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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.

Abstract Iron homeostasis and dyserythropoiesis are poorly investigated in pyruvate kinase deficiency (PKD), the most common glycolytic defect of erythrocytes. Herein, we studied the main regulators of iron balance and erythropoiesis, as soluble transferrin receptor (sTfR), hepcidin, erythroferrone (ERFE), and erythropoietin (EPO), in a cohort of 41 PKD patients, compared with 42 affected by congenital dyserythropoietic anemia type II (CDAII) and 50 with hereditary spherocytosis (HS). PKD patients showed intermediate values of hepcidin and ERFE between CDAII and HS, and clear negative correlations between log-transformed hepcidin and log-EPO (Person’s r correlation coefficient = − 0.34), log-hepcidin and log-ERFE (r = − 0.47), and log-hepcidin and sTfR (r = − 0.44). sTfR was significantly higher in PKD; EPO levels were similar in PKD and CDAII, both higher than in HS. Finally, genotype–phenotype correlation in PKD showed that more severe patients, carrying non-missense/non-missense genotypes, had lower hepcidin and increased ERFE, EPO, and sTFR compared with the others (missense/missense and missense/non-missense), suggesting a higher rate of ineffective erythropoiesis. We herein investigated the main regulators of systemic iron homeostasis in the largest cohort of PKD patients described so far, opening new perspectives on the molecular basis and therapeutic approaches of this disease.

More than half of patients with paroxysmal nocturnal hemoglobinuria (PNH) treated with complement fraction C5 inhibitors experience residual anemia and hemolysis. This is partly due to the persistent activation of the complement cascade upstream C5, resulting in C3 deposition on PNH erythrocytes and extravascular hemolysis in the reticuloendothelial system. Pegcetacoplan is the first proximal C3 inhibitor to be approved for PNH basing on favorable efficacy and safety data in both naïve and eculizumab treated PNH. Here we report the first Italian patient treated with pegcetacoplan in a named patient program. The patient suffered from hemolytic PNH associated with CALR+ myeloproliferative neoplasm and was heavily transfusion dependent despite eculizumab therapy. Treatment with pegcetacoplan induced a dramatic improvement in Hb, along with normalization of unconjugated bilirubin and reticulocytes, as markers of extravascular hemolysis. Sequential laboratory workup showed the disappearance of C3 deposition on erythrocytes by direct anti-globulin test, the increase of PNH clone on erythrocytes, and a peculiar right shift of the ektacytometry curve. The drug was well tolerated, and the patient reported a significant improvement in his quality of life. Overall, pegcetacoplan appears a safe and effective option “ready to use” in the clinic for patients with PNH and suboptimal response to anti-C5 agents.

We investigated by targeted next generation sequencing the genetic bases of hereditary spherocytosis in 25 patients and compared the molecular results with the biochemical lesion of RBC membrane obtained by SDS-PAGE analysis. The HS diagnosis was based on available guidelines for diagnosis of congenital hemolytic anemia, and patients were selected because of atypical clinical presentation or intra-family variability, or because presented discrepancies between laboratory investigation and biochemical findings. In all patients but 5 we identified pathogenic variants in SPTA1, SPTB, ANK1, SLC4A1, EPB42 genes able to justify the clinical phenotype. Interestingly, a correspondence between the biochemical lesion and the molecular defect was identified in only 11/25 cases, mostly with band 3 deficiency due to SLC4A1 mutations. Most of the mutations in SPTB and ANK1 gene didn’t hesitate in abnormalities of RBC membrane protein; conversely, in two cases the molecular lesion didn’t correspond to the biochemical defect, suggesting that a mutation in a specific cytoskeleton protein may result in a more complex RBC membrane damage or suffering. Finally, in two cases the HS diagnosis was maintained despite absence of both protein defect and molecular lesion, basing on clinical and family history, and on presence of clear laboratory markers of HS. The study revealed complex relationships between the primary molecular lesion and the final effect in the RBC membrane cytoskeleton, and further underlines the concept that there is not a unique approach to the diagnosis of HS.

Low-risk myelodysplastic syndromes (LR-MDS) are a very heterogeneous disease, with extremely variable clinical features and outcome. Therapeutic strategies are still limited and mainly consist of erythropoiesis-stimulating agents (ESAs) and transfusion support. The contribution of molecular lesions and of autoimmune phenomena to pathogenesis and clinical course, including leukemic evolution, is a field of open investigation. We analyzed data from a cohort of 226 patients with LR-MDS followed at our center in the last 20 years, focusing on morphological, immunological (antiplatelets and anti-erythrocyte autoantibodies, anti-erythroblast antibodies), and molecular features. Hypoplastic bone marrow was found in 7% of the cases correlating with younger age, deeper cytopenia, lower dysplasia, and worse response to ESAs. A marker of autoimmunity was observed in 46% of the tested cases, who were younger, were less frequent dysplastic changes, and responded better to ESAs and steroids. Finally, 68% of the tested cases displayed at least one somatic mutation, most commonly SF3B1, TET2, ASXL1, and SRSF2, associated with older age, presence of neutropenia, and lower response to ESAs. Leukemic evolution (2.2%) was associated with presence of somatic mutations, and survival was favorably related to response to ESAs and transfusion independence. Overall, granular evaluation and re-evaluation are pivotal in LR-MDS patients to optimize clinical management.

Congenital hemolytic anemias (CHAs) are heterogeneous and rare disorders caused by alterations in structure, membrane transport, metabolism, or red blood cell production. The pathophysiology of these diseases, in particular the rarest, is often poorly understood, and easy-to-apply tools for diagnosis, clinical management, and patient stratification are still lacking. We report the 3-years monocentric experience with a 43 genes targeted Next Generation Sequencing (t-NGS) panel in diagnosis of CHAs; 122 patients from 105 unrelated families were investigated and the results compared with conventional laboratory pathway. Patients were divided in two groups: 1) cases diagnosed with hematologic investigations to be confirmed at molecular level, and 2) patients with unexplained anemia after extensive hematologic investigation. The overall sensitivity of t-NGS was 74 and 35% for families of groups 1 and 2, respectively. Inside this cohort of patients we identified 26 new pathogenic variants confirmed by functional evidence. The implementation of laboratory work-up with t-NGS increased the number of diagnoses in cases with unexplained anemia; cytoskeleton defects are well detected by conventional tools, deserving t-NGS to atypical cases; the diagnosis of Gardos channelopathy, some enzyme deficiencies, familial siterosterolemia, X-linked defects in females and other rare and ultra-rare diseases definitely benefits of t-NGS approaches.

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. [ABSTRACT FROM AUTHOR]

Wilma Barcellini, Bruno Fattizzo, Anna ZaninoniUOC Ematologia, Fondazione IRCCS Ca’ Grande Ospedale Maggiore Policlinico, Milano, ItalyAbstract: Autoimmune hemolytic anemia (AIHA) is increasingly observed after allogeneic hematopoietic stem cell transplantation (allo-HSCT), with a reported incidence between 4% and 6%. The disease is generally severe and refractory to standard therapy, with high mortality, and there are neither defined therapies, nor prospective clinical trials addressing the best treatment. Most of the knowledge on the therapy of AIHAs derives from primary forms, which are highly heterogeneous as well, further complicating the management of post-allo-HSCT forms. The review addresses the risk factors associated with post-allo-AIHA, including unrelated donor, the development of chronic extensive graft-versus-host disease, CMV reactivation, nonmalignant diagnosis pre-HSCT, and alemtuzumab use in conditioning regimens. Regarding therapy, we describe standard treatments, such as corticosteroids, intravenous immunoglobulin, splenectomy, rituximab, cyclophosphamide, and plasma exchange, which have lower response rates than those reported in primary forms. New therapeutic options, including sirolimus, bortezomib, abatacept, daratumumab and complement inhibitors, are promising tools for this detrimental complication occurring after allo-HSCT.Keywords: autoimmune hemolytic anemia, allogeneic hematopoietic stem cell transplantation, rituximab, sirolimus and abatacept, bortezomib and daratumumab

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.

In the last years, a substantial contribution of red blood cells (RBCs) in cardiovascular homeostasis has been evidenced, as these cells are able to regulate cardiovascular function by the export of adenosine triphosphate and nitric oxide as well as to maintain redox balance through a well-developed antioxidant system. Recently a link between high-risk plaque (HRP) features and myocardial ischemia, in the absence of severe lumen stenosis, has been evidenced. Nonobstructive coronary artery disease (nonob CAD) has been associated in fact with a greater 1-year risk of myocardial infarction and all-cause mortality compared with no apparent CAD. This new evidence increases interest in searching new triggers to identify these high-risk patients, in the absence/or on top of traditional hazard markers. In this study, we investigated the existence of any association between RBC morphodynamics and HRP features in individuals with different grades of coronary stenosis detected by coronary computed tomography angiography (CCTA). Ninety-one consecutive individuals who underwent CCTA [33 no CAD; 26 nonobstructive (nonob), and 32 obstructive (ob) CAD] were enrolled. RBC morphodynamic features, i.e., RBC aggregability and deformability, were analyzed by means of Laser Assisted Optical Rotation Cell Analyzer (LoRRca MaxSis). The putative global RBC morphodynamic (RMD) score and the related risk chart, associating the extent of HRP (e.g., the non-calcified plaque volume) with both the RMD score and the max % stenosis were computed. In nonob CAD group only positive correlations between RBC rigidity, osmotic fragility or aggregability and HRP features (plaque necrotic core, fibro-fatty and fibro-fatty plus necrotic core plaque volumes) were highlighted. Interestingly, in this patient cohort three of these RBC morphodynamic features result to be independent predictors of the presence of non-calcified plaque volume in this patients group. The risk chart created shows that only in nonob CAD plaque vulnerability increases according to the score quartile. Findings of this work, by evidencing the association between erythrocyte morphodynamic characteristics assessed by LoRRca and plaque instability in a high-risk cohort of nonob CAD, suggest the use of these blood cell features in the identification of high-risk patients, in the absence of severe coronary stenosis.

Thrombopoietin receptor agonists (TPO-RA) are currently indicated for the treatment of chronic immune thrombocytopenia and relapsed refractory aplastic anemia. However, the off-label use of these drugs is more and more frequent, including in the setting of aplasia secondary to chemotherapy and hemopoietic stem cell transplant (SCT). Growing evidence suggests that mechanisms of action of TPO-RA go beyond the TPO-receptor stimulation and point at the immunomodulating properties of these drugs. Here, we present a case of prolonged bone marrow aplasia secondary to autologous SCT treated with eltrombopag. We describe the clinical efficacy and the immunomodulating effect of this drug on inflammatory cytokine profile and bone marrow histology. Furthermore, we provide a review of the most recent literature highlighting the efficacy and safety of TPO-RA after SCT and chemotherapy for hematologic conditions.

Congenital hemolytic anemias (CHAs) are a heterogeneous group of rare hereditary conditions including defects of erythrocyte membrane proteins, red cell enzymes, and disorders due to defective erythropoiesis. They are characterized by variable degree of anemia, chronic extravascular hemolysis, reduced erythrocyte life span, splenomegaly, jaundice, biliary lithiasis, and iron overload. Although few data are reported on the role of the immune system in CHAs, several immune-mediated mechanisms may be involved in the pathogenesis of these rare diseases. We reported in ~60% of patients with hereditary spherocytosis (HS), the presence of naturally-occurring autoantibodies (NAbs) directed against different membrane proteins (α- and β-spectrin, band 3, and dematin). Positive HS subjects showed a more hemolytic pattern and NAbs were more evident in aged erythrocytes. The latter is in line with the function of NAbs in the opsonization of damaged/senescent erythrocytes and their consequent removal in the spleen. Splenectomy, usually performed to reduce erythrocyte catheresis and improve Hb levels, has different efficacy in various CHAs. Median Hb increase is 3 g/dL in HS, 1.6–1.8 g/dL in pyruvate kinase deficiency (PKD), and 1 g/dL in congenital dyserythropoietic anemias (CDA) type II. Consistently with clinical severity, splenectomy is performed in 20% of HS, 45% of CDAII, and in 60% of PKD patients. Importantly, sepsis and thrombotic events have been registered, particularly in PKD with a frequency of ~7% for both. Furthermore, we analyzed the role of pro-inflammatory cytokines and found that interleukin 10 and interferon γ, and to a lesser extent interleukin 6, were increased in all CHAs compared with controls. Moreover, CDAII and enzymatic defects showed increased tumor necrosis factor-α and reduced interleukin 17. Finally, we reported that iron overload occurred in 31% of patients with membrane defects, in ~60% of CDAII cases, and in up to 82% of PKD patients (defined by MRI liver iron concentration >4 mg Fe/gdw). Hepcidin was slightly increased in CHAs compared with controls and positively correlated with ferritin and with the inflammatory cytokines interleukin 6 and interferon γ. Overall the results suggest the existence of a vicious circle between chronic hemolysis, inflammatory response, bone marrow dyserythropoiesis, and iron overload.

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.

Abstract This study investigated for the first time brain ischemic involvement in 19 consecutive neurologically asymptomatic PNH patients by non-enhanced cerebral MRI, and by intracranial arterial and venous angio-MRI. Eleven cases (58%, 7 aged 5 mm, and 5 cases a score >4 by the age-related white matter changes (ARWMC) scale. Compared with age and sex-matched controls (1:2 ratio), patients showed an increased frequency of periventricular WM vascular degeneration (32% versus 5.2%, p = 0.04) and of severe lesions (ARWMC scale score >4) (26% versus 2.6%, p = 0.05), and a higher overall ARWMC scale score (3.5 ± 1.07 versus 2.0 ± 0.8, mean ± SD, p

Abstract The Gardos channel is a Ca2+ sensitive, K+ selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients’ RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient’s RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca2+ overload in the RBCs of both patients and hypersensitivity of Ca2+ uptake by RBCs to swelling. Altogether these findings suggest that the ‘Gardos channelopathy’ is a complex pathology, to some extent different from the common hereditary xerocytosis.