Belated diagnosis in three patients with rifampicin-induced immune haemolytic anaemia

We report three patients who developed haemolysis following rifampicin treatment. Initially, autoimmune haemolytic anaemia (AIHA) of the warm type and/or an acute haemolytic transfusion reaction (AHTR) was suggested. The direct antiglobulin tests (DAT) were strongly positive for IgG and C3d, and tests for rifampicin-dependent antibodies were positive in all three cases, featuring C-specificity in one case. The outcome was fatal in two out of the three cases, presumably due to belated diagnosis. This shows that rifampicin may stimulate the production of autoantibodies (aab) and/or drug-dependent antibodies (ddab), and that the resulting haemolytic syndrome bears similarities with AIHA and AHTR.     

Massive haemolysis after intramuscular diclofenac in a patient who apparently tolerated oral medication

Background and Objectives  Administration of diclofenac may lead to immune haemolytic anaemia (IHA) owing to the presence of drug-dependent antibodies and/or autoantibodies. A relationship with oral or intramuscular drug administration is unknown. Here, we describe a patient who apparently tolerated oral diclofenac but developed severe IHA following intramuscular injection of the drug.
Patients and Methods  A 66-year-old-female was admitted to hospital because of jaundice and nausea, which were initially presumed to be manifestations of a postcholecystectomy syndrome. The patient soon developed haemolysis and renal failure. Although the symptoms and signs were suggestive of autoimmune haemolytic anaemia (AIHA), the patient had diclofenac-induced IHA.
Results  Serological testing, including detection of drug-dependent antibodies, was performed using standard techniques. The patient's serum was found to contain a highly reactive diclofenac-dependent red cell antibody of the immune complex type (titre 256 000). She recovered after 7 weeks of treatment with prednisolone, blood transfusions, haemodialysis and plasma exchange.
Conclusions  Diclofenac-induced IHA should always be considered when a patient on diclofenac develops haemolysis.     

Detection of drug-dependent, platelet-reactive antibodies by solid-phase red cell adherence assays

We developed a simple modification of the solid-phase red cell adherence (SPRCA) assay system that can be used to identify drug-dependent platelet antibodies (DDPAs) reactive by either the hapten or immune complex reaction mechanisms. Between January 1994 and August 1996 we tested sera from 173 patients [123 (71%) with unexplained thrombocytopenia and 50 (29%) because of poor responses to platelet transfusions not explicable by alloimmunization or the clinical situation] for DDPAs possibly associated with the receipt of 61 different drugs. We correlated positive results with patients' clinical courses. DDPAs were identified in samples from 138 (80%) of the patients tested. Antibodies reactive only by the hapten mechanism were identified in 51 (37%) of those sera exhibiting positive reactions. The clinical courses of 108 (78%) patients were evaluable. Discontinuation of the implicated drug(s) resulted in prompt (<5 d) resolution of the thrombocytopenia or improvement in response to transfusion in all of these patients. In four cases thrombocytopenia returned upon re-exposure to the implicated drug. This adaptation of SPRCA provides a simple means of investigating the possibility of DDPAs and documents a higher frequency of these antibodies than has previously been suspected.     

Red blood cell membrane-bound IgG: demonstration of antibodies in patients with autoimmune haemolytic anaemia and immune complexes in patients with rheumatic diseases

Summary. Immunoglobulin G (IgG) bound in vivo to the surfaces of red blood cells (RBC-IgG) was quantificated by an enzyme-linked immunosorbent assay (ELISA) using the cells themselves as solid phase. The method was applied on RBC from normal subjects, patients with autoimmune haemolytic anaemia (AIHA) and rheumatoid patients with and without circulating immune complexes (CIC). Small amounts of RBC-IgG were detected in normal subjects and rheumatoid patients without CIC. Fifteen out of 16 patients with AIHA had increased RBC-IgG indicating RBC sensitization with IgG antibodies, although only eight patients had a positive direct antiglobulin test (DAT) with anti-IgG. Ten out of 13 rheumatoid patients with a negative DAT and with CIC had increased RBC-IgG suggesting RBC C3 receptor-bound IC. The results provide background for further studies of the significance of RBC-IgG in health and disease.     

Detection of drug-dependent antibodies by the 51cr platelet lysis test: Documentation of immune thrombocytopenia induced by diphenylhydantoin,diazepam, and sulfisoxazole

Sudden, severe thrombocytopenia developed in each of three patients receiving diphenylhydantoin, diazepam, and sulfisoxazole, respectively. Recovery followed discontinuance of the drugs. An antiplatelet antibody requiring the presence of an appropriate drug for interaction with platelets was demonstrated in each case by the ⁵¹Cr platelet lysis test using normal, paroxysmal nocturnal hemoglobinuric, or enzyme-treated normal platelets as target cells. These antibodies could not be detected by techniques that depend on clot retraction inhibition, complement fixation, or platelet factor-3 activation. Quinine- and quinidine-dependent antiplatelet antibodies in the serum of 16 patients who developed acute thrombocytopenia while taking either quinine or quinidine could be demonstrated readily with the ⁵¹Cr platelet lysis test and could also be detected by other methods employed.     

Two cases of immune haemolytic anaemia,associated with anti-piperacillin,detected by the 'immune complex' method

BACKGROUND AND OBJECTIVES: Sera containing antibodies to penicillin and penicillin-related drugs are typically thought to react with drug-coated red blood cells (RBCs) (drug adsorption method), but not when the sera are added to drug and RBCs in the same tube ('immune complex' method). Two cases of immune haemolytic anaemia caused by anti-piperacillin have been previously described. Serological details were given in only one patient. In that subject, the antibody was immunoglobulin (Ig)M + IgG and reacted by both the drug adsorption and 'immune complex' methods. MATERIALS AND METHODS: Two patients with cystic fibrosis developed positive direct antiglobulin tests (DATs) and haemolytic anaemia after 11-12 days of piperacillin therapy. Serological studies were performed with piperacillin, Zosyn (piperacillin + tazobactam) and penicillin by using the drug adsorption and 'immune complex' methods. RESULTS: The first patient's serum contained an IgG, complement-activating anti-piperacillin that reacted by the 'immune complex' method only. The second patient's IgM + IgG, complement-activating anti-piperacillin reacted by the 'immune complex' method and agglutinated piperacillin-treated RBCs. An eluate from the patient's RBCs reacted weakly with all RBCs tested without the presence of drug. This patient had evidence of intravascular haemolysis and died. CONCLUSIONS: We describe the third and fourth examples of immune haemolytic anaemia caused by anti-piperacillin; one was associated with fatal haemolytic anaemia. As piperacillin is commonly used in the treatment of cystic fibrosis, anti-piperacillin should be considered whenever patients with cystic fibrosis develop haemolytic anaemia and/or positive DATs.     

Drug induced red blood cell autoantibodies co-developed with drug specific antibodies causing haemolytic anaemias

Four individuals with anti-glafenine, anti-latamoxef and anti-teniposide antibodies were found to have an associated red blood cell autoantibody. The two components could be separated by selective absorption and showed distinct time course patterns. In three patients a well-defined blood group antigen was recognized as the receptor for both auto- and drug specific antibodies. Similarities between this type of immune response to drugs and the well-known hapten and carrier specificities developed in animals immunized by hapten-carrier conjugates are discussed.     

Fatal immune haemolysis due to a degradation product of ceftriaxone

A 16-year-old girl who was treated with ceftriaxone developed two intravascular haemolytic attacks that led to acute renal failure and death. The direct antiglobulin test was positive only for C3d, and no ceftriaxone-dependent antibodies were detectable. Her serum reacted strongly positive with red blood cells in the presence of ex vivo antigen related to ceftriaxone (urine samples from patients receiving the drug). This is the first reported case in which the causative antibodies appeared to be stimulated solely by a degradation product of ceftriaxone.     

Disappearance of factor VIII autoantibodies preceding autoimmune haemolytic anaemia.

We describe a previously healthy woman who at the age of 44 years developed a factor VIII inhibitor, that over the years increased to a maximum level of 3600 Bethesda units (BU) mL(-1) in 1978. The epitope specificity of the factor VIII inhibitor was investigated and antibodies directed against the A2 and C2 domains of factor VIII were detected. The majority of these antibodies were of subclass IgG4. Over the years, the inhibitor titre gradually decreased and in 1989, the inhibitor could no longer be detected. Shortly after, the patient developed autoimmune haemolytic anaemia. A possible link between the disappearance of factor VIII inhibitors and the development of other autoantibodies may be explained by concomitant development of anti-idiotypic antibodies that neutralize the activity of factor VIII inhibitors. We were unable to detect anti-idiotypic antibodies, which could explain the decline in factor VIII inhibitor titre in this patient.     

Carbimazole-induced immune haemolytic anaemia: role of drug-red blood cell complexes for immunization

We report on a patient who developed acute intravascular immune haemolysis while receiving carbimazole. Serological studies revealed a strongly (3+) and a discretely positive (1+) direct antiglobulin test due to C3d and IgG respectively, and a very weak IgG autoantibody in the eluates. Serum from the patient contained specific carbimazole-dependent red blood cell (RBC) antibodies which reacted with all normal human RBC in the presence of free carbimazole as well as with RBC coated with the drug either in vitro or in vivo, although carbimazole itself is not detectable in plasma after oral administration. The results provide direct evidence for the sequence of the drug-RBC-antibody interaction and show that the RBC (and not plasma proteins) function as 'carrier-like' macromolecules in the immune response.     

Islet cell antibodies and insulin autoantibodies in patients treated with oral hypoglycaemic agents

Clinical and immunological features suggesting Type 1 diabetes were assessed in 202 patients treated with oral hypoglycaemic agents for presumed Type 2 diabetes. Islet cell antibodies (ICA) were detected at a level exceeding 5 JDF units in 5.9% of patients, and complement-fixing ICA were detected in 3.4%. IgG insulin autoantibodies were detected in 8.8% of insulin-naive patients, none of whom were ICA positive. ICA were detected more frequently in patients with shorter duration of diabetes (p = 0.02). Age and relative body weight were similar in ICA positive and negative groups. ICA positive patients were more likely to have lost weight (p less than 0.02) than ICA negative patients, although this may have been attributable to the differing duration of diabetes in the two groups. Other individual clinical features suggesting Type 1 diabetes were not significantly more frequent in ICA positive patients. However, a higher proportion of ICA positive than ICA negative patients had one or more features suggestive of Type 1 diabetes irrespective of the duration of diabetes. Clinical features suggesting Type 2 diabetes were present in a similar proportion of ICA positive and ICA negative patients. Fasting and glucagon stimulated C-peptide levels were similar in ICA positive and matched ICA negative patients.     

Insulin autoantibodies are associated with islet cell antibodies; their relation to insulin antibodies and B-cell function in diabetic children

Summary Blood was drawn from 74 children, 3–16 years old, at diagnosis of Type 1 (insulin-dependent) diabetes and before the first insulin injection. Insulin autoantibodies were detected with a polyethylen-glycol-method in 27/74 (36.4%) and with an immuno-electrophoretic method in 6/74 (8.1%). Islet cell cytoplasmic antibodies detected by indirect immuno-fluorescence were found in 49/74 patients (66.2%), who included as many as 23 of the 27 patients with insulin autoantibodies determined with the polyethylen-glycol-method (p<0.01). The proportion of insulin autoantibody-positive patients who developed insulin antibodies during the first 9 months of insulin treatment was not significantly greater (51.8%) than that of insulin autoantibody-negative patients (44.6%), but patients with both islet cell antibodies and insulin autoantibodies at diagnosis produced more insulin antibodies during the first 9 months (p<0.05). There was no difference in fasting or meal stimulated serum C-peptide after 3, 9 or 18 months as related to occurrence of insulin autoantibodies and/or islet cell antibodies. The correlation between insulin autoantibodies and islet cell antibodies indicates that both types of autoantibodies reflect the same immunological process, although the lack of correlation to C-peptide may indicate that they play a minor causal role. In addition, the results show that patients with an active autoimmune process evidently tend to produce more insulin antibodies during the first months of insulin treatment, but the islet cell antibodies and insulin autoantibodies-positive patients had at least as good residual B-cell function as patients without autoantibodies at diagnosis. If insulin antibodies produced as a response to exogenous insulin do have a negative effect on B-cell function our present results suggest that such mechanisms are of minor importance.     

Immune haemolytic anaemia associated with ampicillin dependent warm antibodies and high titre cold agglutinins in a patient with Mycoplasma pneumonia

A case of severe immune haemolytic anaemia in a 54-year-old man suffering from Mycoplasma pneumonia is presented. A strongly positive direct Coombs test with erythrocyte bound IgG, C₃d and C₄ was demonstrated during the haemolytic process. Further, serologic investigations revealed ampicillin-dependent ‘warm’ antibodies and a cold agglutinin titre > 2000. No firm conclusion can be drawn as to which antibody caused the haemolysis. Treatment including withdrawal of antibiotics, blood transfusions (washed red cells, concentrated erythrocyte suspension) and corticosteroids was successful. It is suggested that tests should be carried out for erythrocyte bound drug-induced antibodies in cases of haemolysis with a history of drug exposure.     

Ontogeny of islet cell antibodies,insulin autoantibodies and insulitis in the non-obese diabetic mouse

Summary The predictive value of insulitis, islet cell cytoplasmic antibodies and insulin autoantibodies for insulin-dependent diabetes was studied in young female non-obese diabetic mice. The ontogeny of the three markers was examined cross-sectionally at days 15, 25, 40 and 90 while islet cell antibodies and insulin autoantibodies were studied longitudinally from day 35 or day 144–168 until approximately day 250. Insulitis was first observed at day 40 (50%) and subsequently at day 90 (70%). Islet cell antibodies and insulin autoantibodies were present at day 15 in 46% and 54% of the animals respectively. The rate of islet cell antibodies was slightly higher at day 25 (60%) than at day 40 (40%) and day 90 (54%) whereas antibodies to insulin were present in all samples from day 25–90. At day 40 and day 90 insulitis and insulin autoantibodies were present together in 42% and 70% of the animals, respectively, while insulitis and islet cell antibodies had a lower rate of concordance (17% and 42%, respectively; diabetes rate, 30%). The concordance rates for islet cell antibodies and insulin autoantibodies were 42% at day 40 and 54% at day 90. Concordance for all three markers was first observed at day 40 (17%) which increased to 38% at day 90. In longitudinal studies, islet cell antibodies and insulin autoantibodies were often present together whether or not diabetes supervened. In the islet cell antibody procedure, immunoreactive cells were shown immunohistochemically to correspond with insulin and/or glucagon cells. However, this staining was not suppressible with insulin- or glucagon- absorbed sera, implying the presence of non-hormonal autoantigens. We conclude that the three markers investigated are expressed early after birth and well before clinical symptoms appear in this animal model. Both islet cell antibodies and insulin autoantibodies preceded insulitis but the prevalence rate for each marker or their degree of concordance was different from the anticipated rate of diabetes in our colony. Consequently, the early expression of the three markers alone is not predictive of diabetes although concordance for the two, or all three markers may be of some value. However, no animal developed diabetes without the prior appearance of both islet cell antibodies and insulin autoantibodies.     

Relationship between serum insulin autoantibodies,islet cell antibodies and Coxsackie-B4 and mumps virus-specific antibodies at the clinical manifestation of Type 1 (insulin-dependent) diabetes

Summary In order to elucidate the possible relationship between insulin autoantibodies (IAA), conventional (ICA-IgG) and complement-fixing (CF-ICA) islet cell antibodies and Coxsackie-B4 and mumps virus-specific antibodies (IgG, IgM and IgA classes), we studied 194 children and adolescents with newly diagnosed Type 1 (insulin-dependent) diabetes. Sixty-one (31.4%) of the subjects were IAA-positive at diagnosis and 73.8% (45/61) of these also had ICA-IgG compared to 51.1% (68/113, p<0.01) of IAA-negative children. CF-ICA showed no significant association with IAA. The levels of IAA were significantly higher in the patients with ICA-IgG compared to those without [5.9±1.6% (SEM) vs 2.5±0.3%, p<0.01]. The patients positive for IAA were younger at diagnosis than the IAA-negative ones; (7.1±0.5 vs 9.3±0.3 years, p<0.001) and this was also true for ICA-IgG-positive children (8.1±0.4 vs 9.4±0.5 years, p<0.05) in comparison to ICA-IgG-negative subjects. No significant associations were found between IAA or ICA on the one hand and a positive family history of Type 1 diabetes or metabolic derangements at diagnosis on the other. Subjects negative for ICA were more frequently positive for mumps virus specific IgG antibodies than the ICA-positive patients (50/80 vs 53/111, p<0.05), and Coxsackie-B4 virus-specific IgA antibodies were more common in the CF-ICA-negative than the CF-ICA-positive children (53/111 vs 29/80, p<0.05). There was no association between the IAA levels and Coxsackie-B4 or mumps virus specific antibodies. However, patients with serological evidence of a recent mumps infection (n=13) had higher IAA levels than the other children (4.4±7.7% vs 2.8±1.4%, p<0.02). Our data suggest a positive association between IAA and ICA-IgG, supporting the view that IAA are like ICA serological markers of autoimmune B cell damage. The inverse associations between autoantibodies and age and between ICA and viral antibodies support the hypothesis that autoimmune mechanisms may play a more crucial role in younger patients contracting Type 1 diabetes while environmental factors may be more important in older ones.     

Rituximab in auto‐immune haemolytic anaemia and immune thrombocytopenic purpura: a Belgian retrospective multicentric study

Objectives. For better characterizing the effect of anti‐CD20 therapy, we analysed the use of rituximab in Belgian patients experiencing auto‐immune haemolytic anaemia (AIHA) and immune thrombocytopenic purpura (ITP). Design. We performed a retrospective multicentric analysis of patients with AIHA and ITP treated with rituximab in Belgium. Setting. Haematological departments were invited to fill in a questionnaire about patient and disease characteristics. Subjects. All patients with AIHA and ITP, both primary and secondary to other diseases, who received one or more courses of rituximab during their disease course were included. Sixty‐eight courses of rituximab in 53 patients with AIHA and 43 courses in 40 patients with ITP were analyzed. Intervention. Response rates, duration of response and factors predictive for response were assessed. Results. All patients were given rituximab after failing at least one previous line of treatment, including splenectomy in 19% and 72.5% of AIHA‐patients and ITP‐patients respectively. Overall response rates were 79.2% in AIHA and 70% in ITP, with a median follow‐up since first rituximab administration of 15 months (range 0.5–62) in AIHA and 11 months (range 0–74) in ITP. Progression free survival at 1 and 2 years were 72% and 56% in AIHA and 70% and 44% in ITP. In this retrospective analysis we were not able to identify pretreatment characteristics predictive for response to rituximab. Nine patients with AIHA and three patients with ITP were given one or more additional courses of rituximab. Most of these patients, who had responded to a previous course, experienced a new response comparable to the previous one, both in terms of quality and of duration of response. Finally, the outcome of patients who failed to respond to rituximab therapy was poor both in terms of response to subsequent therapy and in terms of survival. Conclusions. This study confirms that rituximab induces responses in a majority of previously treated patients with AIHA and ITP. Response duration generally exceeds 1 year. Retreatment with rituximab in responding patients is most often successful. The outcome of patients who fail on rituximab is poor. We were not able to identify pretreatment patient characteristics predicting for response.     

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.