ReviewArticle

Wien Klin Wochenschr (2008) 120/56: 136151 DOI 10.1007/s00508-008-0945-1

WIENER KLINISCHE WOCHENSCHRIFT

The Middle European Journal of Medicine Printed in Austria

Diagnosisandtreatmentofautoimmunehaemolyticanaemiasinadults: aclinicalreview
PeterValentandKlausLechner
Department of Internal Medicine I, Division of Haematology and Haemostaseology, Medical University of Vienna, Austria Received November 26, 2007, accepted after revision February 18, 2008 Springer-Verlag 2008

Diagnose und Therapie von autoimmunhmolytischen Anmien im Erwachsenen: Eine klinische bersicht Zusammenfassung. Die autoimmunhmolytische Anmie (AIHA) ist eine immunologische Erkrankung, bei der es zur Produktion von Antikrpern kommt, die sich gegen patienteneigene Erythrozyten richten und diese zerstren. Man unterscheidet primre (idiopathische) und sekundre Formen der Erkrankung. Die Diagnose basiert auf dem Nachweis einer Anmie, von Hmolysezeichen mit Retikulozytose, vermindertem Haptoglobin, erhhter Laktatdehydrogenase und erhhtem indirekten Bilirubin, sowie einem positiven Coombs Test. In einem Teil der Patienten finden sich nicht alle diese Vernderungen. Die meisten AIHA sind durch Wrme-Antikrper verursacht, nur selten hingegen werden Klte-Antikrper entdeckt. Whrend Wrme-Antikrper in vielen Fllen einer idiopathischen AIHA zuzuordnen sind, findet sich bei Klte-Antikrpern fast immer eine sekundre AIHA. Bei den entsprechenden Grunderkrankungen handelt es sich vor allem um Non Hodgkin Lymphome, systemische Autoimmunerkrankungen, Organtransplantationen, Infektionserkrankungen oder solide Tumore. berdies kann sich eine AIHA nach einer Therapie mit NucleosidAnaloga entwickeln. Die meisten Patienten mit einer AIHA bentigen eine Therapie. Im Fall der Wrme-Antikrper-mediierten AIHA gelten Glucocorticosteroide mit und ohne zustzlichen hochdosierten Immunglobulinen als Erstlinien-Therapie, whrend die Splenektomie als Zweitlinientherapie gilt. Die Ansprechrate einer primren AIHA auf die Erstlinientherapien ist relativ hoch. Nach Erreichen einer Remission sollte das Glucocorticoid langsam und vorsichtig ausgeschlichen werden. Ein Teil der Patienten bentigt eine Dauertherapie. Bei sekundren AIHA sind die Ansprechraten geringer. In vielen refraktren Fllen zeigt Rituximab (anti-CD20) gute Effekte, und bei Lymphomen oder Autoimmunerkrankungen kann die Anmie auf Mycophenolate Mofetil ansprechen. Die Klteantikrpermediierte AIHA ist in der Regel durch Steroide oder Splenektomie nicht beeinflussbar. In cirka 50% der Flle findet sich ein Ansprechen auf Rituximab, obwohl die

Dauer des Ansprechens zumeist kurz ist. In manchen Fllen verschwindet eine Lymphom- oder Tumor-assoziierte AIHA nach einer erfolgreichen Chemotherapie oder nach der chirurgischen Entfernung des Tumors.

Summary. Autoimmune haemolytic anaemia (AIHA) is an immune disorder caused by antibodies directed against unmodified autologous red cells. The disorder may be a primary (idiopathic) or a secondary disease. The diagnosis is based on the presence of anaemia, signs of haemolysis with reticulocytosis, low haptoglobin, increased lactate dehydrogenase, elevated indirect bilirubin, and a positive direct antiglobulin test (Coombs test). Sometimes, not all of these typical features are present. Most AIHA are caused by warm antibodies, whereas cold antibodies are less commonly detected. While half of the warm antibody-based AIHA are idiopathic anaemias, almost all cold antibody AIHA are secondary anaemias. Underlying diseases are Non Hodgkins lymphomas and systemic autoimmune disorders, and less frequently organ transplantation, infections, or solid tumors. Moreover, AIHA is an important complication of treatment with nucleoside analogs. Most patients with AIHA require therapy. In warm antibody AIHA, standard first line therapy are glucocorticosteroids with or without high dose immunoglobulins, whereas splenectomy is considered second-line therapy. Response rates of primary AIHA to corticosteroid therapy are high. After initial remission, the dose should be tapered down slowly and with caution, and in some cases, low-dose maintenance therapy is required. The efficacy of standard therapy is low in secondary AIHA that develops in lymphoma patients, posttransplant patients, or tumor patients. Among other immunosuppressive treatments, rituximab (anti-CD20) appears to be highly effective in patients with warm antibody AIHA refractory to standard therapy. Mycophenolate mofetil is quite effective in AIHA patients with an underlying autoimmune or lymphoproliferative disease. Patients with cold agglutinins are refractory to steroids and splenectomy. Half of these patients may respond to rituximab,

Valent and Lechner, Diagnosis and treatment of autoimmune haemolytic anaemias in adults although responses usually are short-lived. Sometimes, AIHA that is associated with malignant lymphomas or tumors, disappears after successful anti-lymphoma or anti-tumor therapy. Key words: Autoimmune haemolytic anaemia, diagnosis, CLL, treatment, rituximab.

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Introduction
Autoimmune haemolytic anaemia (AIHA) is a term used for anaemias caused by destruction of erythrocytes by antibodies that are directed against membrane antigens of the patients own unmodified (autologous) red cells. AIHA must be differentiated from congenital haemolytic anaemias, drug-dependent haemolytic anaemias, isoimmune anaemias, and acquired non-immune (Coombs negative) haemolytic anaemias. Of all anaemias, AIHA is an uncommon form of anaemia. Even among the groups of haemolytic anaemias, AIHA is not the most common variant. Based on registry data and respective estimates, no more than 5 to 10 patients with AIHA per year are diagnosed in larger haematological centers in the western world [1, 2]. However, AIHA is an increasingly recognized complication of anti-neoplastic therapy in cancer patients, in particular those with chronic lymphocytic leukemia (CLL). In addition, AIHA is quite often seen in patients with infectious diseases, sometimes in collagen disorders (systemic autoimmune disorders), and sometimes also after organ transplantation. Several overview articles have reviewed the pathogenesis, clinical features, and prognosis of AIHA in the past [319]. In the current article, we attempted to summarize previous and current knowledge on AIHA with special reference to triggering factors, the pathogenesis of the disease, clinical symptoms and features, established treatment options as well as emerging new therapeutic approaches.

Epidemiology
The reported incidence of AIHA is 0.611.3 per 100,000 per year in the western world [2], and thus is lower than that of autoimmune thrombocytopenia [20]. However, in practice, AIHA may be diagnosed more frequently than autoimmune thrombocytopenia, since only idiopathic forms of AIHA are usually recorded and reported in registries, whereas the more frequent secondary form of the disease most probably is not as well documented and the data in these patients usually are collected less frequently in data registries. Two thirds of the patients with AIHA are older than 50 years at diagnosis. The majority of patients with idiopathic AIHA are women. By contrast, however, most CLL patients with AIHA [21], and 80% of all patients with fludarabine-induced AIHA are males [22].

ThediagnosisofAIHAafivestepprocedure
The diagnosis of AIHA should be established in a step-wise fashion: In a first step, it has to be demonstrated that the anaemia is a haemolytic anaemia. Haemolytic anaemias

are usually normocytic anaemias, or are macrocytic because of marked reticulocytosis or because of (concomitant) folate deficiency. Usually, there is marked anisocytosis (increased red cell distribution width). Spherocytes may be seen in the blood smear. Sometimes, anemia is rapidly progressing and severe or even life-threatening. Laboratory parameters indicating haemolysis include a reduced serum haptoglobin (marker of increased red cell destruction) and an increased reticulocyte count (as sign of reactive increased erythropoiesis). On the basis of these findings, the diagnosis of haemolytic anaemia can usually be established. Other typical findings are an increase in indirect bilirubin, an increased urobilinogen in the urine, and an elevated lactate dehydrogenase (LDH) level. The latter findings are non-specific, but represent valuable information confirming the diagnosis of haemolytic anaemia. The LDH level is a useful marker related to the severity of haemolysis, and thus also a useful marker for monitoring of treatment responses. Although the diagnosis of haemolytic anaemia using the above mentioned parameters usually is a straight forward approach, there are sometimes pitfalls that have to be considered in special situations. Likewise, haptoglobin is an acute phase protein. Therefore, haptoglobin may be normal or even elevated despite haemolysis in the presence of a co-existing or underlying tumor or a chronic inflammation. On the other hand, haptoglobin may be low in the absence of haemolysis, for example in chronic liver diseases or in case of intramedullary haemolysis. A rare but potentially important cause of low haptoglobin is congenital ahaptoglobinaemia or hypohaptoglobinaemia. There are several known genotypes of haptoglobin. Patients with the genotype Hp0/Hp0 have undetectable haptoglobin levels in their serum. The Hp0/Hp0 genotype is relatively common in the Korean population (4%), but is rare in Europe and North America (1:4,000). However, patients with the Hp2/Hp0 genotype [23, 24] also have reduced serum haptoglobin concentrations (mean 17.8 mg/ dl), and the same holds true for patients with the Hp1/Hp0 genotype (median haptoglobin 42.6 mg/dl). Thus, low haptoglobin alone is not always diagnostic for haemolysis. Reticulocytosis is not always present at diagnosis in haemolytic anaemias. In one study, 25% of patients with proven haemolytic anaemia had a normal reticulocyte count at presentation [25]. The main reason for the absence of reticulocytosis may be a delayed bone marrow response. In a majority of these patients, reticulocytosis can be demonstrated after a certain time-interval (several days). However, there are other AIHA patients who never have reticulocytosis (e.g. in some cold antibody haemolytic anaemia patients). Absence of reticulocytosis may also be observed in haemolytic patients with reduced bone marrow function, for example in AIHA occurring during or after chemotherapy, or in patients with an underlying disease that infiltrates the bone marrow (e.g. leukaemias or lymphomas). When the reticulocyte count is very low (almost undetectable) in a patient who otherwise displays signs of haemolytic anaemia (and did not receive chemotherapy), the diagnosis of pure red cell aplasia (PRCA) is almost certain. PRCA in a patient with haemolytic anaemia may result from an infection with parvovirus or from antibodies directed against erythro-

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poietic progenitor cells, reticulocytes, or both (in addition to antibodies against mature red cells). In fact, PRCA may occur concurrently with warm or cold antibodies, in particular in T-cell lymphomas [22] or in posttransplant patients who are severely immunosuppressed [26]. In such cases, it may be difficult to know to which extent impaired erythropoiesis and haemolysis contribute to anaemia. Low haptoglobin and/or increased LDH are important markers of haemolysis in these patients. The second step in the diagnostic algorithm is the differentiation of immune- from non-immune haemolytic anaemias. This is best performed by the direct antiglobulin test (DAT, Coombs test). Using this test, the presence of immunoglobulins (in particular IgG, but also IgA or IgM) and/or of degradation products of complement (C3d or C3c) on the surface of red cells can be demonstrated. These proteins are present on normal red cells in small amounts, but are elevated in patients with immune haemolysis. When the DAT is positive for immunoglobulins and/or complement fragment(s) in a patient with typical signs of haemolytic anaemia (step one), the diagnosis of immune haemolytic anaemia can usually be established. However, the significance of a positive or negative DAT has to be carefully evaluated by the clinician. A positive DAT alone is not sufficient for the diagnosis of immune haemolytic anaemia, as 0.007%0.1% of the healthy population and 0.3%8% of hospitalized patients without haemolytic anaemia have a positive DAT. One reason for a false positive DAT is hypergammaglobulinaemia, for example after high dose immunoglobulin therapy. In case of a negative DAT, the presence of immune haemolytic anaemia is unlikely, but cannot be excluded with certainty. Thus, a false negative DAT may occur in 1%10% of all patients with AIHA. A major reason for false negative test results in older studies may have been a less sensitive laboratory test [27]. Today, however, most laboratories employ rather sensitive test assays, so that the frequency of false negative results may be quite low. On the other hand, there may be cases of AIHA with a truly negative DAT test result. Examples are some patients with AIHA after therapy with fludarabin, cyclophosphamide, or rituximab for CLL [28], patients after treatment of AIHA with rituximab who become DAT-negative despite ongoing hemolysis [29], and patients with AIHA and solid tumors [30]. In these cases it must be assumed that the numbers of immunoglobulin- or complement molecules on red cells was too low to produce a positive DAT test result. The sensitivity of the DAT test may increased from various modifications in the procedure [27]. An unresolved question is whether the concentration (titre) of AIHA-antibodies is of prognostic significance. In fact, some of the AIHA patients with weakly positive or even negative DAT may have severe haemolysis, while other rare patients with a strongly positive DAT may even present without overt anaemia. In general, however, a strongly positive DAT is more likely to be associated with severe AIHA and thus severe anaemia [31, 32]. Once the diagnosis of immune haemolytic anaemia has been established, the next diagnostic step is the identification of the type of antibody. Notably, four types of antibodies can be detected in AIHA patients. The most

common type are warm antibodies (75%90%), and the AIHA is then called warm antibody autoimmune haemolytic anemia (WA-AIHA). In these patients, the Coombs antibody reacts strongly with IgG(+/ complement)-loaded red cells at 37 C, but reacts much weaker with IgGloaded red cells at 4 C. In case of WA-AIHA, the Coombs test is positive with IgG in 20%66%, with IgG plus complement (C3d) in 24%64%, and with complement alone in 7%14% of all cases. IgG antibodies are panreactive antibodies which are directed against Rh antigens, glycophorin A, or other red cell antigens. Warm antibodies usually are polyclonal antibodies, but in rare cases may be monoclonal antibodies. In very rare cases, warm antibodies are of the IgM type (+/ C3d). These rare patients suffer from severe haemolysis [33]. Similarly, patients with IgA antibodies (often found together with IgG antibodies) seem to have more severe haemolysis [34]. In patients with cold antibodies, the antibody is usually an IgM which is directed against polysaccharide antigens and reacts either with adult (anti I) or fetal (anti i) red cells. The antibody reacts with the antigen at 4 C, whereas red cell lysis occurs at 37 C. The antibody activity can be measured by the cold agglutinin assay. The DAT is positive with complement (alone) in 74% of all cases, with IgG and C3 in 22.4%, and with IgG (alone) in 3.4% [9]. Cold antibodies are frequently monoclonal antibodies (primary cold agglutinin disease, lymphomas) but may also be polyclonal antibodies (infections and possibly T-cell lymphomas). For the clinician, not only the titre, but also the thermal amplitude of cold agglutinin is of importance. Patients with high antibody titres with a narrow thermal amplitude may have intermittent attacks of severe haemolytic anaemia. Patients with haemolytic anaemia usually have titres in excess of 1:1000, whereas haemolysis may occur at much lower titres when the thermal amplitude is high. The haemolysis in cold agglutinin disease is mainly an intravascular haemolysis (in contrast to paroxysmal cold hemoglobinuria). The clearance of red cells in these patients occurs in the liver. In a subgroup of patients (8%), mixed antibodies (warm and cold antibodies) are detectable [35]. Such mixed antibodies may also occur in patients with AIHA after fludarabine therapy [36]. These patients have severe haemolysis but show (in contrast to patients with cold agglutinins alone) a good clinical response to therapy with glucocorticosteroids. A rare but clinically important cold antibody is the so called Donath-Landsteiner antibody. This is an IgGantibody that reacts with red cells in the cold, whereas haemolysis occurs at 37 C. This antibody is polyclonal and is directed against the P-antigen. The DAT is negative or weakly positive with IgG in these patients. The condition is characterized by intravascular haemolysis with haemoglobinuria which poorly responds to treatment, but usually resolves spontaneously (self-limited process). The last step in the diagnostic algorithm is to define whether the AIHA is an idiopathic (primary) disease or developed on the basis of an underlying disease (secondary AIHA). The spectrum of underlying diseases in warm antibody AIHA and cold antibody AIHA is similar but is not identical.

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To differentiate between primary and secondary AIHA, additional laboratory tests (apart from those discussed above) are required. The selection of markers and tests depends on a careful case history, taking all possible causes (triggers) of immune-mediated haemolysis into consideration, such as drug exposure, infections, or autoimmune diseases. In addition, the results from routine laboratory tests (acute phase reaction, electrophoresis) and the type of antibody (warm or cold) must be considered. A careful examination of the blood smear is essential to document or to exclude a malignant haematologic disorder, in particular a lymphoproliferative disease. A bone marrow examination is recommended in all cases of suspected lymphoma or other haematological neoplasms. An abdominal CT scan is indicated in otherwise unexplained AIHA to exclude intra-abdominal lymphomas [22] or a solid tumor [30]. Quantitative determination of immunoglobulins and immunofixation should be performed in all cases in order to detect monoclonal gammopathy or/and immunoglobulin deficiency. Immunophenotyping of lymphocytes may be a sensitive method to detect smaller amounts of monoclonal peripheral or bone marrow lymphocytes. A screen test for lupus anticoagulant (APTT with a lupus sensitive reagent) should also be performed, since coexistence of AIHA with a lupus anticoagulant is not uncommon [37, 38]. Such patients may be at high risk of thrombosis [39]. Anticardiolipin or antinuclear antibodies are often elevated in patients with AIHA [40], but such test results have no predictive value they should thus only be determined when clinically indicated.

occur in patients with paroxysmal cold haemoglobinuria and in those with paroxysmal nocturnal haemoglobinuria.

CausesofAIHA
AIHA may occur as idiopathic AIHA, may be associated with an underlying or concomitant disease, or may be a drug-induced anaemia. Although both types of antibodies (warm and cold) may occur in most associated disorders, a specific underlying condition is usually associated with predominant occurrence of either warm or cold antibodies. Therefore, the causes of warm antibody AIHA and cold antibody AIHA will be discussed separately below.

CausesofwarmantibodyAIHA(WA-AIHA)

Idiopathic WA-AIHA
There are no robust data describing the relative prevalence of idiopathic warm antibody AIHA and of secondary warm antibody AIHA. The few studies that have dealt with this topic were retrospective studies, had no predefined diagnostic criteria (with regard to the presence or absence of an underlying disease), and the number of patients studied was rather small (no more than 100 cases). In one well-conducted but retrospective study [45], half of the patients with WA-AIHA were found to have idiopathic WA-AIHA. In the secondary patients the underlying diseases were lymphomas (20% of all cases, mostly CLL or other NHL) and connective tissue diseases (another 20%). In the remaining patients (10%), infections, drugs, and solid tumors were described (among other rare causes) as underlying disorders.

ClinicalsymptomsinAIHA
The most common symptoms in warm and cold antibody AIHA are signs of anaemia, such as fatigue, dizziness and breathlessness. The onset of symptoms may be insidious, subacute or acute [1]. Peracute haemolysis with very low haemoglobin levels may develop in patients with fludarabine-induced haemolysis. In such patients, myocardial ischemia may occur and may be triggered by a low haemoglobin concentration, but may also (in addition) result from NO inhibition caused by an excess of free (circulating) haemoglobin [41]. In warm antibody AIHA patients, an increased risk (tendency) of venous thromboembolic events has been described [42], in particular in those who have a concurrent lupus anticoagulant [39] or systemic lupus erythematosus [43]. In patients with a chronic course of disease, the severity of anaemia may wax and wane, sometimes without a recognisable trigger. In CA-AIHA, anaemia may be mild and thus may not need medical treatment or transfusion. In other cases, in particular in patients with high antibody titres with a narrow thermal amplitude, haemolytic crises with a precipitous drop in haemoglobin may occur. A typical symptom of CA-AIHA is acrocyanosis, a bluish discoloration of local skin in finger tips, nose or ears exposed to the cold. These skin changes usually disappear after warming without a reactive hyperaemia. In rare severe forms of AIHA, necrosis of the extremities has been reported [44]. Intravascular haemolysis with haemoglobinuria is not a feature of CA AIHA, but can

WA-AIHA associated with malignancies (Table 1)

Lymphomas
The most obvious and one of the most frequent association is that between AIHA and CLL (Table 1). In six larger studies, the prevalence of AIHA in CLL ranged between 4.3% and 9% [11, 21, 4649]. In the vast majority of these patients (80%90%), warm antibodies were detected (WA-AIHA) [21]. Although the exact impact of anti-neoplastic therapy on AIHA-development in these patients remains uncertain, it can be assumed that spontaneous as well as treatment-related AIHA-cases were included (combined) in most studies. In fact, only one study [21] determined the prevalence of AIHA in (definitively) untreated patients (prevalence: 3%). From recent prospective studies, we also know, that drug-induced AIHA may well occur after the end of drug treatment e.g. for CLL [28, 50], but it is difficult to prove a relationship in individual patients, and there is no consensus about the time interval (end of treatment to occurrence of AIHA) that would allow for the conclusion AIHA was a druginduced event. In lymphomas other than CLL, AIHA is less commonly observed. The prevalence of AIHA in non-CLL type NHL patients reportedly ranges between 0.13% and 2.1% [46, 49, 51], and is even lower in patients with Hodgkins disease (0.19%2%) [51]. WA-AIHA is ob-

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Valent and Lechner, Diagnosis and treatment of autoimmune haemolytic anaemias in adults Table1. Frequency of AIHA in various diseases

Underlying disease CLL Makroglobulinemia NHL, all types, except CLL AILD Hodgkins disease Systemic lupus erythematosus Antiphospholipid antibody syndrome Ulcerative colitis

Frequency 4.3%9% 16% 0.23%2.6% 17.8% 0.19%1.7% 6.6%7.5% 9.7% 1.41.7%

References 10, 21, 46, 47, 49 49 46, 48, 51, 170, 171 172 51, 171 62, 63 37 65, 174

tumor resection was followed by a complete resolution of AIHA in all cases, after a latency period of 313 weeks.

Drug induced WA-AIHA

A number of drugs have been described to induce warm antibody-based anaemia (WA-AIHA). WA-AIHA has particularly been observed after treatment with purine analogs (fludarabine, cladribine), but also after chlorambucil [53], pentostatin [54], and even after rituximab therapy [55]. Almost all treatment-induced AIHA are caused by warm antibodies. Most of these patients had CLL and either received fludarabine or cladribine as monotherapy or received combinations of nucleoside-analogs and other drugs such as cyclophosphamide, prednisone, or rituximab (Table 2). While the primary goal of these combinations was an increase of efficacy, one may also have expected that the risk of AIHA is reduced since these additional drugs (prednisone, rituximab) are effective also in AIHA. However, this is obviously not the case, since with one exception [56], AIHA occurred in about the same frequency as with fludarabine or cladribine monotherapy. In one study combining fludarabine, cyclophosphamide and rituximab [28], the rate of AIHA was particularly high. Interestingly, however, 80% of AIHA that occurred in this study were DAT negative AIHA. A comparison of the frequency of AIHA after treatment of CLL with various drugs and drug combinations is difficult, as these studies differ not only in treatment schedules, but also with regard to patient populations (untreated, pretreated), definition of AIHA, and inclusion or exclusion of AIHA cases after the end of treatment. AIHA is a definitive but rare complication of interferon-alpha treatment for hepatitis C, lymphoproliferative disorders, chronic myeloid leukaemia, or renal cell cancer [5761]. In these patients, AIHA usually occurs after long term treatment (median time to development of AIHA: 10 months) [61]. The onset may be earlier in those patients who have a positive DAT test before therapy [57].

served in various subtypes of Non Hodgkins lymphomas, such as hairy-cell leukaemia, follicular lymphoma, marginal cell lymphoma, or diffuse large cell lymphoma, but has not been reported in mantle cell lymphoma patients so far [22]. While AIHA in NHL is commonly associated with active disease, AIHA has been described in patients who had been treated successfully for Hodgkins disease, i.e. at the time of complete remission.

Solid tumors
Rarely, AIHA may develop as a paraneoplastic (immunological) syndrome in patients with solid tumors. Sokol et al. [52] reported that this association is a more frequent event than has been assumed previously. Two thirds of the affected patients were found to have warm antibodies, and one third of them exhibited cold antibodies. A causal relationship between the tumor and the development of AIHA is well established for ovarian dermoid cysts. In particular, Baker [30] described 15 patients in his review, and reported that treatment with prednisone or splenectomy showed no effect, whereas

Table2. Frequency of AIHA after drug exposure Disease CLL CLL CLL CLL CLL CLL CLL CLL CLL CML Solid tumors Drug Fludarabine Fludarabine oral Fludarabine, Cyclophosphamide Fludarabine, Cyclophosphamide Rituximab Cladribine Cladribine, Cy Cladribine, Cy, Mito Cladribine + prednisone Chlorambucil (+/ prednisone) CAP, ChOP Interferon alpha Interleukin 2 Incidence 210% 5.1% 2.74% 7.1%, 5.67%, 8% 6% 5.6% (all during treatment) 13%, 2% 04.8%, 0 very rare very rare References 50, 56, 173, 175 176 50, 56 28, 177 178,179 179 179 180 50, 180 173 181

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WA-AIHA associated with collagen disorders

The second large group of underlying conditions (apart from neoplasms) are autoimmune and collagen disorders. Likewise, autoimmune haemolytic anaemia occurs in 5.2% to 7.5% of all patients with systemic lupus erythematosus (SLE) [43, 62, 63], which is a significant number, but is less frequent than autoimmune thrombocytopenia in these patients. In most cases, AIHA occurs at onset or in an early phase of the disease [43, 62]. All patients with SLE in whom AIHA was diagnosed, were found to have the WA-AIHA subtype [43]. Interestingly, 41%90% of the patients were already on immune suppressive therapy when developing AIHA. In patients who responded to steroids, the recurrence rate is low. About 85% of the patients are recurrence-free after 5 years [43], and about 73% are recurrence-free after 15 years of follow up [64]. AIHA is highly associated (9.7% of cases) with the antiphospholipid antibody syndrome [37]. The prevalence of AIHA is particularly high in patients with SLE who also have antiphospholipid antibodies [38] There is a definitive association between WA-AIHA and ulcerative colitis [65, 66], and a less well established association between AIHA and Crohns disease [67]. Patients with ulcerative colitis and AIHA show a male preponderance and have a more severe disease course [65]. AIHA is only rarely associated with other immune disorders. These associations may be coincidental.

ceived intensive immunosuppression (CD52 antibody before and after HSCT) [74]. The frequency of AIHA was high (15.3% at three years), and in six of nine cases, AIHA was associated with a molecular or a cytogenetic relapse. After treatment with donor lymphocyte infusions, three of five patients had a complete resolution of AIHA and a molecular remission of their CML.

AIHA after transplantation of solid organs

Severe WA-AIHA, often in combination with pure red cell aplasia (PRCA] and/or thrombocytopenia, occurs in about 5.6% of patients who underwent pancreas- or kidney transplantation and received intensive immunosuppressive therapy (alemtuzumab, daclizumab, mycophenolate mofetil) [26]. In these patients, AIHA developed after 13 to 23 months following transplantation. All of these patients developed WA-AIHA [26]. Reduction of the dose of immunosuppressive therapy led to improvement of AIHA and PRCA. AIHA may also occur after renal transplantation [75], cardiac transplantation [76], and liver transplantation [77].

AIHA after blood transfusions

AIHA may occur after repeated blood transfusions [78]. In fact, AIHA has been observed in patients with sickle cell anaemia as well as in patients with thalassemia who received multiple transfusions. It has been suggested that the transfusion-induced formation of alloantibodies is commonly associated with autoantibodies and rarely also with AIHA [79]. The exact impact of chronic transfusions on the development of AIHA remains uncertain. Notably, also, AIHA is less commonly observed in patients with myelodysplastic syndromes, although many of these patients also are chronically transfused patients.

AIHA after transplantation and blood transfusion

AIHA following allogeneic or autologous haematopoietic stem cell transplantation (HSCT)
AIHA may be a serious complication of allogeneic HSCT. It is believed that this form of AIHA is due to donor red cell-directed antibodies produced by the donors immune system. This type of haemolytic anaemia must be differentiated from haemolysis due to major ABO incompatibility and the lymphocyte passenger syndrome [16]. In three larger studies performed in adults, the frequency of AIHA after allogeneic HSCT ranged between 3.0% and 4.4% [6870], and amounted to 5% at three years in a paediatric study [71]. A high frequency of AIHA (19.5%) was observed in children with severe combined immunodeficiency (SCID) who underwent T-celldepleted haploidentical HSCT [72]. The patient populations were different in these studies with regard to the underlying disease, T-cell depletion, and type of donor. The onset of AIHA was usually late after HSCT, the median time from HSCT to onset of AIHA ranging between 4 and 10 months [6872]. Most of these patients were in CR at the time of AIHA. The AIHA were of the warm or cold antibody type. Cold antibodies occurred earlier than warm antibodies in one study [69], whereas this was not found in another study [70]. Risk factors were T-cell depletion, unrelated donor, and extensive graft versus host disease. Most patients were refractory to standard treatment, and the mortality was found to be high and to result from severe infections. Rituximab may be a good treatment option in these patients [70, 73]. The study of Cwynarski [74] differed from the other studies in several aspects. In particular, all patients had CML, they received T-cell depleted allografts from unrelated donors and re-

AIHA associated with infections

In a group of patients with AIHA, infectious diseases are found and are considered to contribute to the initiation of haemolytic anaemia. Such infection-associated forms of AIHA may occur in children or in adults, may occur in immunocompetent or immunosuppressed patients, and are caused by cold antibodies, warm antibodies, or mixed antibodies. A number of viruses and other infectious agents have been implicated as potential trigger of AIHA. Warm and mixed antibodies have also been described in HIV positive patients [80], and in immunocompetent adults with cytomegaly virus infection [81]. AIHA occurred concurrently with the infectious disease in these cases [81]. Also, WA-AIHA is a rare complication of treatment-naive hepatitis. Ramos Casals [82] collected 35 cases from their department and from the literature, and found that most of these patients had advanced disease (liver cirrhosis), and all had other immunological abnormalities (apart from AIHA), in particular immune thrombocytopenia. Despite an initial response to steroids, the prognosis was found to be grave in these patients.

AIHA in pregnancy
A positive DAT is only rarely seen in pregnancy (1:50,000), and only one third of these women have anae-

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mia [83]. AIHA may occur in the first or in the third trimester of pregnancy [84, 85]. Some of these patients may require steroid treatment. Newborns of mothers with AIHA may have a positive DAT, but do not have haemolytic anaemia [85].

CausesofcoldantibodyAIHA(CA-AIHA)

Clonal disorders
AIHA caused by cold antibodies is far less frequently diagnosed than WA-AIHA. The prevalence of idiopathic, primary cold AIHA is 1.6/100.000, and the incidence is 0.1/100,000/year [86]. Primary (idiopathic) chronic cold agglutinin disease (CAD) is defined as a condition with chronic haemolysis induced by cold antibodies in the absence of clinical signs of overt lymphoma or another underlying disease. However, in fact, most of these patients have occult (subclinical) NHL or monoclonal gammopathy of uncertain significance (MGUS). In a large study from Norway, 94% of patients with CAD had monoclonal immunoglobulins, 90% of these paraproteins belonging to the subclass IgM kappa [86]. Bone marrow histology and immune phenotyping revealed the presence of a non-Hodgkin lymphoma in 76% of these patients [86]. The most common lymphoma types were lymphoplasmacytic lymphoma (50%). The remaining patients had marginal cell or small lymphocytic lymphomas [86]. Some of these cases may rather be classified as MGUS (subclass IgM), depending on the definition of MGUS and of lymphomas [19, 86, 87]. About 3.5% of patients with CAD and indolent NHL progressed to diffuse large cell lymphoma within a median observation time of 5 years. Interestingly, trisomy 3 was found in some lymphomas associated with cold agglutinins [88]. Haemolytic anaemias due to cold antibodies may occur in almost all other subtypes of NHL, including diffuse large cell lymphoma and T-cell lymphomas [22]. The titre of cold agglutinins can be used as a marker of lymphoma progression or regression following therapy [22]. CA-AIHA, often combined with PRCA, occurs relatively frequently in angioimmunoblastic lymphadenpathy (AILD). In this lymphoma type, cold agglutinins may be reactive (polyclonal). CA-AIHA may also be associated with solid tumors, in particular renal cell cancer [89]

In patients with rubella infection, cold IgG or IgM antibodies with anti-Pr specificity have been described, although such cases are unusual [91]. CA-AIHA may also be triggered by an adenovirus infection, e.g. after allogeneic HSCT [92]. Cold antibodies without anaemia have been detected in a considerable number of patients with advanced HIV-infection [93]. CA-AIHA caused by a Donath-Landsteiner antibody is very rare. Originally, it was detected in patients with syphilis. It may also occur in children after viral infections.

TreatmentofAIHA
Treatment recommendations for AIHA patients are based on results from relatively small cohort studies (with heterogeneous patient samples), on results from case reports, or from expert opinions. In principle, there are four treatment strategies: (1) Immunosuppressive therapy or immuno-modulating therapy (recommended in most cases), (2) Treatment of the underlying condition (intensive chemotherapy, surgery, antimicrobial agents), (3) Elimination of drugs or other causes (drug induced AIHA), and (4) no specific treatment except transfusions (viral AIHA). There are no established criteria and definitions for responses and for complete remission in AIHA. In case reports and several studies, a complete remission was usually assumed when haemoglobin increased to a stable value of at least 12 g/dl (irrespective of gender). However, complete remission of haemolysis would also require normalization of haemolysis-related parameters (LDH, haptoglobin, reticulocyte count). Some patients with a haematological CR still have a positive DAT, whereas others have a negative DAT (serological remission). Rarely, patients have a pure serological, but no haematologic, remission. The predictive value of a pure serological remission is unknown. Since there are profound differences in the therapy of WA-AIHA and CA-AIHA, treatment of these two conditions are discussed separately below:

TreatmentofwarmantibodyAIHA

First line treatment

The treatment of warm antibody AIHA is much more difficult than the treatment of autoimmune thrombocytopenia (AITP). One reason may be that AIHA is less responsive to immunosuppressive drugs and splenectomy. However, the most likely reason is that in AITP, the clinical goals of treatment are easier to achieve than in AIHA, since in AITP, a platelet level of 20.00030.000/l (which is less than a partial remission) is sufficient to keep the patient symptom-free, whereas in AIHA, a stable haemoglobin level of at least 10 g/dl needs to be reached to guarantee a constant improvement in clinical symptoms. The third reason may be the high prevalence of secondary AIHA. When these patients are treated with prednisone or other immunosuppressive drugs, the treatment is often not well tolerated, because high doses are required, and many of these already immunosuppressed patients may succumb infectious diseases. For most cases of idiopathic and secondary AIHA, prednisone at a dose of 1 mg/kg/day is the first line treat-

Cold antibodies associated with infection

Several viral or other infections may be associated with an increase of the cold agglutinin titer, although anaemia is relatively rare in these patients [13]. A typical example is mycoplasma pneumonia, which may be associated with various immune reactions, including CA-AIHA. Hemolysis usually occurs 23 weeks after the onset of symptoms. The antibody has an anti-I specificity. CAAIHA may also be triggered by EBV infections. In these cases the cold antibody has an anti-i specificity. The temporal relationship of AIHA and EBV infection is less predictable than in mycoplasma infection. Although AIHA usually develops 12 weeks after the onset of infectious symptoms, it may also occur at the onset of symptoms or with a delay of up to 2 months [4]. The course of AIHA is usually mild and self-limited in these patients, but in rare cases may even be life-threatening [90].

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ment of choice [94]. Usually, it takes some days until an increase in haemoglobin is seen. Therefore, many patients require transfusion support. It would appear logical to reduce the antibody level by pretransfusion plasma exchange before starting transfusions in these patients in order to improve transfusion efficacy, but recent data have shown that such pretransfusion manoeuvre has no effect [95]. After three weeks of treatment with prednisone, about 8090% of all patients have a clear clinical response (i.e. hemoglobin > 10.0 g/dl). Patients who have no clinical response at that time are unlikely to improve from continuation of steroids, and should thus be switched to alternative treatments [96]. After 3 months, two thirds of the patients are in CR and about 2123% are in partial remission [1, 45]. About 10% of all patients are non-responders. When a response is achieved, it is extremely important to taper the prednisone dose slowly. It is recommended to reduce it by 20 mg/ day every two weeks down to a daily dose of 20 mg/day. In those patients in whom the haemoglobin response can be maintained, the dose of prednisone should then be reduced slowly, i.e. only by 5 mg or 2.5 mg/day every month. The reason for this strategy which is in contrast to that in AITP is, that it takes a relatively long time to know whether the actual dose of prednisone is sufficient to keep the haemoglobin level within an acceptable target-range (long half life of erythrocytes). While in children the rate of spontaneous remissions is high and prednisone can be stopped at a relatively early time point, most adult patients require a maintenance dose. Without such maintenance, the median remission duration is 912 months [45, 97]. About 20% of the patients remain in sustained remission without further therapy, 4050% of the patients require low maintenance therapy, and 1520% require a high maintenance dose of prednisone [96]. Thus, one particular problem of steroid therapy in AIHA are the relative high doses of steroids required, and the prolonged treatment periods. In addition, many of these patients have an underlying disease. Many responding patients, e.g. 23% in one study [45], died from infections or from the underlying disease. Patients with AIHA associated with systemic lupus erythematodes appear to respond well to steroid therapy. The initial response rate is high and the recurrence rate relatively low in these patients [43, 64]. However, some patients may also exhibit a primary resistance against steroid therapy, especially those who are suffering from an NHL [22], those with a post-transplant lymphoma [16], and patients with ovarian dermoid cysts [30]. For these patients, other first-line treatment-approaches may be required [98]. The value of therapy with high dose immunoglobuline (HdIgG) alone or in combination with prednisone is controversial. While in one study [99], primary treatment with HdIgG was ineffective in five patients, Flores et al. [100] reported a response to this treatment in as much as 39.5% of patients. Besa et al. [101] reported good responses in 5 patients with recurrent AIHA in CLL, and concluded that recovery of hemoglobin is faster when prednisone and high dose immunoglobulins are combined. Since patients with chronic haemolytic anaemia often have folate deficiency, it is recommended to give low dose folate as treatment as long as AIHA persists.

Second line therapies

For patients who have no satisfactory response to initial prednisone, who relapse after having responded or need more than 1015 mg prednisone per day to maintain an acceptable haemoglobin level, splenectomy is probably the most effective second-line treatment, although the efficacy of this therapy has never been compared to that of other second-line therapies. Today, splenectomy is performed via laparoscopy in most centers. The surgical risk of laparoscopic splenectomy is low compared to conventional surgery, provided that the size of the spleen is not extensive. Patient with massive splenomegaly (for example in CLL) have a high postoperative risk of splenic portal vein thrombosis [102]. The risk for the most feared complication of splenectomy, overwhelming pneumococcal septicaemia, is considered to be reduced by preoperative vaccination, although this has not been formally proven. The response rate to splenectomy is 3870% (1, 45, 103105]. However, in contrast to autoimmune thrombocytopenia, there are no good long term data on remission duration. In earlier studies, late relapses have been described [94]. In a recent study on patients with AIHA and CLL, 7/9 patients had a complete response, and 6 of these patients, a prolonged response after a median observation time of 2 years was observed [105]. Splenectomy is also an effective therapy for patients with AIHA associated with splenic marginal cell lymphoma [22]. For patients, who are unwilling or unfit to undergo splenectomy, do not respond to splenectomy or relapse after response to splenectomy, a number of immunosuppressive agents are available including cyclophosphamide, azathioprine, cyclosporin A, mycophenolate mofetil (MMF), and more recently monoclonal antibodies (rituximab and alemtuzumab). However, so far, no randomised clinical trials have been performed with these agents in patients with AIHA. Rather, the efficacy of these agents has documented only in single cases or in small series of (probably preselected) patients. Low dose cyclophosphamide (100 mg/days) and azathioprine (100 mg/days) are two drugs used for the treatment of refractory AIHA since many years. Most of the data on efficacy were generated in the sixties of the last century. Improvement was reported in 2540% of these patients [45, 106, 107]. More recently, high dose cyclophosphamide (50 mg/kg/days for 4 days) was used in 8 patients with highly refractory WAAIHA [108]. These patients became severely aplastic (median time until ANC > 500/ul: 16 days), but all patients survived. Five achieved complete remission (CR), and three of these patients a partial remission (PR) which lasted for 429+ months [108]. Favourable results have also been obtained in a few patients with autologous stem cell transplantation [109]. Vincristine-loaded platelets is another old form of treatment of AIHA, that despite efficacy in AITP and AIHA, was not used in later years. However, more recently, Shvidel et al. [110] described that this treatment is effective in 3 out of 4 refractory patients with AIHA. Two of these patients achieved a remission lasting for 8+ and 9+ years, respectively. Cyclosporin A is a potent immunosuppressive drug that exhibits high efficacy in pure red cell aplasia associated with CLL. In AIHA, complete responses were ob-

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tained in 3/3 patients with refractory disease [111]. Liu et al. [112] found a high response rate (88.9%) in 18 AIHA patients treated with a combination consisting of cyclosporine, prednisone, and danazol. In a subsequent nonrandomized study they showed that patients who received cyclosporine A in addition to prednisone as maintenance therapy had a lower relapse rate compared to those that did not receive cyclosporine A as maintenance therapy [97]. In patients with AIHA and CLL (some of them after fludarabin, 40% previously untreated), an excellent haemoglobin response was obtained with cyclosporine A, i.e. in 56% of all patients [113]. The inosine monophosphate dehydrogenase (IMPDH) inhibitor mycophenolate mofetil (MMF) is another immunomodulatory drug used in clinical medicine. Treatment with MMF was successful used in a few patients with refractory AIHA [114116]. The time to remission was relatively long (28 months) and continuous treatment was required to maintain the response. MMF has been proven particularly effective in autoimmune lymphoproliferative syndrome (ALPS) of children and young adults. ALPS is caused by an inherited mutation of the TNF receptor or of the FAS ligand, and is associated with a high frequency of immune cytopenias, including AIHA. In a recent study, Rao et al. [117] showed that 12 out 13 heavily pretreated ALPS patients with AIHA responded to MMF in terms of maintenance of an adequate blood count and/or reduction or cessation of other immunosuppressive drugs. The median follow up time in the responders was 49 weeks. MMF treatment was well tolerated in these patients. The monoclonal antibody rituximab (anti-CD20; B cell-targeting antibody) has probably the best benefit/risk ratio for treatment of refractory WA-AIHA. At least 28 cases have been reported in whom rituximab (375 mg/ sqm weekly for 4 weeks) was given as monotherapy (sometimes along with tapered steroid doses) to adults with refractory primary or secondary AIHA or Evans syndrome. Responses (CR or PR) were achieved in most patients with idiopathic AIHA [118121], in 8/9 patients with underlying CLL or PLL (6 CR, 2 PR) [122129], in 1/1 with myeloma [130], in 5/5 with Hodgkins disease or non-Hodgkin lymphomas (4 CR, 1PR) [125, 128, 131, 132], in 1/1 with SLE [133], in 1/1 with drug induced AIHA [134], in one patient with AIHA after liver transplantation [135], and in 3/3 (all 3 CR) in AIHA after stem cell transplantation (HSCT) [136, 137]. Overall (taking all case reports into account), 75% of all patients achieved a CR, and 21% a PR. The time to complete remission ranged from 2 to 13 weeks. In 5/10 patients with CR, and in 1/3 with a PR, a negative post-therapy DAT was found. The duration of remission ranged from 5 to 20+ months. In case series [29, 138, 139] with at least 5 cases, the results were less impressive. Among 30 patients (most with CLL), 8 (27%) achieved a CR, 11 a PR, and 8 had a treatment failure (5 deaths). Rituximab monotherapy seems to be an especially attractive therapy for children. Zecca et al. [140] treated 15 children with refractory AIHA with rituximab monotherapy and achieved a hemoglobin response in 13 cases. The duration of remission ranged from 9.6 to 27+ months. Using a combination of dexamethasone, cyclophosphamide and rituximab, Gupta [141] re-

ported on a high remission rate in 8 patients with CLL associated with AIHA (7 CR and 1 PR, 5 patients became DAT negative). The median number of cycles to CR/PR was 3 (range 14), the median duration of hemoglobin responses 13 (range 723) months. The high response rate that can be calculated when taking all single case reports together is most probably an overestimation due to a reporting bias, but there is no doubt that the drug is effective in a subgroup of patients and does not produce major toxicities. Five patients with refractory WA-AIHA or pure red cell aplasia have been successfully treated with the anti CD52 antibody alemtuzumab [142147]. Interestingly, one patient was refractory to rituximab but responded to alemtuzumab both in terms of lymphoma-regression and remission of AIHA [143]. In one case, AIHA could be controlled by an antibody directed against the interleukin 6 receptor [148]. Treatment of the underlying disease may be an effective therapy in some cases. Interestingly, in CLL, drugs that otherwise might induce AIHA, may also be a successful treatment in spontaneous (CLL-associated) AIHA. In particular, fludarabine was found to be an effective and well-tolerated therapy of AIHA associated with previously untreated CLL [149151]. It is noteworthy in this regard that several patients with CLL-associated AIHA have been successfully treated with cladribine (+/ prednisone) [152154]. In these studies, 11 out of 34 patients (32.3%) achieved a CR (among them 6 with a negative DAT after therapy), and 11 patients entered PR, whereas four patients died from haemolytic crisis [153]. However, despite favourable results in some patients, it has become common practice to regard the presence of AIHA as a contraindication to treatment with purine analogs. In some lymphomas associated with WA-AIHA, chemotherapy or surgical removal of a localized lymphoma has induced durable remissions of lymphoma and AIHA [22]. In a patient with Crohn`s disease and associated refractory AIHA, a subtotal colectomy and resection of the terminal ileum led not only to improvement of Crohns disease but also to a sustained remission of AIHA [67]. A suggested algorithm for treatment of WA-AIHA is given in Table 3.

TreatmentofcoldantibodyAIHA
Many patients with chronic cold agglutinin disease (CAD) do not require treatment (except folate replacement), since these patients usually have only mild anaemia, and exacerbations of AIHA can be avoided by prevention of infections and by keeping patients at warm temperature. However, the efficacy of the latter measure remains questionable [8]. It is of importance to be aware that patients with CAD or CA-AIHA who require therapy (because of anaemia) do not respond to prednisone or splenectomy. These treatments are not only ineffective, but potentially dangerous in CA-AIHA. In case of an underlying lymphoma, specific therapy of the lymphoma (cytoreduction, chemotherapy) may improve AIHA, although not all patients may respond [86]. In a few reports [155, 156], patients with CAD were found to responded to interferonalpha. A drug with well documented efficacy in CA-AIHA

Valent and Lechner, Diagnosis and treatment of autoimmune haemolytic anaemias in adults Table3. Suggested treatment algorithm in warm antibody AIHA* Type of AIHA Idiopathic, young, good condition Idiopathic, older, poor condition CLL-associated Fludarabine/cladribine induced High malignant NHL (B-and T-cell) Autoimmune lymphoproliferative syndrome Ovarian dermoid cysts, renal cancer Autoimmune disorders Post-transplant Viral infections First line Steroids +/ HdIgG Steroids +/ HdIgG Steroid +/ HdIgG Steroid +/ HdIgG Chemotherapy (including steroids) Mycophenolate Mofetil Surgery Steroids/HdIgG Rituximab None (transfusion) Second line Splenectomy Rituximab Splenectomy Rituximab Third line

145

Cy, Aza, MMF, CyA, Rituximab Cy, Aza, MMF, CyA, Rituximab, Campath

Cy, Aza, CyA

HdIgG High dose immunoglobulin; Cy Cyclophosphamide; Aza Azathioprine; MMF Mycophenolate Mofetil; CyA Cyclosporin A. * The suggested algorithm is based on published data [1, 29, 30, 43, 45, 67, 94154] and expert recommendations.

is rituximab. The beneficial effects of rituximab monotherapy in CAD/CA-AIHA (using the same schedule as in follicular lymphoma patients) has been demonstrated in single patients [129, 157161] as well as in two prospective phase II trials [162, 163]. Berentsen [162] treated 27 previously untreated (n = 12) or pretreated (n = 15) CA-AIHA patients with underlying low grade lymphoma and low tumor burden with rituximab. Fourteen of these patients (52%) responded, and a majority of them (n = 13) achieved a partial remission (hemoglobin increase of > 2.0 g/dl). The median time to response was 1.5 months, the medium duration of remission was 11 months. Eight patients relapsed and were again treated with rituximab (+/ interferon), and five of these patients achieved a second PR. Very similar results with the same treatment protocol were obtained in a Danish study [163], although there were some differences in the study population and definition of PR. This study included untreated and pretreated patients with either idiopathic CAD (12/20) or with underlying lymphomas. The definition of PR was an increase in haemoglobin by 1.0 g/dl. In 9/20 patients (45%), a response was documented, one had a CR, and eight patients a PR. The median time to response was 3 months and the median duration of response 6 months, with only a few long term responders. Side effects of rituximab were mild in both studies. Rituximab may also be combined with conventional chemotherapy [164] and thus may be preferable for those NHL patients who need chemotherapy and develop CAD/CA-AIHA. In patients with AILD or other T-cell lymphomas, chemotherapy of the underlying lymphoma successfully controlled CA-AIHA in some cases [22]. Patients with infection-associated CA-AIHA rarely present with severe anaemia. However, after mycoplasma infections, severe anaemia may occur. These patients require transfusions without additional drugs, as spontaneous (rapid) recovery with resolution of anaemia can be expected. Treatment of the infection may shorten the time to recovery in these patients.

When patients with (viral) infection-associated CAAIHA or WA-AIHA are treated with glucocorticosteroids, the immunosuppressive effect of the drug and the related risk (e.g. reactivation of viral disease) must be considered [165]. Special attention has to be paid to transfusions in patients with CA-AIHA. Notably, the determination of the blood group may be difficult in such cases. Prewarming of transfused blood is absolutely necessary. Special attention has to be drawn to CA-AIHA patients who undergo surgery where cooling is required, for example in lungheart surgery. There is no established standard therapy for paroxysmal cold haemoglobinuria. Theoretically, these patients should receive P negative blood in case of severe anaemia, but in practice it appears that transfusion of P positive blood is also a safe approach. In some patients with AIHA presenting with cold antibodies, with the Donath-Landsteiner antibody, but also in cases with WA-AIHA, plasmapheresis has been suggested as a therapeutic manoeuvre [95, 166169]. In some instances, responses have been described [166169]. Therefore, plasmapheresis may be an alternative treatment (experimental therapy) for patients who did not show a response to first- and second line therapies. However, the exact value of plasmapheresis (plasma-exchange) in the treatment of AIHA remains unknown.

Concludingremarks
AIHA is an important hematologic diagnosis that is based on the presence of anaemia, signs of haemolysis, and detection of red cell-reactive antibodies. The management of AIHA requires exact knowledge about the underlying disease and triggering factors, and the nature and biologic behaviour of the related antibody. In idiopathic AIHA, glucocorticosteroids remain first line therapy, and splenectomy remains accepted second line therapy. In refractory patients and those in whom standard therapy is

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Valent and Lechner, Diagnosis and treatment of autoimmune haemolytic anaemias in adults 19. Crisp D, Pruzanski W (1982) B-cell neoplasms with homogeneous cold-reacting antibodies (cold agglutinins). Am J Med 72: 915922 20. Lechner K, Weltermann A, Pabinger I (2006) Autoimmunthrombozytopenie (AITP) des Erwachsenen: Klinik, Diagnose und Therapie. Wien Klin Wochenschr 118: 255264 21. Mauro FR, Foa R, Cerretti R, Giannarelli D, Coluzzi S, Mandelli F, et al (2000) Autoimmune hemolytic anemia in chronic lymphocytic leukemia: clinical, therapeutic, and prognostic features. Blood 95: 27862792 22. Hauswirth AW, Skrabs C, Schutzinger C, Gaiger A, Lechner K, Jger U (2007) Autoimmune hemolytic anemias, Evans syndromes, and pure red cell aplasia in non-Hodgkin lymphomas. Leuk Lymphoma 48: 11391149 23. Park KU, Song J, Kim JQ (2004) Haptoglobin genotypic distribution (including Hp0 allele) and associated serum haptoglobin concentrations in Koreans. J Clin Pathol 57: 10941095 24. Delanghe J, Langlois M, De Buyzere M (1998) Congenital anhaptoglobinemia versus acquired hypohaptoglobinemia. Blood 91: 3524 25. Liesveld JL, Rowe JM, Lichtman MA (1987) Variability of the erythropoietic response in autoimmune hemolytic anemia: analysis of 109 cases. Blood 69: 820826 26. Elimelakh M, Dayton V, Park KS, Gruessner AC, Sutherland D, Howe RB, et al (2007) Red cell aplasia and autoimmune hemolytic anemia following immunosuppression with alemtuzumab, mycophenolate, and daclizumab in pancreas transplant recipients. Haematologica 92: 1029 1036 27. Garratty G (2005) Immune hemolytic anemia associated with negative routine serology. Semin Hematol 42: 156 164 28. Borthakur G, OBrien S, Wierda WG, Thomas DA, Cortes JE, Giles FJ, et al (2007) Immune anaemias in patients with chronic lymphocytic leukaemia treated with fludarabine, cyclophosphamide and rituximab incidence and predictors. Br J Haematol 136: 800805 29. DArena G, Laurenti L, Capalbo S, DArco AM, De Filippi R, Marcacci G, et al (2006) Rituximab therapy for chronic lymphocytic leukemia-associated autoimmune hemolytic anemia. Am J Hematol 81: 598602 30. Baker LR, Brain MC, Azzopardi JG, Worlledge SM (1968) Autoimmune haemolytic anaemia associated with ovarian dermoid cyst. J Clin Pathol 21: 626630 31. Wheeler CA, Calhoun L, Blackall DP (2004) Warm reactive autoantibodies: clinical and serologic correlations. Am J Clin Pathol 122: 680685 32. Wikman A, Axdorph U, Gryfelt G, Gustafsson L, Bjorkholm M, Lundahl J (2005) Characterization of red cell autoantibodies in consecutive DAT-positive patients with relation to in vivo haemolysis. Ann Hematol 84: 150 158 33. Sokol RJ, Booker DJ, Stamps R, Sobolewski S, Haynes AP (1998) Autoimmune hemolytic anemia caused by warm-reacting IgM-class antibodies. Immunohematol 14: 5358 34. Sokol RJ, Booker DJ, Stamps R, Booth JR, Hook V (1997) IgA red cell autoantibodies and autoimmune hemolysis. Transfusion 37: 175181 35. Shulman IA, Branch DR, Nelson JM, Thompson JC, Saxena S, Petz LD (1985) Autoimmune hemolytic anemia with both cold and warm autoantibodies. JAMA 253: 17461748

unlikely to induce a long lasting response, new immunosuppressive agents such as rituximab or mycophenolate mofetil may be considered. In many cases, therapy can control AIHA. In other cases, however, especially in coldantibody-mediated disease, AIHA remains a clinical challenge. For these patients, new more effective drugs (antibodies) and drug combinations need to be established in the future.

Acknowledgments
We thank Dr. Esther Jimenez Boj, Department of Medicine III, Medical University of Vienna, for translating spanish papers, Prof. Dr. Li Yen for translating chinese papers, and Ms. Yen-An Chen for assistance in the preparation of the manuscript.

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