Negative Autoimmune Hemolytic Anemia Research Articles

Challenges in the diagnosis and management of autoimmune hemolytic anemia: A case-based approach. Experience from a tertiary care hospital in the Haryana region.

Autoimmune hemolytic anemia (AIHA) is a rare immune disorder which occurs when antibodies are directed against self red blood cells (RBCs) leading to hemolysis. AIHA is widely classified as warm autoimmune hemolytic anemia, cold agglutinin syndrome, mixed AIHA, paroxysmal cold hemoglobinuria and rarely drug induced AIHA. The pathogenesis of AIHA is complex interplay between genetic predisposition, immune dysregulation and enviornmental triggers. A direct antiglobulin test can be used to assess the immunological origin of the hemolysis in order to diagnose AIHA after identifying laboratory and clinical symptoms of hemolysis. The objective is to understand underlying mechanism in AIHAs, and usage of targeted therapies to modulate specific components of the immune response. We are hereby presenting a case series of 11 clinically suspected cases of AIHA in collaboration with their clinical features, immuno-hematological and other laboratory parameters, Flow cytometric analysis of lymphocyte subset in relevant cases, underlying etiology as well as serological subtype are also included. Majority of the patients were categorized as secondary AIHA (7/11, 63.63%). Out of 11 cases 7 were serologically subtyped as warm AIHA (7/11, 63.63%) ,2 cases were DaaT negative AIHA (2/11;18.18%), 2 cases were characterized as mixed AIHA subtype (2/11, 18.18%). Accurate subtyping of AIHA requires a systematic immunohematological approach coupled with comprehensive evaluations of clinical, hematological, and biochemical parameters.

Direct antiglobulin test-negative autoimmune hemolytic anemia associated with erythropoiesis stimulating agent resistance in a CKD patient with IgG4-related disease: A case report

Rationale: Direct antiglobulin test (DAT)-negative autoimmune hemolytic anemia (AIHA) is an important differential diagnosis for erythropoiesis-stimulating agents-resistant renal anemia with hemolysis. Patient concerns: An 82-year-old female with a past medical history of diabetes, end-stage kidney disease (estimated glomerular filtration rate: 6.9 mL/min/1.73 m2), post-transcatheter aortic valve implantation, and IgG4-related retroperitoneal fibrosis on prednisolone therapy was referred to our department for assessment of severe anemia. The patient underwent immunohematological testing for serological evaluation. Diagnoses: DAT-negative AIHA and IgG4-RD was established based on DAT-negative and immunohematological tests. Interventions: Glucocorticoid was administered to the patient. Outcomes: The patient’s condition including hemoglobin level was relieved. Lessons: In chronic kidney disease, AIHA may contribute to the development of severe anemia and erythropoiesis-stimulating agents-resistance, irrespective of the results of Coombs test. Reticulocytosis, undetectable haptoglobin and a fall in hemoglobinA1c despite sustained hyperglycemia may be a clue to the diagnosis of hemolysis.

COVID-19 and Immune-Mediated RBC Destruction.

ObjectivesTo summarize the epidemiologic, clinical, and laboratory characteristics of autoimmune hemolytic anemia (AIHA) secondary to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination.MethodsWe conducted a systematic review using standardized keyword search to identify all reports of SARS-CoV-2 infection or vaccination and AIHA across PubMed, Web of Science, Scopus, and Google Scholar through September 24, 2021.ResultsFifty patients (mean [SD] age, 50.8 [21.6] years) diagnosed with coronavirus disease 2019 (COVID-19) and AIHA were identified. AIHA subtypes and number of patients were as follows: cold AIHA (n = 18), warm AIHA (n = 14), mixed-type AIHA (n = 3), direct antiglobulin test (DAT)–negative AIHA (n = 1), DAT-negative Evans syndrome (n = 1), Evans syndrome (n = 3), and subtype not reported (n = 10). Mean (SD) hemoglobin at AIHA diagnosis was 6.5 [2.8] g/dL (95% confidence interval, 5.7-7.3 g/dL). Median time from COVID-19 symptom onset to AIHA diagnosis was 7 days. In total, 19% (8/42) of patients with COVID-19–associated AIHA with reported outcomes were deceased. Four patients (mean [SD] age, 73.5 [16.9] years) developed AIHA following SARS-CoV-2 vaccination: Pfizer-BioNTech BNT162b2 vaccine (n = 2); Moderna mRNA-1273 vaccine (n = 1); undisclosed mRNA vaccine (n = 1). AIHA occurred after 1 dose in 3 patients (median, 5 days).ConclusionsSARS-CoV-2 infection and vaccination are associated with multiple AIHA subtypes, beginning approximately 7 days after infectious symptoms and 5 days after vaccination.

Single Nucleotide Polymorphisms of Cytokine Genes in Warm Autoimmune Hemolytic Anemias: Relationship with Clinical and Hematological Parameters

Background: Autoimmune hemolytic anemia (AIHA) is due to an increased immune peripheral destruction of red blood cells. The disease is characterized by high heterogeneity regarding anemia severity, treatment requirement and occurrence of complications. It has been shown that Hb levels at baseline, presence of complement activation, and the degree of bone marrow compensation are the main predictors of outcome. Moreover, multi-relapsing cases show bone marrow dysmyelopoiesis, and fibrosis suggesting an immune attack against bone marrow precursors. An interesting area to investigate is the genetic landscape of AIHA, including cytokine gene signature and other molecular mechanisms of refractoriness.Aim: To evaluate the frequency of cytokine single nucleotide polymorphisms (SNPs) in warm AIHA and their correlation with clinical and hematological parameters, including the degree of anemia, treatment refractoriness and number of therapies lines.Methods: We investigated SNPs of five cytokine genes [tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, interleukin (IL)-6 and IL-10] in 72 Italian warm AIHA patients, using the Cytokine Tray (One Lambda, Canoga Park, CA) according to the manufacturer's recommendations. Allele and genotype frequency results were compared to those described in the Italian population by Uboldi de Capei et al. (2003) and Poli et al. (2002).Results: Seventy-two warm AIHA patients have been evaluated: median age 63 years (range 17-90), classified as per DAT positivity in IgG+ (N=48), IgG+C (N=17), IgA+ (N=1), and DAT negative AIHA (N=6). Demographic and hematological characteristics of patients are described in Figure 1A. Regarding cytokine SNP frequencies, we observed that AIHA patients had a significantly higher prevalence of the TNF-alpha-308 high producers genotypes (G/G and G/A) compared to controls (40.3% vs 16% or 23%, p=0.0003 and p=0.006, respectively). No differences were detected regarding genotype frequencies of the other cytokine SNPs (Figure 1B). By dividing patients according to anemia severity at onset, a more frequent low producer genotype of IFN-gamma (A/A) was observed in patients with Hb levels >7.4 g/dL (median value of patients cohort) compared to the other group (p=0.05). Finally, considering the number of relapses or therapy lines, an increased frequency of TNF-alpha -308 high producers genotypes were found among multi-relapsing (> 3 relapses, 70.5% vs 56%) and multi-treated patients (> 3therapy lines, 52% vs 35%), although not significantly.Conclusions: These preliminary data show that wAIHA patients display a more pro-inflammatory cytokine signature at molecular level as compared to the Italian control population. This is highlighted by an increased prevalence of TNF-alpha high producer genotype that is also associated with multiple relapses and greater treatment requirement. Additionally, cytokine SNPs may be also associated with anemia severity at onset, as observed for IFN-gamma low producer genotype that was more frequent in less severe cases. [Display omitted] DisclosuresFattizzo: Alexion, Amgen, Annexon, Apellis, Grifols, Novartis, Momenta: Consultancy, Speakers Bureau. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Barcellini: Agios: Honoraria, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Alexion Pharmaceuticals: Honoraria; Incyte: Membership on an entity's Board of Directors or advisory committees.

Comparison of Polybrene DAT and Conventional DAT in Cases of Suspected DAT Negative Autoimmune Hemolytic Anemia (AIHA)

Abstract Introduction/Objective A negative Direct Antiglobulin Test (DAT) is seen in approximately 1-5% of patients presenting with signs and symptoms of immune hemolysis. A well-performed ‘standard’ DAT detects ~100-500 molecules of bound IgG per red cell. A more sensitive polybrene DAT may be helpful in order to confirm a diagnosis of AIHA in patients whose samples have tested negative via standard DAT. However, there has been little reported on utility of the polybrene DAT in evaluating such cases with modern DAT reagents. We hypothesized that the polybrene DAT would not contribute substantially to the analysis of Coombs-negative hemolytic anemia (C-NHA), based primarily on our anecdotal observation. As such, we undertook a study to assess results of polybrene DAT in cases evaluated for possible (C-NHA). Methods/Case Report Two study sites were used for data analysis (Yale-New Haven Hospital, Site A, and VA Connecticut, Site B) over a five year period (2016-2021). During this time, standard DATs were performed at both study sites by the tube method using polyspecific antiglobulin and, if positive, reflex to anti-IgG and -C3. For cases of suspected C-NHA (which are reported to Blood Bank via a consult mechanism), conventioned DAT- samples are referred to our regional immunohematology laboratory (American Red Cross, Farmington, CT) for manual hexadimethrine bromide (Polybrene) DAT. Polybrene DAT is reported as negative with two sources of polyspecific AHG. Subsets of patients also underwent acid elution studies (Gamma ELU-KITII) as part of a C-NHA algorithm developed during the study period. Results of standard DAT, acId elution, and polybrene DAT were extracted from Blood Bank electronic records at both study sites. Results (if a Case Study enter NA) Evaluation for C-NHA was performed in 32 patients/cases over the study period. Amongst these individuals, 96.8% (31/32) underwent polybrene DAT assessment and none (0%; 0/31) demonstrated a positive polybrene DAT result. Notably, acid elution studies were performed in 90.6% (29/32) of traditional DAT negative cases. Of these, 10.3% (3/29) had reactive eluates. Conclusion Performance of the polybrene DAT appeared to be of no value in the assessment of suspected C-NHA. Given that nearly 10% of individuals with a negative conventional DAT had a positive acid elution, this testing step appears to be of greater value in potentially identifying an autoantibody in suspected cases of C-NHA.

Difficult Cases of Autoimmune Hemolytic Anemia: A Challenge for the Internal Medicine Specialist.

Autoimmune hemolytic anemia (AIHA) is diagnosed in the presence of anemia, hemolysis, and direct antiglobulin test (DAT) positivity with monospecific antisera. Many confounders of anemia and hemolytic markers should be included in the initial workup (i.e., nutrients deficiencies, chronic liver or kidney diseases, infections, and cancers). Besides classical presentation, there are difficult cases that may challenge the treating physician. These include DAT negative AIHA, diagnosed after the exclusion of other causes of hemolysis, and supported by the response to steroids, and secondary cases (infections, drugs, lymphoproliferative disorders, immunodeficiencies, etc.) that should be suspected and investigated through careful anamnesis physical examination, and specific tests in selected cases. The latter include autoantibody screening in patients with signs/symptoms of systemic autoimmune diseases, immunoglobulins (Ig) levels in case of frequent infections or suspected immunodeficiency, and ultrasound/ computed tomography (CT) studies and bone marrow evaluation to exclude hematologic diseases. AIHA occurring in pregnancy is a specific situation, usually manageable with steroids and intravenous (iv) Ig, although refractory cases have been described. Finally, AIHA may complicate specific clinical settings, including intensive care unit (ICU) admission, reticulocytopenia, treatment with novel anti-cancer drugs, and transplant. These cases are often severe, more frequently DAT negative, and require multiple treatments in a short time.

Small Paroxysmal Nocturnal Hemoglobinuria Clones in Autoimmune Hemolytic Anemia: Clinical Implications and Different Cytokine Patterns in Positive and Negative Patients.

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.

Outcomes of splenectomy in relapsed/refractory autoimmune hemolytic anemia

目的评估脾切术治疗复发/难治自身免疫性溶血性贫血（AIHA）患者的疗效。方法回顾性分析中国医学科学院血液病医院30例行脾切除术治疗的复发/难治自身免疫性溶血性贫血（AIHA）患者的临床资料，评估手术疗效。结果30例复发/难治AIHA患者中，单纯AIHA 20例（温抗体型13例，温冷双抗体型2例，Coombs阴性AIHA 5例），Evans综合征10例。脾切除术后10~14 d，20例患者可评价疗效，总有效率（ORR）为90%（完全缓解12例，部分缓解6例）。13例患者有长期随访资料，中位随访14（4~156）个月，截至随访终点，除3例Evans综合征患者死亡外（2例无效，1例复发），10例复发/难治AIHA患者术后6个月9例（90%）有效，术后12个月7例（70%）有效，其中3例患者已持续完全缓解3年以上。结论脾切除术作为二线方案治疗复发/难治AIHA近期疗效好，1年远期ORR达到70%，约三分之一患者可维持长期持续缓解。

Common presentation of an uncommon anaemia in a resource-limited setting: A diagnostic challenge

A 38-year-old female in the reproductive age group presented with anaemia in a resource-poor setting. The patient was given multiple transfusions during the course of her treatment. After a proper work-up she was diagnosed with Coomb's negative autoimmune haemolytic anaemia (AIHA). She was later treated with steroid and she showed significant improvement. Early diagnosis in her case at the primary care level with other causes of anaemia in mind could have prevented unnecessary transfusions and morbidity. Proper history and approach to anaemia is the key in a resource-poor setting.

A case of Direct Coombs test (DAT)-negative autoimmune hemolytic anemia associated with low-affinity autoantibodies

A case of Direct Coombs test (DAT)-negative autoimmune hemolytic anemia associated with low-affinity autoantibodies

Thirteen Year Retrospective Analysis Of Adult Patients With Autoimmune Hemolytic Anemia At The Cleveland Clinic: Responses To Therapy

Background and ObjectiveTreatment of autoimmune hemolytic anemia (AIHA) is complicated by disease heterogeneity and a high propensity for relapse. Most cases of AIHA are mediated by warm and cold-reactive autoantibodies, as defined by the direct antiglobulin test (DAT), and the treatment approach differs depending on the subtype. Evidence supporting use of specific AIHA treatments is limited to retrospective studies, small series, or case studies. To address this issue, a retrospective analysis of all patients with AIHA treated at the Cleveland Clinic from January 2000 to April 2013 was performed. MethodsPatients were included if they were ≥ 18 years old and presented with a new diagnosis or relapse of AIHA. Frequencies of complete responses (CR) or partial responses (PR) were recorded, and the median time to relapse measured using the Kaplan-Meier method. PR was defined as a nontransfused hemoglobin of ≥ 10 mg/dL with a hemoglobin increase of ≥ 2 mg/dL, while CR was defined as a nontransfused hemoglobin of ≥ 12 mg/dL, hemoglobin increase ≥ 2 mg/dL, and normalization of hemolytic markers. The duration of high dose and total corticosteroid therapy was recorded. Results180 patients were identified. 122 (69%) presented with warm antibody-mediated AIHA, 42 (24%) with cold antibody-mediated AIHA, 6 (3%) with mixed AIHA, and 8 (4%) with DAT negative AIHA. 97 (54%) were females. 61% of patients were between the ages of 50 and 80. Steroids were used in 82 patients with warm AIHA, 14 patients with cold AIHA, 3 patients with mixed AIHA, and 5 patients with DAT negative AIHA. Steroid response rates were 81% (27% CR, 54% PR) in warm AIHA, 85% (21% CR, 64% PR) in cold AIHA, 66% (33% CR, 33% PR) in mixed AIHA, and 80% (40% CR, 40% PR) in DAT negative AIHA. Median time to relapse was 14.3 months in warm AIHA, 3.6 months in cold AIHA, and 13 months in mixed AIHA; median time to relapse could not be determined in DAT negative AIHA. The mean duration of steroid use was 6.3 months in warm AIHA, 4.3 months in cold AIHA, 5.4 months in mixed AIHA, and 1.5 months in DAT negative AIHA. Rituximab was used in 23 patients with warm AIHA and 14 patients with cold AIHA, with overall response rates of 74% (17% CR, 57% PR) and 80% (27% CR, 53% PR), respectively. The median time to relapse was 8.5 months for warm and 55.2 months for cold AIHA after Rituximab treatment. In 15 patients with warm AIHA, splenectomy induced responses in 83% (33% CR, 50% PR) with a median time to relapse of 37.8 months. In 24 patients with warm AIHA and 7 patients with cold AIHA, treatment of the underlying disorder induced responses in 87% (33% CR, 54% PR) and 86% (29% CR, 57% PR), respectively. Median time to relapse was 22.1 months in warm AIHA and 16.5 months in cold AIHA. Other less commonly used treatments included immunosuppressants, intravenous immunoglobulin, and erythropoietin derivatives, with bortezomib and cold avoidance used in the cold AIHA group. When comparing warm and cold AIHA, no significant difference was found in total response rates to various treatments, including steroids (81% vs. 85%, p = 0.486), Rituximab (74% vs. 80%, p = 0.49), and treatment of the underlying condition (87% vs. 86%, p = 0.662). ConclusionsThis analysis demonstrated a surprisingly high response rate for corticosteroids in all subtypes of AIHA, though most responses in cold AIHA were partial and of relatively short duration. Rituximab was effective in both warm and cold antibody-mediated AIHA, with a high incidence of durable responses in the latter. Definitive treatment of the underlying condition was used less commonly, but was a highly effective approach for treating secondary AIHA. Splenectomy yielded durable remissions in most patients with warm AIHA. Disclosures:No relevant conflicts of interest to declare.

A Case of Non-Hodgkin's Lymphoma in Patient with Coombs' Negative Hemolytic Anemia and Idiopathic Thrombocytopenic Purpura

Coombs' negative autoimmune hemolytic anemia (AIHA) is a rare disease which shares similar clinical and hematological features with Coombs' positive AIHA, but its exact frequency remains unknown. There have been few reports of idiopathic thrombocytopenic purpura (ITP) and Coombs' negative AIHA associated with other lymphoproliferative disorders (LPDs). Since there is a well known association between LPDs and autoimmune phenomena, it is important to investigate the possibility of an underlying malignancy. We report a case of ITP and Coombs' negative AIHA associated with diffuse large B-cell lymphoma.

Role of gel test and flow cytometry in diagnosis of Coombs’ negative autoimmune haemolytic anaemia

This study evaluated the use of both gel test (GT) and flow cytometry (FC) techniques in the detection of red blood cell-bound immunoglobulin G (IgG) and, hence, the diagnosis of autoimmune haemolytic anaemia (AIHA) in cases that were negative by the conventional tube technique (CTT) direct antiglobulin test (DAT). The study enrolled 50 clinically diagnosed AIHA patients with CTT Coombs' negative test and 55 control subjects. GT and FC were performed on both groups. Ninety-two percent (46/50) of patients were positive by GT. As for FC assay, the Receiver Operating Characteristic curve determined that a cut-off of 17.5% fluorescence was the best value for interpreting FC-DAT positively in the group of patients with haemolytic anaemia (100% sensitivity and specificity), while the best cut-off for mean fluorescence intensity (MFI) was 1.74, with 76% specificity and 96% sensitivity. Both mean percent fluorescence and MFI were significantly higher among patients when compared to controls (P < 0.001). FC assay results showed no statistically significant correlations with patients' laboratory data or GT grades (P > 0.05). Flow cytometry is more sensitive than GT for assessing CTT-DAT-negative AIHA. We propose that FC percent fluorescence cut-off values should be employed to determine the Coombs' negative AIHA cases.

IgA‐mediated autoimmune hemolytic anemia in an infant

Autoimmune hemolytic anemia (AIHA) is characterized by the presence of autoantibodies, most frequently of the IgG isotype, directed against erythrocyte surface antigens. The direct antiglobulin test (DAT) is the critical laboratory test for the diagnosis of AIHA, but is negative in 3-11% of cases. In these cases of DAT negative AIHA, a wider spectrum of clinical data including more specialized testing for erythrocyte autoantibodies may be required. We describe the unique and challenging case of an infant with corticosteroid-responsive, DAT negative AIHA, in which specialized gel card testing identified an isolated IgA autoantibody on the erythrocyte surface.

Cut‐off value of red‐blood‐cell‐bound IgG for the diagnosis of Coombs‐negative autoimmune hemolytic anemia

Direct antiglobulin test (DAT)-negative autoimmune hemolytic anemia (Coombs-negative AIHA) is characterized by laboratory evidence of in vivo hemolysis, together with a negative DAT performed by conventional tube technique (CTT) in clinically suspected AIHA patients. The immunoradiometric assay (IRMA) for red-blood-cell-bound immunoglobulin G (RBC-IgG) can be used to diagnose patients in whom CTT does not detect low levels of red cell autoantibodies. We investigated the diagnostic cutoff value of the IRMA for RBC-IgG in Coombs-negative AIHA and calculated its sensitivity and specificity. Of the 140 patients with negative DAT by CTT referred to our laboratory with undiagnosed hemolytic anemia, AIHA was clinically diagnosed in 64 patients (Coombs-negative AIHA). The numbers of Coombs-negative AIHA and non-AIHA patients changed with age and gender. The cutoff values were determined from receiver operating characteristic (ROC) curve according to age and gender. The IRMA for RBC-IgG proved to be sensitive (71.4%) and specific (87.8%) when using these cutoffs. Using these cutoffs for 41 patients with negative DAT referred to our laboratory in 2006, all the pseudonegative cases were treated with steroids before the test. The 31 untreated cases could be grouped using one cutoff value of 78.5 and showed 100% sensitivity and 94% specificity, independent of gender and age. Results indicate that RBC-IgG could become a standard approach for the diagnosis of Coombs-negative AIHA, when measured before treatment.

Coombs’-Negative Autoimmune Hemolytic Anemia in a Patient With Small Lymphocytic Lymphoma Involving the Bone Marrow

1. What are some possible causes of hemolytic anemia and into which 2 broad categories can they be divided? 2. What is the only hemolytic anemia caused by an acquired intrinsic defect in the RBC cell membrane? 3. What is Coombs’ testing? 4. What are some of the possible etiologies of autoimmune hemolytic anemia? 5. What is an appropriate workup for a Coombs’-negative hemolytic anemia that is suspected to be autoimmune?

An unusual case of autoimmune hemolytic anemia in treatment naïve hepatitis C virus infection

Hepatitis C virus (HCV) infection is emerging as a common and insidiously progressive liver condition. In more than one third of the cases, extrahepatic manifestations are seen in the course of the disease. Over the past decade, authors have reported membranous nephropathy, cutaneous vasculitis, idiopathic thrombocytopenic purpura, porphyria cutanea tarda and diabetes mellitus, among other extrahepatic manifestations of HCV infection. Recently, there have been a growing number of reports relating HCV infection to autoimmune cytopenias. Here, we report an unusual case of Coombs'-negative autoimmune hemolytic anemia (AHA) with severe autoimmune leukopenia and neutropenia, occurring simultaneously, in a patient with untreated hepatitis C infection. Mild cytopenias during chronic hepatitis C have been reported widely in the medical literature; however, severe cytopenias are seldom described and are usually seen only after or simultaneously with therapy.

Application of flow cytometry in detection of red cell bound IgG in Coombs negative AIHA

Coombs negative autoimmune hemolytic anemia (AIHA) is characterized by laboratory evidence of in vivo hemolysis along with a negative direct antiglobulin test (DAT) performed by conventional tube technique (CTT) in clinically suspected AIHA patients. The sensitive gel test (GT) and flow cytometry (FC) can effectively diagnose such patients where CTT does not detect low level of red cell autoantibodies. We investigated the use of FC in the serological evaluation of CTT DAT negative AIHA and its comparison with GT DAT. Of the 50 patients with suspected AIHA, CTT DAT was negative in 5 patients (Coombs negative AIHA). GT DAT could detect red cell autoantibodies in 4 of these 5 patients. Monospecific GT DAT showed IgG and/or C3d as the responsible autoantibody. FC was considered as reactive when MFI was > 3.6 (mean of 20 healthy negative volunteers +2SD). FC was reactive in all five Coombs negative AIHA patients. The mean MFI in five known CTT DAT positive samples taken for comparison was significantly higher compared to 5 DAT negative AIHA (18.3 ± 7.78 vs. 7.88 ± 1.35, p < 0.05). There was poor correlation between strength of GT DAT and MFI by FC. We conclude that FC is more sensitive test than the CTT and helps in the serological diagnosis of Coombs negative AIHA. However, in resource poor settings, GT DAT can be a good alternative to FC.

Anémie hémolytique auto-immune et dysérythropoïèse révélatrices d'un déficit de l'apoptose Fas chez 3 enfants

Defective apoptosis caused by mutations of the Fas gene can lead to an autoimmune lymphoproliferative syndrome (ALPS). The main autoimmune manifestations are haematological: hemolytic anemia, thrombocytopenia and neutropenia. We described 3 patients with ALPS presenting as a lymphoproliferative syndrome associated with a Coomb's negative autoimmune hemolytic anemia and dyserythropoiesis predominating on the more mature erythroblasts. Fas apoptosis deficiency was evidenced in the 3 patients by the demonstration of an increased number of CD4 –CD8 –TCRαβ + T cells, a decreased apoptotic response of activated T lymphocytes to anti-Apo 1-3 monoclonal antibody and the presence of a heterozygous mutation of the Fas receptor gene.

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.