Transfusions in autoimmune hemolytic anemias: Frequency and clinical significance of alloimmunization.

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.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients: A proof-of-concept study.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients. Data showed decreased glycolytic activity in red blood cells of 2/3 of patients with lower-risk MDS. These results highlight a potential effect of the PK activator in this setting.

Changing trends of splenectomy in hereditary spherocytosis: The experience of a reference Centre in the last 40 years.

We describe the clinical/haematological characteristics of 446 patients with hereditary spherocytosis diagnosed in the last 40 years in a reference centre. The frequency of splenectomy decreased over time (44% before 1990 to 7% in 2011-2020), notwithstanding a confirmed good efficacy. Age at splenectomy progressively increased (63% in children before 1990 to 88% in patients aged ≥20 years in 2011-2020). Our real-life experience showed that even a fraction of patients in the trait/mild categories (19/92, 21%) were splenectomised, whilst 30/78 (38%) in the moderate/severe groups were not. Overall, these data pinpoint to the increasing awareness about post-splenectomy thromboses and infections.

Iron overload in congenital haemolytic anaemias: role of hepcidin and cytokines and predictive value of ferritin and transferrin saturation.

Iron overload (IO) is poorly investigated in the congenital haemolytic anaemias (CHAs), a heterogeneous group of rare inherited diseases encompassing abnormalities of the erythrocyte membrane and metabolism, and defects of the erythropoiesis. In this study we systematically evaluated routine iron parameters and cardiac and hepatic magnetic resonance imaging, together with erythropoietin, hepcidin, non-transferrin bound iron (NTBI), and cytokine serum levels in patients with different CHAs. We found that 40% of patients had a liver iron concentration (LIC) >4 mg Fe/g dry weight. Hepatic IO was associated with ferritin levels (P = 0·0025), transferrin saturation (TfSat, P = 0·002) and NTBI (P = 0·003). Moreover, ferritin >500 µg/l plus TfSat >60% was demonstrated as the best combination able to identify increased LIC, and TfSat alteration as more important in cases with discordant values. Possible confounding factors, such as transfusions, hepatic disease, metabolic syndrome and hereditary haemochromatosis-associated mutations, had negligible effects on IO. Erythropoietin and hepcidin levels were increased in CHAs compared with controls, correlating with LIC and ferritin, respectively. Regarding cytokines, γ-interferon (IFN-γ) was increased, and both interleukin 6 and IFN-γ levels positively correlated with ferritin and hepcidin levels. Overall, these findings suggest the existence of a vicious cycle between chronic haemolysis, inflammatory response and IO in CHAs.

Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients.

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.

Detection of erythroblast antibodies in mitogen-stimulated bone marrow cultures from patients with myelodysplastic syndromes.

BACKGROUND: Low-risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial. STUDY DESIGN AND METHODS: We studied the presence of erythroblast antibodies in mitogen-stimulated bone marrow (BM) cultures of 70 patients with early-stage MDS (refractory anemia and refractory anemia with ringed sideroblasts). RESULTS: Sixty-six percent of patients showed positive erythroblast antibodies, along with BM erythroid hyperplasia and a hemolytic picture in the peripheral blood. Supernatants from positive cultures induced an increase of overall cellularity, the appearance of erythroblastic clustering, and dyserythropoietic signs in normal BM. We identified CD45(dim) Gly-A(dim) CD71(bright) cells (red blood cell precursors at different maturation stage) as the target of the antibodies. Erythropoietin (EPO) levels were reduced and EPO receptors (EPO-R) increased in BM culture supernatants from positive patients. However, flow cytometric analysis showed that neither EPO nor EPO-R was involved in an abnormal stimulation driven by these autoantibodies. Values of the proapoptotic protein Bax were increased in positive patients and Bcl-2 levels were decreased, although not significantly. CONCLUSION: MDS patients with anti-erythroblast autoimmunity showed increased BM apoptosis, suggesting that the autoimmune reaction may contribute to an unfavorable BM microenvironment for optimal erythropoiesis.

Detection of red blood cell antibodies in mitogen-stimulated cultures from patients with hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS. STUDY DESIGN AND METHODS: RBC antibody-bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect. RESULTS: A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p < 0.0001), although lower than that observed in autoimmune hemolytic anemia (AIHA; 634 ± 371 ng/mL vs. 236 ± 192 ng/mL, p < 0.0001). Western blot experiments showed that purified IgG fraction from MS-DAT-positive patients bind to α- and ß-spectrin, Band 3, and Band 4.9. Positive cases displayed increased reticulocytosis and slightly reduced hemoglobin (Hb) values compared to negative ones. Patients displaying RBC-bound IgG of more than 250 ng/mL (the positive threshold of AIHA) showed increased number of spherocytes and mainly had spectrin deficiency. RBC-bound IgG and free Hb increased over time after storage at 4°C, a surrogate of ex vivo aging, more evidently in HS than controls, and particularly in Band 3 deficiency. CONCLUSION: RBC autoantibodies were detected by MS-DAT in more than a half of HS patients. Positive cases showed a more evident hemolytic pattern suggesting a pathogenic role of these autoantibodies in RBC opsonization and splenic removal.

Low-dose rituximab in adult patients with idiopathic autoimmune hemolytic anemia: clinical efficacy and biologic studies.

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.

Recombinant erythropoietin in autoimmune hemolytic anemia with inadequate bone marrow response: a prospective analysis.

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.

Sustained response to low-dose rituximab in idiopathic autoimmune hemolytic anemia.

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.

Cytokine polymorphisms in patients with autoimmune hemolytic anemia.

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-ß1, 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-ß 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-ß and IL-10 high gene expression was higher in patients with more severe anemia, and TGF-ß high gene expression was higher in patients with active disease. Considering treatment, TNF-α and TGF-ß 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-ß 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.

Evaluation of the main regulators of systemic iron homeostasis in pyruvate kinase deficiency.

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.