Fatigue scores at treatment assessment timepoint. Sutimlimab normalized mean bilirubin by week 1. Improvements correlated with near-complete inhibition of the classical complement pathway (2.3% mean activity at week 1) and C4 normalization. Twenty-one (96%) sutimlimab patients and 20 (100%) placebo patients experienced ≥1 treatment-emergent adverse event. Headache, hypertension, rhinitis, Raynaud phenomenon, and acrocyanosis were more frequent with sutimlimab vs placebo, with a difference of ≥3 patients between groups. Three sutimlimab patients discontinued owing to adverse events; no placebo patients discontinued. These data demonstrate that sutimlimab has potential to be an important advancement in the treatment of CAD. This trial was registered at www.clinicaltrials.gov as #NCT03347422.

Targeted next-generation sequencing revealed a novel homozygous mutation in the LRBA gene causes severe haemolysis associated with Inborn Errors of Immunity in an Indian family.

OBJECTIVES: LPS-responsive beige-like anchor protein (LRBA) deficiency abolishes LRBA protein expression due to biallelic mutations in the LRBA gene that lead to autoimmune manifestations, inflammatory bowel disease, hypogammaglobulinemia in early stages, and variable clinical manifestations. MATERIALS AND METHODS: Mutational analysis of the LRBA gene was performed in Indian patients using targeted Next Generation Sequencing (t-NGS) and confirmed by Sanger sequencing using specific primers of exons 53. Then, bioinformatics analysis and protein modeling for the novel founded mutations were also performed. The genotype, phenotype correlation was done according to the molecular findings and clinical features. RESULTS: We report an unusual case of a female patient born of a consanguineous marriage, presented with severe anaemia and jaundice with a history of multiple blood transfusions of unknown cause up to the age of 5 yrs. She had hepatosplenomegaly with recurrent viral and bacterial infections. Tests for hemoglobinopathies, enzymopathies, and hereditary spherocytosis were within the normal limits. The t-NGS revealed a novel homozygous missense variation in exon 53 of the LRBA gene (chr4:151231464C > T; c.7799G > A) (p.C2600Y), and the parents were heterozygous. The further immunological analysis is suggestive of hypogammaglobulinaemia and autoimmune haemolytic anaemia. The bioinformatics tools are suggestive of deleterious and disease-causing variants. CONCLUSION: This study concludes the importance of a timely decision of targeted exome sequencing for the molecular diagnostic tool of unexplained haemolytic anaemia with heterogeneous clinical phenotypes.

Immunoglobulin subtyping and quantification in direct antiglobulin test: positive haemolysis in an HIV-prevalent setting.

AIMS: Positive direct antiglobulin tests (DATs) are valuable in identifying the aetiology of autoimmune haemolysis and in guiding therapeutic intervention. However, in HIV-positive individuals with background polyclonal gammopathy, a positive DAT in the absence of haemolysis is common. In this setting, IgG quantification and subtyping may be of value, as this is possible with the recently introduced gel cards. There is paucity of literature evaluating the diagnostic usefulness of IgG subtyping and quantification in HIV-positive individuals who are investigated for autoimmune haemolytic anaemia (AIHA). This study evaluated the usefulness of IgG quantification and subtyping in the diagnostic work-up of AIHA in patients with a positive DAT, with and without HIV infection. METHODS: This retrospective, cross-sectional study included patients investigated for AIHA in a quaternary care hospital. Those with a positive DAT had their IgG subtyped and quantified using the ID-Card DAT IgG1/IgG3 and IgG-dilution cards (Bio-Rad, Cressier, Switzerland). RESULTS: Ninety patients admitted from December 2019 to March 2020 were investigated for AIHA. Forty-four (49%) patients had a positive DAT of whom 26 (59%) had evidence of haemolysis, and 16 (36%) were HIV positive. Concurrent HIV and haemolysis were present in eight patients, two of whom had IgG1 although none had an IgG antibody titre >1:30. None of the HIV-positive patients without features of haemolysis had IgG1/IgG3 or IgG antibody titres >1:30. CONCLUSION: In our clinical setting, IgG quantification and subtyping were found to be of limited value in the diagnostic characterisation of AIHA in HIV-positive patients with false-positive DAT.

Circulating bioactive bacterial DNA is associated with immune activation and complications in common variable immunodeficiency.

Common variable immunodeficiency (CVID) is characterized by profound primary antibody defects and frequent infections, yet autoimmune/inflammatory complications of unclear origin occur in 50% of individuals and lead to increased mortality. Here, we show that circulating bacterial 16S rDNA belonging to gut commensals was significantly increased in CVID serum (P < 0.0001), especially in patients with inflammatory manifestations (P = 0.0007). Levels of serum bacterial DNA were associated with parameters of systemic immune activation, increased serum IFN-γ, and the lowest numbers of isotype-switched memory B cells. Bacterial DNA was bioactive in vitro and induced robust host IFN-γ responses, especially among patients with CVID with inflammatory manifestations. Patients with X-linked agammaglobulinemia (Bruton tyrosine kinase [BTK] deficiency) also had increased circulating bacterial 16S rDNA but did not exhibit prominent immune activation, suggesting that BTK may be a host modifier, dampening immune responses to microbial translocation. These data reveal a mechanism for chronic immune activation in CVID and potential therapeutic strategies to modify the clinical outcomes of this disease.

Severe autoimmune hemolytic anemia following receipt of SARS-CoV-2 mRNA vaccine.

BACKGROUND: Large clinical trials have demonstrated the overall safety of vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, reports have emerged of autoimmune phenomena, including vaccine-associated myocarditis, immune thrombocytopenia, and immune thrombotic thrombocytopenia. CASE PRESENTATION: Here we present a novel case of a young woman who developed life-threatening autoimmune hemolytic anemia (AIHA) after her first dose of a SARS-CoV-2 mRNA vaccine. Notably, initial direct antiglobulin testing was negative using standard anti-IgG reagents, which are "blind" to certain immunoglobulin (IgG) isotypes. Further testing using an antiglobulin reagent that detects all IgG isotypes was strongly positive and confirmed the diagnosis of AIHA. The patient required transfusion with 13 units of red blood cells, as well as treatment with corticosteroids, rituximab, mycophenolate mofetil, and immune globulin. CONCLUSION: As efforts to administer SARS-CoV-2 vaccines continue globally, clinicians must be aware of potential autoimmune sequelae of these therapies.

Prospective validation of an automatic reflex test for identifying spurious elevations of mean corpuscular haemoglobin concentration due to the presence of cold agglutinins.

Cold agglutinins (CA) in blood may cause false reduction in red blood cell (RBC) count and false increases of RBC indices, such as mean corpuscular haemoglobin concentration (MCHC). Preheating at 37 °C for 2 h is used to overcome this problem. We previously proposed the integration in a total laboratory automation (TLA) setting of a customized reflex test in the presence of MCHC >385 g/L for identifying spurious elevations due to CA. Here, we prospectively evaluate this approach after its introduction in our clinical practice. We evaluated 73 consecutive blood samples from 34 adult patients. Short heating (<1 min) at 41 °C using the reticulocyte channel of Sysmex XN-9000 platform was followed by calculation of optical parameters by the instrument software to ensure quick solution of the CA-dependent problems. After the reflex test in the reticulocyte channel, MCHC dropped below 385 g/L in 50 samples. The reflex markedly corrected the RBC number in eight samples obtained from three patients with CA condition. Two samples from markedly anaemic patients had low blood haemoglobin and RBC count before and after reflex. The remaining 13 samples were obtained from 12 patients, most of whom were on antiretroviral therapy or suffered severe electrolyte disorders, known conditions associated to increased MCHC. The implementation of the proposed automatic reflex by reticulocyte channel on the Sysmex XN-9000 platform in a TLA setting may solve the problem of spuriously high MCHC due to RBC agglutination for CA in a few minutes instead of waiting hours for sample preheating.

Resolution of serologic problems due to cold agglutinin mediated autoimmune hemolytic anemia and its transfusion decision.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is a rare disease characterized by hemolysis caused by autoantibodies against erythrocyte surface antigen. These antibodies can be classified as warm, cold, or mixed types. METHODS: We report two cases of cold agglutinin disease (CAD), which were eventually diagnosed owing to blood group discrepancy. Resolution was achieved after washing the red blood cells (RBCs) with warm saline and absorbing the autoantibodies at 4°C with the washed RBCs. We also assessed the patient's condition and discussed the strategy of blood transfusion. RESULTS: The first case occurred after postoperative chemotherapy for rectal cancer, and the other manifested with anemia from the outset. Direct antiglobulin tests were positive and revealed autoantibodies against C3d only. Cold agglutinin titration was performed, and the titers of both were 1:1024. Eventually, the patient's condition stabilized without blood transfusion. CONCLUSION: The serological discrepancies observed in the blood transfusion department can successfully guide blood transfusion decisions in cases of CAD.

Analysis of multiple organ damage and clinical immunological characteristics in systemic lupus erythematosus patients with hematologic involvement.

Objective: To investigate clinical immunological characteristics and imaging findings of multiple organ damage of systemic lupus erythematosus (SLE) patients with hematologic involvement. Methods: SLE patients diagnosed in the Second Affiliated Hospital of Nanchang University from June 2015 to March 2019 were selected, including 93 SLE patients with hematologic involvement and 68 SLE patients without hematologic involvement. Immunological indicators such as autoantibodies, immunoglobulin G (IgG), complement 4 (C4) and imaging data of several organs were measured respectively. The results were statistically analyzed. Results: SLE patients with hematologic involvement were more likely to have autoimmune hemolytic anemia (AIHA) (20.43%, P<0.05). The erythrocyte sedimentation rate (ESR) of SLE patients with hematologic involvement was 75.82 (±35.33) mm/h, IgG was 28.84 (±6.00) g/L and C4 was 0.073 (±0.031) g/L (P< 0.05). The area under the curve (AUC) of IgG was the highest among the above indicators (P<0.01). The positive anti-RO-52 antibody (OR=15.926, P<0.05) was an independent risk factor for pulmonary inflammatory lesions in SLE patients with hematologic involvement. Conclusion: Compared with the control group, abnormal immunological indicators and multiple organs damage are more obvious. Positive anti-RO-52 antibody may play an important role in the pathogenesis of pulmonary inflammation in SLE patients.

Sutimlimab in Cold Agglutinin Disease.

BACKGROUND: Cold agglutinin disease is a rare autoimmune hemolytic anemia characterized by hemolysis that is caused by activation of the classic complement pathway. Sutimlimab, a humanized monoclonal antibody, selectively targets the C1s protein, a C1 complex serine protease responsible for activating this pathway. METHODS: We conducted a 26-week multicenter, open-label, single-group study to assess the efficacy and safety of intravenous sutimlimab in patients with cold agglutinin disease and a recent history of transfusion. The composite primary end point was a normalization of the hemoglobin level to 12 g or more per deciliter or an increase in the hemoglobin level of 2 g or more per deciliter from baseline, without red-cell transfusion or medications prohibited by the protocol. RESULTS: A total of 24 patients were enrolled and received at least one dose of sutimlimab; 13 patients (54%) met the criteria for the composite primary end point. The least-squares mean increase in hemoglobin level was 2.6 g per deciliter at the time of treatment assessment (weeks 23, 25, and 26). A mean hemoglobin level of more than 11 g per deciliter was maintained in patients from week 3 through the end of the study period. The mean bilirubin levels normalized by week 3. A total of 17 patients (71%) did not receive a transfusion from week 5 through week 26. Clinically meaningful reductions in fatigue were observed by week 1 and were maintained throughout the study. Activity in the classic complement pathway was rapidly inhibited, as assessed by a functional assay. Increased hemoglobin levels, reduced bilirubin levels, and reduced fatigue coincided with inhibition of the classic complement pathway. At least one adverse event occurred during the treatment period in 22 patients (92%). Seven patients (29%) had at least one serious adverse event, none of which were determined by the investigators to be related to sutimlimab. No meningococcal infections occurred. CONCLUSIONS: In patients with cold agglutinin disease who received sutimlimab, selective upstream inhibition of activity in the classic complement pathway rapidly halted hemolysis, increased hemoglobin levels, and reduced fatigue. (Funded by Sanofi; CARDINAL ClinicalTrials.gov number, NCT03347396.).

Serologic characteristics of oxaliplatin antibodies in 15 patients with drug-induced immune hemolytic anemia.

BACKGROUND: Oxaliplatin, a third-generation platinum derivative is commonly used in combination treatment of metastatic colorectal cancer. Since 2008, it is the second most common cause of drug-induced immune hemolytic anemia (DIIHA) investigated in our laboratory. STUDY DESIGN AND METHODS: Samples from fifteen patients including nine (60%) with intravascular hemolysis, suspected of having DIIHA were studied for the presence of anti-oxaliplatin. Direct antiglobulin tests (DATs) and tests with oxaliplatin-treated red blood cells (RBCs) or untreated and enzyme-treated RBCs in the presence of oxaliplatin were performed. A pool of normal AB sera with no unexpected antibodies was used as a control for nonimmunologic protein adsorption (NIPA). RESULTS: Eleven (73%) of the fifteen patients had antibodies to oxaliplatin that reacted with drug-treated RBCs and untreated RBCs in the presence of drug by tube and/or gel method. Lower-titer reactivity (<20) obtained with four patients' sera and the corresponding pooled normal sera was most likely due to NIPA. Eighty seven percent (13/15) of the patients had positive DAT either with anti-IgG only (33%), IgG + C3d (40%), or C3d only (13%). Two patients had a negative DAT. No directly agglutinating antibody was observed with the pools of normal donor's sera in the presence of oxaliplatin. CONCLUSION: Anti-oxaliplatin can cause severe intravascular hemolysis. Complement can usually be detected on the patient's RBCs and anti-oxaliplatin can be detected in the patient's serum. RBC-bound albumin detection with anti-human albumin needs to be performed to confirm NIPA which could have contributed to the patient's hemolytic anemia.

Hematologic autoimmune disorders in the course of COVID-19: a systematic review of reported cases.

OBJECTIVE: As COVID-19 is a new emerging disease, the hematological/immunological changes that develop in the infected patients remain unknown. This study aims to systematically review the hematologic autoimmune complications in these patients. METHOD: Data from three online databases including Medline (via PubMed), Scopus and Web of Science were searched on 19 December 2020, and after excluding duplicate, irrelevant and inappropriate records, eligible documents were identified. Afterwards, information such as patients' history, presentations, paraclinical data, treatment course and outcome were extracted from the records. RESULTS: A total of 58 documents were considered to be eligible for data extraction which described 94 patients with COVID-19 who developed hematologic autoimmune disorder in their course of infection. Of these patients with COVID-19, the most common hematologic autoimmune disorder was immune thrombocytopenic purpura (55 cases) followed by autoimmune hemolytic anemia (22 cases). Other hematologic autoimmune disorders include antiphospholipid syndrome, thrombotic thrombocytopenic purpura, Evans syndrome and autoimmune neutropenia. CONCLUSION: The current study would help us to always consider an autoimmune etiology for cases with abnormal hematologic finding which further lead to an appropriate treatment of the patients, especially when the symptoms present in about 1-2 weeks after the first manifestation of the infection symptoms. Maybe, at least in this pandemic, it should be recommended to evaluate patients with unexpected and unexplained decrease in their hemoglobulin or platelet count for COVID-19. Another challenging issue is the treatment options. Given the multiorgan involvement and multifaceted nature of the infection, an individualized approach should be taken for each patient.

Features of serum complement C3 and C4 levels in autoimmune hemolytic anemia patients.

INTRODUCTION: Abnormally activated complement system induces erythrolysis in a part of autoimmune hemolytic anemia (AIHA) patients. However, the alterations in serum complement levels in these patients are seldom reported. In this study, we aimed to evaluate the serum complement features of AIHA patients according to different clinical and laboratory characteristics and to find relationships between complement levels and hemolysis-associated laboratory indexes. METHODS: A retrospective analysis of 146 AIHA patients was performed, and serum complement C3 and C4 levels were compared between control subjects and AIHA patients with different subtypes. Correlations of serum C3/C4 levels with titers of cold agglutinin test (CAT), direct antiglobulin test (DAT), and serological indexes were assessed. Spearman correlation analysis was performed to analyze the relationship between serum complement levels and other laboratory indexes. RESULTS: Autoimmune hemolytic anemia patients showed reduced serum C3 levels, while serum C4 levels tended to be lower in DAT-positive AIHA patients but not in DAT-negative AIHA patients. Patients with warm AIHA secondary to connective tissue diseases and cold agglutinin disease/cold agglutinin syndrome had the lowest serum C3/C4 levels. Serum C4 levels were negatively correlated with CAT (P = .004) and DAT (anti-C3d) (P = .007) titers. In patients with positive CAT and/or DAT (anti-C3d) but negative DAT (anti-IgG), serum C3/C4 levels were negatively correlated with indirect bilirubin (P = .017 and =.026, respectively). CONCLUSION: The study findings may be helpful in not only unraveling the mechanism underlying hemolysis in AIHA but also diagnosing AIHA and selecting targeted treatment strategies.

Prognostic significance of hemoglobin level and autoimmune hemolytic anemia in SARS-CoV-2 infection.

Higher levels of D-dimer, LDH, and ferritin, all have been associated with the poor prognosis of COVID-19. In a disease where there are acute inflammation and compromised oxygenation, we investigated the impact of initial hemoglobin (Hgb) levels at Emergency Department (ED) triage on the severity and the clinical course of COVID-19. We conducted a cross-sectional study on 601 COVID-19 patients in a COVID-19 national referral center between 13 and 27 June 2020. All adult patients presented at our hospital that required admission or hotel isolation were included in this study. Patients admitted to the intensive care unit (ICU) had a lower initial Hgb than those admitted outside the ICU (12.84 g/dL vs. 13.31 g/dL, p = 0.026) and over the course of admission; the prevalence of anemia (Hgb < 12.5 g/dL) was 65% in patients admitted to ICU, whereas it was only 43% in non-ICU patients (odds ratio of 2.464, 95% CI 1.71-3.52). Anemic ICU patients had a higher mortality compared with non-anemic ICU patients (hazard ratio = 1.88, log-rank p = 0.0104). A direct agglutination test (DAT) for all anemic patients showed that 14.7% of ICU patients and 9% of non-ICU patients had autoimmune hemolytic anemia (AIHA). AIHA patients had significantly longer length of hospital stay compared with anemic patients without AIHA (17.1 days vs. 14.08 days, p = 0.034). Lower Hgb level at hospital presentation could be a potential surrogate for COVID-19 severity.