Three different pathways of IgM-antibody-dependent hemolysis are mainly regulated by complement.

Antibodies to red blood cells (RBCs) may hemolyze erythrocytes via Fc-mediated phagocytosis or complement-dependent. Complement activation on RBCs can be detected by C3d-direct antiglobulin test (DAT), which is the only test in immune hematology that directly targets complement. However, a positive DAT with anti-C3d cannot distinguish between C3b-mediated extravascular hemolysis, C5b-C9-mediated intravascular hemolysis and C5b-C8-mediated eryptosis. Furthermore, DAT is not suitable to estimate the strength of hemolysis. Autoimmune hemolytic anemia (AIHA) is a rare disease that is caused by autoantibodies to red blood cells that is divided in warm AIHA and in cold agglutinin disease (CAD). The causative antibodies in CAD and sometimes in warm AIHA are from the IgM class. Depending on strength of complement activation they can induce extravascular hemolysis, intravascular hemolysis and eryptosis. We studied the three types of hemolysis by use of sera from patients with CAD under various conditions. We found that additionally to the routinely applied C3d-DAT, indirect tests for complement activity (free hemoglobin and Annexin V-binding to phosphatidylserine-exposing RBCs) should be used to determine the portion of extravascular, intravascular and eryptotic hemolysis. Eryptotic hemolysis may have a significant share in clinical relevant CAD or IgM warm AIHA, which should be considered for successful treatment.

Post-treatment haemolysis is common following oral artemisinin combination therapy of uncomplicated malaria in travellers.

BACKGROUND: Artemisinin-based combination therapy (ACT) for the treatment of malaria is highly effective, well tolerated and safe. Episodes of delayed haemolysis occur in up to 57.9% of patients with severe malaria treated with intravenous artesunate, mainly caused by 'pitting' of infected red blood cells in the spleen and the delayed loss of these once-infected RBCs (oiRBCs). Several reports indicate that post-treatment haemolysis (PTH) also occurs in uncomplicated malaria treated with oral ACT, calling for systematic investigation. METHODS: A prospective observational study to identify the incidence of PTH after oral ACT, defined as increased lactate dehydrogenase activity and low haptoglobin level on Day 14 after treatment. Patients were enrolled at two study centres in Germany and Italy. Study visits took place on Days 1, 3, 7, 14 and 28. Laboratory investigations included extended clinical routine laboratory tests, quantitative PfHRP2, anti-RBC antibodies and oiRBCs. The state of semi-immunity to malaria was assessed from childhood and ongoing exposure to Plasmodium spp. as per patient history. RESULTS: A total of 134 patients with uncomplicated malaria and 3-day ACT treatment were recruited. Thirty-seven (37.4%) of 99 evaluable patients with Pf and none of 9 patients with non-Pf malaria exhibited PTH on d14. Patients with PTH had higher initial parasitaemia, higher oiRBC counts on d3 and a 10-fold decrease in oiRBCs between d7 and d14 compared with patients without PTH. In patients with PTH, loss of haemoglobin was 4-fold greater in non-Africans than in Africans (-1.3 vs -0.3 g/dl). Semi-immune African patients with PTH showed markedly increased erythropoiesis on d14 compared with not semi-immune African and non-African patients with PTH. CONCLUSIONS: PTH is common in patients with uncomplicated malaria and oral ACT. While the observed loss of haemoglobin will often not be clinically relevant, it could aggravate pre-existing anaemia, warranting follow-up examinations in populations at risk.

Silvernanoparticle-induced hemolysis confounded with direct antiglobulin test-negative autoimmune hemolytic anemias diagnosis.

BACKGROUND: We describe here the first patient with recurrent hemolysis related to disinfectant containing silver nanoparticles (AgNps). METHODS: A 58-year-old chemist repeatedly experienced DAT-negative (Coombs-negative) hemolysis during the last 5 years. He was treated with a number of immunosuppressive drugs including 18 times rituximab. The attempt to treat him with cyclosporine A served only to increase the rate of hemolysis. Only by chance, we revealed that the patient regularly used a hand disinfectant containing AgNps. Serological testing was performed using standard techniques. Eryptosis was measured by binding annexin to exposed phosphatidylserine (PS) of the circulating red blood cells (RBCs). RESULTS: Antiglobulin tests remained negative, and PS exposing RBCs were detected two times during the last hemolytic episodes. Hemolysis completely disappeared following discontinuation of AgNp containing products. CONCLUSION: AgNps are increasingly being used in a large variety of products. Recently, it was reported that they induce in vitro prohemolytic and procoagulant effects via oxidative stress and eryptosis. The clinical findings imply the hemolysis was provoked by the patient's regular use of cleansing products containing AgNps. Our finding might help to explain the etiology of hemolytical disorders that may remain obscure in many cases.

A fluorometric erythrophagocytosis assay using differentiated monocytic THP-1 cells to assess the clinical significance of antibodies to red blood cells.

BACKGROUND AND OBJECTIVES: The significance of antibodies to red blood cells (RBCs) is variable and cannot be predicted solely by serological testing. A flow cytometry-based erythrophagocytosis assay was established using phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells and RBCs labelled with PKH26 to assess allo- and autoantibodies to RBCs. MATERIALS AND METHODS: THP-1 cells were differentiated into macrophage-like cells by treatment with PMA. RBC samples coated with alloantibodies or autoantibodies were obtained from 16 patients with autoimmune haemolytic anaemia of warm type (wAIHA) as well as from five pregnant women with warm autoantibodies. RBCs from healthy blood donors were used as controls. RBCs were labelled with the red lipophilic fluorescent dye PKH26 and incubated with PMA-treated THP-1 cells. After removal of nonadherent RBCs by washing and haemolysis of adherent RBCs, erythrophagocytosis was quantified by flow cytometry. RESULTS: We observed significant phagocytosis of RBCs coated with clinically relevant alloantibodies (i.e. anti-D and anti-K) or autoantibodies from patients with active wAIHA, but not of those coated with alloantibodies (anti-Ch) or autoantibodies from patients and pregnant women without haemolysis. CONCLUSION: The flow cytometry-based erythrophagocytosis test described here is quantitative, highly reliable, and may be helpful for the assessment of the clinical significance of antibodies to RBCs.

Disruption of the tumour-associated EMP3 enhances erythroid proliferation and causes the MAM-negative phenotype.

The clinically important MAM blood group antigen is present on haematopoietic cells of all humans except rare MAM-negative individuals. Its molecular basis is unknown. By whole-exome sequencing we identify EMP3, encoding epithelial membrane protein 3 (EMP3), as a candidate gene, then demonstrate inactivating mutations in ten known MAM-negative individuals. We show that EMP3, a purported tumour suppressor in various solid tumours, is expressed in erythroid cells. Disruption of EMP3 by CRISPR/Cas9 gene editing in an immortalised human erythroid cell line (BEL-A2) abolishes MAM expression. We find EMP3 to associate with, and stabilise, CD44 in the plasma membrane. Furthermore, cultured erythroid progenitor cells from MAM-negative individuals show markedly increased proliferation and higher reticulocyte yields, suggesting an important regulatory role for EMP3 in erythropoiesis and control of cell production. Our data establish MAM as a new blood group system and demonstrate an interaction of EMP3 with the cell surface signalling molecule CD44.

Risk of piperacillin-induced hemolytic anemia in patients with cystic fibrosis and antipseudomonal treatment: a prospective observational study.

BACKGROUND: Drug-induced immune hemolytic anemia (DIIHA) is a rare but severe side effect caused by numerous drugs. Case reports and case series suggest that piperacillin-related DIIHA may be more common among patients with cystic fibrosis (CF). However, the prevalence is speculative. The aim of this prospective, observational study was determine the prevalence of DIIHA in such affected patients. METHODS AND MATERIALS: Patients with CF hospitalized for parenteral antibiotic therapy at Charité Universitätsmedizin Berlin, who had previously been exposed to IV antibiotics, were enrolled. Blood samples were collected on Days 3 and 12 of antibiotic treatment courses. Serological studies were performed using standard techniques with gel cards. Screening for drug-dependent antibodies (ddab) was performed in the presence of the drugs and their urinary metabolites. RESULTS: A total of 52 parenteral antibiotic cycles in 43 patients were investigated. Ddab against piperacillin were detected in two patients (4.7%). The direct AHG was positive with anti-IgG only in both patients. However only one of these patients developed mild immune hemolytic anemia. Both patients had been repeatedly treated with piperacillin without any evident hemolysis. There was no correlation between the exposure to piperacillin and the prevalence of ddab. CONCLUSION: Our prospective study indicates that piperacillin-induced ddab occur more frequently in patients with CF than previously suggested. The question related to the significance of piperacillin-dependent antibodies may reflect new aspects in this field.

Sublytic Terminal Complement Components Induce Eryptosis in Autoimmune Haemolytic Anaemia Related to IgM Autoantibodies.

BACKGROUND/AIMS: Eryptosis, the suicidal death of red blood cells (RBCs), is characterized by phosphatidylserine (PS) exposure at the cell surface. It can be catalysed by a variety of abnormal conditions and diseases. Until now, the many questions surrounding the physiology and pathophysiology of eryptosis have not been sufficiently answered. Recently, we demonstrated IgM and IgA autoantibodies (aab) to induce PS exposure on circulating RBCs of patients with autoimmune haemolytic anaemia (AIHA). However, it remained unclear how these aab lead to eryptosis. METHODS: Serum and plasma samples from patients with clinically relevant AIHA of cold type were used to induce eryptosis in O RBCs. Serum containing fresh complement from healthy donors, antibodies to complement component, and complement factor depleted sera were added to examine the influence of the complement on PS-exposure. RBC bound annexin V PE were analysed by flow cytometry. RESULTS: Eryptosis related to IgM aab was found to be dependent on complement activation and could be effectively inhibited by EDTA, serum heat inactivation and anti-C5. PS exposure increased with sequential activation of the sublytic terminal complement components C5b6, C5b-7 and was most significant at the C5b-8 stage. A decrease was observed following the formation of the lytic membrane attack complex C5b-9, either because of lysis of eryptotic RBCs or because of inhibition of eryptosis by C9. CONCLUSION: Our findings reflect new aspects on RBC destruction in AIHA as well the impact of the terminal complement complexes on the RBC membrane. The striking differences to nucleated cell apoptosis may even have physiological meaning of RBC acting as a buffer of the complement system.

Efficacy of Antenatal Intravenous Immunoglobulin Treatment in Pregnancies at High Risk due to Alloimmunization to Red Blood Cells.

BACKGROUND: Alloimmunization to red blood cells (RBCs) may result in fetal anemia prior to 20 weeks gestation. The question as to whether early commencement of antenatal treatment with high-dose intravenous immunoglobulins (IVIG) may prevent or at least delay the development of fetal anemia in the presence of alloantibodies to RBCs is highly relevant. PATIENTS AND RESULTS: Here we describe a patient with high-titer anti-K and two other severely affected pregnant women with a history of recurrent pregnancy loss due to high-titer anti-D or anti-D plus anti-C. Early commencement of treatment with IVIG (1 g/kg/week) resulted in prevention of intrauterine transfusion (IUT) in the former two cases, and in a significant delay of development of fetal anemia in the remaining case (26 weeks gestation). CONCLUSION: Based on our findings and of previously published cases, early initiation of treatment of severely alloimmunized women with IVIG (1 g/kg/week) could potentially improve the outcome of fetuses at risk.

Evaluation of the New Lateral Flow Card MDmulticard® Basic Extended Phenotype in Routine Clinical Practice.

BACKGROUND: Transfusion emergencies and critical situations require specifically designed devices to simplify and optimize the standard procedures. In addition, matching antigens over and above ABO-Rh-K would be beneficial. METHODS: Routine blood samples were collected in four immunohematology centers and tested with the new MDmulticard Basic Extended Phenotype for the simultaneous detection of the Duffy, Kidd, and Ss antigens, according to the principle of the lateral flow. Results were compared with those obtained using routine serology methods. Discrepancies were analyzed by molecular techniques/genotyping. RESULTS: 310 samples were tested (167 donors; 75 patients; 28 subjects with positive direct antiglobulin test (DAT); 15 newborns; 25 previously transfused patients). The 285 samples with non-mixed-field reaction yielded 1,710 antigen results with 8 discrepancies (0.47%) six of which in DAT-positive subjects: three false-positive (Fya) for MDmulticard, and two false-positive (Fya) plus three false-negative (Fyb) for the reference methods (MDmulticard PPA for donors/patients/newborns: 99.82%; negative percent agreement: 100%; sensitivity: 100%; specificity: 99.39%, positive predictive value: 99.75%; negative predictive value: 100%). The MDmulticard detected mixed-field in 15 antigen reactions from 13 transfused patients, undetected by the comparative method, with the opposite result in 8 antigens (5 patients). CONCLUSION: The MDmulticard Basic Extended Phenotype met the criteria prescribed for the testing of donor, patient, DAT-positive, and newborn samples in transfusion laboratory routine.

Ceftriaxone-induced hemolytic anemia with severe renal failure: a case report and review of literature.

BACKGROUND: Drug induced immune hemolytic anemia (DIIHA) is a rare complication and often underdiagnosed. DIIHA is frequently associated with a bad outcome, including organ failure and even death. For the last decades, ceftriaxone has been one of the most common drugs causing DIIHA, and ceftriaxone-induced immune hemolytic anemia (IHA) has especially been reported to cause severe complications and fatal outcomes. CASE PRESENTATION: A 76-year-old male patient was treated with ceftriaxone for cholangitis. Short time after antibiotic exposure the patient was referred to intensive care unit due to cardiopulmonary instability. Hemolysis was observed on laboratory testing and the patient developed severe renal failure with a need for hemodialysis for 2 weeks. Medical history revealed that the patient had been previously exposed to ceftriaxone less than 3 weeks before with subsequent hemolytic reaction. Further causes for hemolytic anemia were excluded and drug-induced immune hemolytic (DIIHA) anemia to ceftriaxone could be confirmed. CONCLUSIONS: The case demonstrates the severity of ceftriaxone-induced immune hemolytic anemia, a rare, but immediately life-threatening condition of a frequently used antibiotic in clinical practice. Early and correct diagnosis of DIIHA is crucial, as immediate withdrawal of the causative drug is essential for the patient prognosis. Thus, awareness for this complication must be raised among treating physicians.

Efficacy and tolerability of old and new drugs used in the treatment of immune thrombocytopenia: Results from a long-term observation in clinical practice.

BACKGROUND: Many patients with immune thrombocytopenia (ITP) may require special attention and long-term treatment. Little is known on the efficacy and tolerability of the drugs used in practice. MATERIAL AND METHODS: We retrospectively reviewed the results of therapy of 400 patients with chronic ITP. All Patients were treated at our institution between 1996-2016 under consideration of guidelines, general recommendations, and individual aspects, including gender, age, weight, comorbidity, patient's medical history and bleeding risk. RESULTS: Treatment was not required in 25% of patients (n = 100) during observation. In treated patients (n = 300), the rate of patients that responded and tolerated treatment with prednisolone was 59% (52/88), with azathioprine 32% (29/90), with eltrombopag 49% (31/63), with romiplostim 59% 27/45, with IVIG (intravenous immunoglobulines) 75% (94/126), with anti-D 37% (19/52) and with dexamethasone 60% (25/42) patients. Eighteen treated patients (6%) entered sustained remission after treatment with various drugs. Twenty-six patients underwent splenectomy (Splx) resulting in sustained remission in 15 cases (60%). Only two patients remained refractory to Splx and to all used drugs. DISCUSSION: None of the currently available drugs used in the treatment of ITP are invariably safe and effective. Responses, the duration of response, intolerability, and the course of disease are unpredictable. Although the treatment of ITP has considerably improved in the recent years, the currently available drugs may rarely cure affected patients. The need for safe and effective therapy in ITP is evident. Optimal treatment decisions for each patient remains a challenge in many cases.

Eryptosis in autoimmune haemolytic anaemia.

OBJECTIVE: Haemolysis and anaemia related to autoimmune haemolytic anaemia (AIHA) of warm type (wAIHA) and of cold type (cAIHA) are believed to be solely due to antibody and/or complement-mediated destruction and clearance of red blood cells (RBCs). There is evidence that RBCs of affected patients may also undergo eryptosis, the suicidal death of RBCs. METHOD: RBCs from 24 patients with wAIHA, 7 patients with chronic cAIHA and one patient with AIHA of mixed type were analysed for exposed phosphatidylserine (PS) by treatment with phycoerythrin-labelled Annexin V, and cell-associated fluorescence was measured using a MACSQuant flow cytometer. RESULTS: PS-exposing RBCs were detected in 7 of 13 patients with clinically significant wAIHA. Haemolysis was mostly related to IgM or IgA autoantibodies (aab) in those patients. In contrast, PS exposure in 11 patients with wAIHA in complete remission was comparable to that in healthy blood donors. All patients with chronic cAIHA and the patient with AIHA of mixed type showed haemolytic activity and high numbers of PS-exposing RBCs. Patients with decompensated AIHA appear to respond to treatment with erythropoietin, which is a known inhibitor of eryptosis. CONCLUSION: Eryptosis may frequently occur in AIHA related to IgM or IgA aab. Inhibition of eryptosis with erythropoietin may represent a new therapeutic option in the treatment of AIHA.

Bidirectional inefficacy or intolerability of thrombopoietin receptor agonists: new data and a concise review.

BACKGROUND: There is evidence that the thrombopoietin-receptor agonists romiplostim and eltrombopag may have different therapeutic values and adverse reaction profiles. Here we present new data and provide a review of all studies dealing with switching between these two drugs. MATERIALS AND METHODS: A total of 89 patients (38 males and 51 females, aged between 14-87 years) were treated with eltrombopag and/or romiplostim between 2007 and 2016 at our institution. Eltrombopag was switched to romiplostim or vice versa in 32 patients. In addition, all published data concerning patients treated sequentially with different thrombopoietin-receptor agonists were identified via a computer-assisted search and summarised in this article. RESULTS: Thirty-two of 89 patients treated with a thrombopoietin-receptor agonist in our institution were given both eltrombopag and romiplostim sequentially. Therapy was switched to the alternate thrombopoietin-receptor agonist 36 times, due to inefficacy (n=21), adverse reactions (n=14), and a patient's preference (n=1). In addition, data from 126 patients who have been treated with both agonists have been published by other groups. In total eltrombopag was replaced by romiplostim in 56 cases due to poor or no response or to adverse reactions. Forty-five patients responded to treatment with romiplostim, 11 patients shared cross-resistance and nine had adverse reactions to romiplostim. In contrast, romiplostim was replaced by eltrombopag in 106 cases. Seventy-eight patients responded to eltrombopag, 27 shared cross-resistance and 19 had adverse reactions to eltrombopag. DISCUSSION: Eltrombopag and romiplostim often share bidirectional cross-resistance and/or adverse reactions. Both drugs appear to cause more adverse reactions than have been previously reported.

Successful treatment of bleeding with tranexamic acid in a series of 12 patients with immune thrombocytopenia.

BACKGROUND AND OBJECTIVES: The clinical significance of immune thrombocytopenia (ITP) is mainly reflected by bleeding and/or bleeding risks, which, in some cases, cannot be adequately controlled by standard therapy. Tranexamic acid (TA) is increasingly used in preventing and reducing bleeding in several medical settings. There is little information on whether TA may also be useful in the management of ITP. MATERIALS AND METHODS: Twelve patients with ITP were treated with TA (0·5-3 g/day) due to recognizable bleeding. Ten of the 12 patients were under regular treatment for ITP. The remaining two patients did not require additional therapy. RESULTS: Cessation or, at least, significant improvement of bleeding was achieved shortly after the initiation of TA in all cases. TA was well tolerated and discontinued after cessation of bleeding. CONCLUSIONS: We recommend the use of TA in ITP patients with bleeding and/or an increased bleeding risk. Ultimately, cessation of bleeding plays a key role in the management of such affected patients. However, future studies are required to optimize dose and administration routes (intravenous or oral).

Piperacillin-induced mild haemolytic anaemia in a 44-year-old patient with cystic fibrosis.

Piperacillin-tazobactam is an antipseudomonal antibiotic frequently used in patients with cystic fibrosis (CF) to treat pulmonary exacerbations. Drug-induced immune haemolytic anaemia is a rare complication during treatment with piperacillin. So far, piperacillin-induced immune haemolytic anaemia (PIHA) is regarded as an acute and severe haemolytic anaemia resulting into life-threatening events. Here we report on a patient with mild PIHA, which did not result in any clinical symptoms or necessity for treatment. To the best of our knowledge, this is the first case report of PIHA without an acute severe haemolytic anaemia. Further research is needed to clarify if this case is a solitary clinical manifestation of PIHA or if mild clinical courses of PIHA might be under-reported. Cases of PIHA have been largely reported in patients with CF. This unequal distribution maybe due to the frequent administration of piperacillin for pulmonary exacerbation in patients with CF or due to CF-related cofactors of yet unknown aetiology.

Hemolysis after Oral Artemisinin Combination Therapy for Uncomplicated Plasmodium falciparum Malaria.

Episodes of delayed hemolysis 2-6 weeks after treatment of severe malaria with intravenous artesunate have been described. We performed a prospective observational study of patients with uncomplicated malaria to investigate whether posttreatment hemolysis also occurs after oral artemisinin-based combination therapy. Eight of 20 patients with uncomplicated malaria who were given oral artemisinin-based combination therapy met the definition of posttreatment hemolysis (low haptoglobin level and increased lactate dehydrogenase level on day 14). Five patients had hemolysis persisting for 1 month. Patients with posttreatment hemolysis had a median decrease in hemoglobin level of 1.3 g/dL (interquartile range 0.3-2.0 g/dL) in the posttreatment period, and patients without posttreatment hemolysis had a median increase of 0.3 g/dL (IQR -0.1 to 0.7 g/dL; p = 0.002). These findings indicate a need for increased vigilance for hemolytic events in malaria patients, particularly those with predisposing factors for anemia.

Harmless Pregnancy-Induced Warm Autoantibodies to Red Blood Cells.

BACKGROUND: There is little information concerning the development and significance of autoantibodies (aab) to red blood cells (RBCs) during pregnancy. METHODS: Unselected pregnant women were routinely screened for the presence of unexpected antibodies to RBCs using standard techniques. RESULTS: Between 2009 and 2013, 153,612 pregnant women were tested. The antibody screening test was positive in 1,721 women (1.12%). In 1,602 (1.04%) cases, immune and/or non-immune alloantibodies and cold-reactive aab were detected, whereas warm-reactive aab were found in 119 women (0.08%). In almost all cases, warm-reactive aab belonged to the IgG class. No evidence of the presence of significant haemolysis in affected women was observed. CONCLUSION: Pregnant women may rarely develop aab to RBCs, which do not appear to cause haemolytic anaemia. Further clarification is required on the reasons behind the development of these aab and their clinical insignificance.

Evidence Suggesting Complement Activation and Haemolysis at Core Temperature in Patients with Cold Autoimmune Haemolytic Anaemia.

BACKGROUND: It is unclear why haemolysis may somewhat persist in patients with cold autoimmune haemolytic anaemia (cAIHA) at 37 °C (core temperature). METHODS: Seven patients with cAIHA were included in this study. Serological testing was performed using standard techniques. Bound autoantibodies (aab) on patients' RBCs were analysed by the direct antiglobulin test (DAT), dual antiglobulin test (DDAT) and flow cytometry (FC) using pre-warmed RBCs (37 °C). Temperature-dependent complement binding was determined by incubation of patients' serum samples with group O RBCs and fresh serum complement. RESULTS: The DAT was strongly positive with anti-C3d in all cases, independent of season and outside temperature. Haemolysis usually improved during warm periods of time, but decompensated following febrile infections, and persisted throughout the year, though exposure to the cold was strictly avoided. In addition, trace amounts of IgM aab were infrequently detectable on patients' RBCs even at 37 °C, and complement activation was demonstrated following incubation of RBCs with the causative aab at 37 °C. CONCLUSIONS: Binding of trace amounts of IgM aab at 37 °C may provide an explanation for the durable C3d-positive DAT and haemolysis in patients with cAIHA.

Variability of Findings in Drug-Induced Immune Haemolytic Anaemia: Experience over 20 Years in a Single Centre.

BACKGROUND: Drug-induced immune haemolytic anaemia (DIHA) is difficult to diagnose, and its true incidence remains obscure. Here, we present cases of DIHA identified at our institute over the last two decades. METHODS: Serological tests were performed according to standard procedures. Detection of drug-dependent antibodies was performed in the presence and absence of the relevant drug and/or their ex vivo antigens. RESULTS: Over the last 20 years, 73 patients have been identified with DIHA in our institute, which was related to 15 different drugs. The most common single drugs identified were diclofenac (n = 23), piperacillin (n = 13), ceftriaxone (n = 12) and oxaliplatin (n = 10). As far as data were available, haemolysis was acute in all patients, and signs of intravascular haemolysis were present in 90% of the cases. Haemolysis resulted in death in 17 patients (23%). The remaining patients recovered, but haemolysis was complicated by transitory renal and/or liver failure or shock in 11 patients. Upon initial evaluation, the antibody screening test was positive in 36 cases. A positive direct antiglobulin test (DAT) at least with anti-C3d was found in 65 cases, with anti-IgG only in 6 cases, and with anti-IgA only in 1 case. CONCLUSION: DIHA is a rare but potentially life-threatening disorder that should be considered if a patient develops haemolysis under drug treatment. The main serological finding is a positive DAT, primarily with anti-C3d.