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Abstract: Up to 30% of patients with autoimmune hemolytic anemia (AIHA) show inadequate bone marrow (BM) compensatory response with inappropriately low levels of reticulocytes and endogenous erythropoietin. Ineffective BM compensation is associated with more severe anemia, transfusion need, and hospital admission, and treatment with recombinant erythropoietin (rEPO) may be beneficial. Here, we prospectively analyzed the efficacy and safety of rEPO in a single-center cohort of 47 patients with AIHA with inadequate reticulocytosis and endogenous erythropoietin at baseline. Epoetin alpha 40 000 international units per week were administered subcutaneously until hemoglobin (Hb) >11 g/dL and then tapered off. Overall response was 55% at 15 days, 74% at 1 month, 74% at 3 months, 80% at 6 months, and 91% at 12 months. Consistently, Hb values significantly increased from baseline to each subsequent time point (P<.001) with a median increase of +1.4, +2.4, +3.4, +3.8, and +4.4 g/dL, respectively. Transfusion needs reduced from 30% to <10% at 15 days and thereafter (P < .001). Concomitant medications included prednisone or methylprednisolone (N = 40, stable since >2 weeks from enrollment), mycophenolate mofetil (N = 1, ongoing since >3 months from enrollment), and rituximab (N = 7 patients with cold agglutinin disease from day 8). No association between concomitant medications and response to rEPO was found. Treatment was generally safe without rEPO-related severe adverse events. The comparison with an AIHA population not treated with rEPO showed a significant benefit of rEPO at 15 days and 1 month on response and Hb increase. These data support the use of rEPO as an add on to standard immunosuppression in AIHA with inadequate BM compensation. This trial was registered at www.clinicaltrials.gov as #NCT05931718., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Autoimmune hemolytic anemia (AIHA) may be associated with transfusion reactions and risk of alloimmunization., Objectives: To evaluate the transfusion policy and rate of alloimmunization and its clinical significance in AIHA., Methods: Data from 305 AIHA patients followed at a reference hematologic Center in Milan, Italy from 1997 to 2022 were retrospectively/prospectively collected (NCT05931718)., Results: Overall, 33% patients required transfusions with a response rate of 83% and eight transfusion reactions (7%), none hemolytic. Alloantibodies were detected in 19% of patients, being associated with higher transfusion burden (p = 0.01), lower Hb increase post-transfusion (p = 0.05), and transfusion reactions (p = 0.04). Along decades, the rate of RBC transfusions decreased from 53% to 20% and that of alloimmunization dropped from 30% to 6% likely due to the adoption of prestorage leukoreduction, the use of more restrictive Hb thresholds, and the implementation of molecular typing., Conclusions: Severe symptomatic AIHA may be safely transfused provided appropriate matching of patients and donors., (© 2023 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.)

Autoimmune hemolytic anemia (AIHA) is due to autoantibodies with or without complement activation and involves cellular and cytokine dysregulation. Here, we investigated cytokine single-nucleotide polymorphisms (SNPs) of TNF-α, TGF-β1, IL-10, IL-6, and IFN-γ, along with their serum levels. The former were related to hematological parameters, therapy, and clinical outcome. The study included 123 consecutive patients with primary AIHA [77 warm AIHA and 46 cold agglutinin disease (CAD)], followed up for a median of 49 months. Results show that the allelic frequency of TNF-α -308 G/A polymorphisms was significantly lower in patients versus controls. Moreover, the genotypic frequency of TNF-α -308G/A and TGF-β gene codon 25 G/C genotypes was significantly lower in patients versus controls. Considering cytokine SNP genotypes associated with different gene expression levels, TNF-α high gene expression was significantly more frequent in patients, TGF-β and IL-10 high gene expression was higher in patients with more severe anemia, and TGF-β high gene expression was higher in patients with active disease. Considering treatment, TNF-α and TGF-β high gene expression was more frequent in multitreated patients and particularly in CAD. It may be speculated that this genetic predisposition to a stronger inflammatory response may result in a greater immune dysregulation and in a relapsed/refractory disease. Regarding cytokine serum levels, TNF-α and TGF-β were significantly lower, and IL-10 and IL-6 were significantly higher in patients versus controls, underlying the complex interplay between genetic background and disease features., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zaninoni, Fattizzo, Pettine, Vercellati, Marcello and Barcellini.)

Background: Thrombosis may complicate autoimmune hemolytic anemia (AIHA), but its predictors are still lacking, and no clear-cut indications for anticoagulant prophylaxis are available., Objectives: To characterize frequency and severity of thromboses in AIHA patients and identify risk factors for thrombosis that may advise primary anticoagulant prophylaxis., Patients/methods: A total of 287 consecutive AIHA patients diagnosed and followed from 1978 at a tertiary Italian center were retrospectively studied; 174 of them were prospectively evaluated from January 2020 until December 2021. AIHA relapse, thrombosis occurrence, and primary anticoagulant prophylaxis were evaluated., Results: Thirty-three AIHA patients (11.4%) experienced thrombosis, 70% of whom hospitalized. The cumulative thrombosis incidence was higher in patients with lactate dehydrogenase (LDH) ≥ 1.5 (hazard ratio [HR] 3.22), in those experiencing infections (HR 3.57), receiving transfusions (HR 3.06), rituximab (HR 3.3), or cyclophosphamide (HR 2.67). By multivariable analysis, LDH, transfusions, rituximab, and cyclophosphamide treatment emerged as independent factors associated with thrombosis. Among 174 patients prospectively followed in the past 2 years, we observed 70 acute hemolytic episodes in 45 patients; 33/45 displayed LDH ≥1.5 × upper limit of normal, and 17 received anticoagulant prophylaxis with low molecular weight heparin for a median of 70 days (30-300). In those receiving prophylaxis no thrombotic complications occurred, whereas five thrombotic episodes were registered in the remaining 16 cases., Conclusions: Thrombosis was observed in about 11% of AIHA patients, mainly grade 3, and associated with intravascular hemolysis, need of transfusions, multitreatment, and infections, advising primary anticoagulant prophylaxis in these settings., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Evans syndrome (ES) is a rare condition, defined as the presence of 2 autoimmune cytopenias, most frequently autoimmune hemolytic anemia and immune thrombocytopenia (ITP) and rarely autoimmune neutropenia. ES can be classified as primary or secondary to various conditions, including lymphoproliferative disorders, other systemic autoimmune diseases, and primary immunodeficiencies, particularly in children. In adult ES, little is known about clinical features, disease associations, and outcomes. In this retrospective international study, we analyzed 116 adult patients followed at 13 European tertiary centers, focusing on treatment requirements, occurrence of complications, and death. ES was secondary to or associated with underlying conditions in 24 cases (21%), mainly other autoimmune diseases and hematologic neoplasms. Bleeding occurred in 42% of patients, mainly low grade and at ITP onset. Almost all patients received first-line treatment (steroids with or without intravenous immunoglobulin), and 23% needed early additional therapy for primary refractoriness. Additional therapy lines included rituximab, splenectomy, immunosuppressants, thrombopoietin receptor agonists, and others, with response rates >80%. However, a remarkable number of relapses occurred, requiring ≥3 therapy lines in 54% of cases. Infections and thrombotic complications occurred in 33% and 21% of patients, respectively, mainly grade ≥3, and correlated with the number of therapy lines. In addition to age, other factors negatively affecting survival were severe anemia at onset and occurrence of relapse, infection, and thrombosis. These data show that adult ES is often severe and marked by a relapsing clinical course and potentially fatal complications, pinpointing the need for high clinical awareness, prompt therapy, and anti-infectious/anti-thrombotic prophylaxis., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Daratumumab is a monoclonal antibody directed against the transmembrane glycoprotein CD38 expressed on plasma cells and lymphoplasmocytes, with a proven efficacy in multiple myeloma. Here we show its clinical efficacy in a patient with cold agglutinin disease (CAD) relapsed after multiple lines of therapy. CAD is caused by cold reactive autoantibodies that induce complement mediated hemolysis and peripheral circulatory symptoms. The disease is also characterized by the presence of monoclonal IgM gammopathy and of a lymphoid bone marrow infiltration that benefits from B-cell targeting therapies (i.e., rituximab) but also from plasma cell directed therapies, such as proteasome inhibitors. In the patient described, we also show that daratumumab therapy influenced the dynamics of several immunoregulatory cytokine levels (IL-6, IL-10, IL-17, IFN-γ, TNF-α, TGF-β) indicating an immunomodulatory effect of the drug beyond plasma cell depletion. In addition, we provide a literature review on the use of daratumumab in autoimmune conditions, including multi-treated and refractory patients with autoimmune hemolytic anemia (both CAD and warm forms), Evans syndrome (association of autoimmune hemolytic anemia and immune thrombocytopenia) and non-hematologic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zaninoni, Giannotta, Gallì, Artuso, Bianchi, Malcovati, Barcellini and Fattizzo.)

Autoimmune hemolytic anemia (AIHA) is characterized by immune mediated erythrocytes destruction by autoantibodies with or without complement activation. Additional pathologic mechanisms include cellular cytotoxicity, cytokline dysregulation, and inadequate bone marrow compensation with fibrosis/dyserythropoiesis. The latter resembles that of bone marrow failures, namely aplastic anemia and myelodysplastic syndromes. Paroxysmal nocturnal hemoglobinuria (PNH) clones are increasingly recognized in bone marrow failure syndromes, and their selection and expansion are thought to be mediated by immune mechanisms. In this study, we aimed to evaluate the prevalence of PNH clones in 99 patients with primary AIHA, and their correlations with disease features and outcomes. Moreover, in the attempt to disclose the physiopathology of PNH positivity in AIHA, serum levels of several immunomodulatory cytokines were tested. A PNH clone was found in 37 AIHA patients (37,4%), with a median size of 0.2% on granulocytes (range 0.03-85). Two patients showed a large clone (16 and 85%) and were therefore considered as AIHA/PNH association and not included in further analysis. Compared to PNH negative, PNH positive cases displayed a higher hemolytic pattern with adequate bone marrow compensation. AIHA type, response to therapy, complications and outcome were comparable between the two groups. Regarding cytokine levels, IFN-γ and IL-17 were lower in PNH positive vs. PNH negative AIHAs (0.3 ± 0.2 vs. 1.33 ± 2.5; 0.15 ± 0.3 vs. 3,7 ± 9.1, respectively, p = 0.07 for both). In PNH positive AIHAs, IFN-γ positively correlated with reticulocytes ( r = 0.52, p = 0.01) and with the bone marrow responsiveness index ( r = 0.69, p = 0.002). Conversely, IL-6 and IL-10 showed the same pattern in PNH positive and PNH negative AIHAs. IL-6 levels and TGF-β positively correlated with clone size ( r = 0.35, p = 0.007, and r = 0.38, p = 0.05, respectively), as well as with LDH values ( r = 0.69, p = 0.0003, and r = 0.34, p = 0.07, respectively). These data suggest testing PNH clones in AIHA since their prevalence is not negligible, and may correlate with a prominent hemolytic pattern, a higher thrombotic risk, and a different therapy indication. PNH testing is particularly advisable in complex cases with inadequate response to AIHA-specific therapy. Cytokine patterns of PNH positive and negative AIHAs may give hints about the pathogenesis of highly hemolytic AIHA., (Copyright © 2020 Fattizzo, Giannotta, Zaninoni, Kulasekararaj, Cro and Barcellini.)

Introduction: Autoimmune hemolytic anemia (AIHA) is a heterogeneous disease mainly due to autoantibody-mediated destruction of erythrocytes but also involves complement activation, dysregulation of cellular and innate immunity, and defective bone marrow compensatory response. Several drugs targeting these mechanisms are under development in addition to standard therapies. Areas covered: The following targeted therapies are illustrated: drugs acting on CD20 (rituximab, alone or in association with bendamustine and fludarabine) and CD52 (alemtuzumab), B cell receptor and proteasome inhibitors (ibrutinib, bortezomib), complement inhibitors (eculizumab, BIVV009, APL-2), and other drugs targeting T lymphocytes (subcutaneous IL-2, belimumab, and mTOR inhibitors), IgG driven extravascular hemolysis (fostamatinib), and bone marrow activity (luspatercept). Expert opinion: Although AIHA is considered benign and often easy to treat, chronic/refractory cases represent a challenge even for experts in the field. Bone marrow biopsy is fundamental to assess one of the main mechanisms contributing to AIHA severity, i.e. inadequate compensation, along with lymphoid infiltrate, the presence of fibrosis or dyserythropoiesis. The latter may give hints for targeted therapies (either B or T cell directed) and for new immunomodulatory drugs. Future studies on the genomic landscape in AIHA will further help in designing the best choice, sequence and/or combination of targeted therapies.

The clinical outcome, response to treatment, and occurrence of acute complications were retrospectively investigated in 308 primary autoimmune hemolytic anemia (AIHA) cases and correlated with serological characteristics and severity of anemia at onset. Patients had been followed up for a median of 33 months (range 12-372); 60% were warm AIHA, 27% cold hemagglutinin disease, 8% mixed, and 5% atypical (mostly direct antiglobulin test negative). The latter 2 categories more frequently showed a severe onset (hemoglobin [Hb] levels ≤6 g/dL) along with reticulocytopenia. The majority of warm AIHA patients received first-line steroid therapy only, whereas patients with mixed and atypical forms were more frequently treated with 2 or more therapy lines, including splenectomy, immunosuppressants, and rituximab. The cumulative incidence of relapse was increased in more severe cases (hazard ratio 3.08; 95% confidence interval, 1.44-6.57 for Hb ≤6 g/dL; P < .001). Thrombotic events were associated with Hb levels ≤6 g/dL at onset, intravascular hemolysis, and previous splenectomy. Predictors of a fatal outcome were severe infections, particularly in splenectomized cases, acute renal failure, Evans syndrome, and multitreatment (4 or more lines). The identification of severe and potentially fatal AIHA in a largely heterogeneous disease requires particular experienced attention by clinicians., (© 2014 by The American Society of Hematology.)

Objectives: To evaluate the sustained response to low-dose (LD) rituximab in autoimmune hemolytic anemia (AIHA), the ex vivo effect on anti-RBC antibody production by mitogen-stimulated direct antiglobulin test (MS-DAT), and the in vitro dose effect of the drug on the production of anti-RBC antibodies., Methods: Thirty two patients, 18 warm (W) AIHA and 14 cold hemagglutinin disease (CHD), were treated with LD rituximab (100 mg fixed dose ×4 weekly infusions) along with a short course of oral prednisone. Complete clinical examination, blood counts, and hemolytic markers were performed at enrollment and at month 6, 12, 24, and 36., Results: Hematological parameters significantly improved at all time points compared to enrollment. The overall response was 90%, 100%, 100%, and 89% and the relapse-free survival 87%, 79%, 68%, and 68% at 6, 12, 24, and 36 months, respectively. Response rates were slightly better in WAIHA than in CHD, and relapse risk was greater in cold than warm forms (HR 2.1, 95% CI 0.6-7.9). Four patients were retreated (one patient twice), all achieving a response, lasting a median of 18 months (range 9-30). Treatment was well tolerated without adverse events or infections. Anti-RBC antibody production by MS-DAT significantly decreased over time. In vitro studies showed that rituximab effectively inhibited anti-RBC antibody production at 50 μg/mL, 1/6 of the drug concentration after therapy with standard doses., Conclusions: These data confirm that LD rituximab treatment is effective and induces sustained responses in AIHA, and that a lower dose of the drug is enough to down-regulate autoantibody production., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This prospective study investigated the efficacy, safety, and response duration of low-dose rituximab (100 mg fixed dose for 4 weekly infusions) together with a short course of steroids as first- or second-line therapy in 23 patients with primary autoimmune hemolytic anemia (AIHA). The overall response was 82.6% at month +2, and subsequently stabilized to ∼ 90% at months +6 and +12; the response was better in warm autoimmune hemolytic anemia (WAIHA; overall response, 100% at all time points) than in cold hemagglutinin disease (CHD; average, 60%); the relapse-free survival was 100% for WAIHA at +6 and +12 months versus 89% and 59% in CHD, respectively, and the estimated relapse-free survival at 2 years was 81% and 40% for the warm and cold forms, respectively. The risk of relapse was higher in CHD and in patients with a longer interval between diagnosis and enrollment. Steroid administration was reduced both as cumulative dose (∼ 50%) and duration compared with the patient's past history. Treatment was well tolerated and no adverse events or infections were recorded; retreatment was also effective. The clinical response was correlated with amelioration biologic markers such as cytokine production (IFN-γ, IL-12, TNF-α, and IL-17), suggesting that low-dose rituximab exerts an immunomodulating activity. This study is registered at www.clinicaltrials.gov as NCT01345708.

The diagnosis of autoimmune hemolytic anemia (AIHA) is based on a positive direct antiglobulin test (DAT), which is performed using various methods with different sensitivities. Recently, mitogen-stimulated (MS)-DAT was suggested to be able to identify latent anti-erythrocyte autoimmunity. Traditional methods (tube, microcolumn, and solid phase) and MS-DAT were compared in 54 consecutive cases of suspected AIHA, 28 idiopathic AIHA in clinical remission, and 12 difficult-to-diagnose cases of DAT-negative AIHA, and the results (all cases) were correlated with hematologic and hemolytic parameters. DAT tube was confirmed as the gold standard to diagnose AIHA since almost all positive cases showed hemolytic anemia and positive eluates; 10 out of 26 tube-negative cases were positive on microcolumn and solid phase antiglobulin tests, and 22 out of 26 using MS-DAT, although only half of them showed clear signs of hemolysis. Mitogen stimulation increased the amount of IgG bound to red blood cells in all groups; moreover, MS-DAT was the only positive test in 10 cases of AIHA, and mitogen stimulation facilitated the identification of autoantibody specificity in culture supernatants. We conclude that a battery of tests rather than a single test is useful for the diagnosis of AIHA, including MS-DAT as an additional test for selected cases, although the results have to be cautiously interpreted based on the overall clinical context.