Expert Review of Hematology

ISSN: 1747-4086 (Print) 1747-4094 (Online) Journal homepage: https://www.tandfonline.com/loi/ierr20

Autoimmune hemolytic anemia in adults: primary

risk factors and diagnostic procedures

Wilma Barcellini, Juri Giannotta & Bruno Fattizzo

To cite this article: Wilma Barcellini, Juri Giannotta & Bruno Fattizzo (2020): Autoimmune
hemolytic anemia in adults: primary risk factors and diagnostic procedures, Expert Review of
Hematology, DOI: 10.1080/17474086.2020.1754791

To link to this article: https://doi.org/10.1080/17474086.2020.1754791

Accepted author version posted online: 10

Apr 2020.

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 Publisher: Taylor & Francis & Informa UK Limited, trading as Taylor & Francis Group

Journal: Expert Review of Hematology

DOI: 10.1080/17474086.2020.1754791
Review

Autoimmune hemolytic anemia in adults: primary risk factors and diagnostic procedures

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Wilma Barcellini1, Juri Giannotta1,2 & Bruno Fattizzo1,2

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1
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

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Università degli Studi di Milano, Milan, Italy

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Corresponding author:
Wilma Barcellini
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UOC Ematologia, UOS Fisiopatologia delle Anemie - Padiglione Granelli, Fondazione IRCCS Ca’ Granda
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Ospedale Maggiore Policlinico, Via F. Sforza 35 - 20122 Milano - Italy
Tel.: +39 0255 033 256
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Fax: +39 0255 033 439

e-mail wilma.barcellini@policlinico.mi.it
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 Abstract
Introduction: Autoimmune hemolytic anemia (AIHA) is due to autoantibodies against erythrocytes that may
arise either because of primary tolerance breakage or along with several associated conditions, including
genetic predispositions, congenital syndromes, environmental triggers, autoimmune diseases,
immunodeficiencies, and neoplasms.

Areas covered: This review evaluated the risk of AIHA development in associated conditions and
summarized disease-intrinsic risk factors for relapse and outcome. Diagnostic procedures were analyzed to

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properly identify primary and secondary forms. A Medline including clinical trials, meta-analyses,

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guidelines, consensus, and case reports, published in the last 30 years was performed.

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Expert opinion: The several associated conditions listed above constitute a risk for AIHA development and

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should be considered since disease course and therapy may be different. Particularly, AIHA developing after
transplant or novel checkpoint inhibitors is an emerging complex entity whose proper therapy is still an

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unmet need. Concerning intrinsic risk factors, the severity of anemia at onset correlated with recurrence of
relapses, refractoriness, and fatal outcome. This finding reflects the presence of several mechanisms
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involved in AIHA, i.e. highly pathogenic antibodies, complement activation, and failure of marrow
compensation. With the advent of novel target therapies (complement and various tyrosine kinase
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inhibitors) a risk-adapted therapy for AIHA is becoming fundamental.

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Keywords

warm autoimmune hemolytic anemia, cold agglutinin disease, autoantibodies, bone marrow
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responsiveness index, checkpoint inhibitors, complement, direct antiglobulin test, risk factors, rituximab,
secondary autoimmune hemolytic anemia.
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 Article highlights box
• Autoimmune hemolytic anemia (AIHA) may be primary or secondary to several conditions including
autoimmune and lymphoproliferative diseases, immunodeficiencies, infections, drugs, congenital
syndromes, and neoplasms.
• Associated conditions represent a risk factor for the development of AIHA and should be properly
diagnosed to harness therapy.
• Several immunopathologic mechanisms are involved in determining the severity of the disease

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including the type and thermal characteristics of antibodies, the degree of complement activation,

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and the failure of bone marrow compensation.
• The most important disease specific risk factor for refractoriness is the severity of anemia at onset.

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Evans’ syndrome, acute renal failure, and infections have been associated with increased mortality.

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• AIHA developing after transplant or novel anti-cancer drugs such as checkpoint inhibitors is an
emerging complex setting requiring proper diagnosis and therapy.
• A risk adapted therapy is an unmet need for severe and refractory AIHAs.

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 1. Introduction

Autoimmune hemolytic anemia (AIHA) is a decompensated acquired hemolysis mainly caused by

autoantibodies against autologous red blood cells, with or without complement activation. It comprises
several entities, warm forms (wAIHA), the most common, cold (cAIHA), mixed AIHA (with both
characteristics of warm and cold forms), and the very rare paroxysmal cold hemoglobinuria (PCH). wAIHA is
due to autoantibodies of the IgG class that weakly activate complement (generally IgG1 and IgG3

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subclasses only), determining extravascular hemolysis through the antibody-dependent cellular cytotoxicity

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(ADCC) in the spleen and lymphoid organs.
cAIHA is determined by anti-RBC antibodies of the IgM class, which lead to erythrocyte aggregation and

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complement activation at low temperature. Final complement activation may induce intravascular

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hemolysis and extravascular RBC destruction mainly in the liver. All these forms may be primary or
associated with a variety of diseases, including lymphoproliferative neoplasms, autoimmune and infectious
diseases, immunodeficiencies, drugs, solid tumors, and transplants [1]. AIHA is a greatly heterogeneous

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condition, from mild/compensated to life threatening, mainly due to the strength of the immune attack.
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Moreover, the severity of the disease depends on other pathogenic mechanisms, i.e. the degree of
erythrocyte destruction in the spleen and the bone marrow compensatory activity [2-4]. The latter has
gained increasing attention in the recent years, revealing subclinical monoclonal lymphoproliferation,
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dyserythropoiesis, erythropoietin (EPO) insufficiency, and fibrosis, particularly in relapsed/refractory (R/R)

cases [5,6].
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AIHA may be looked at as a multifactorial condition where both genetic, immunologic and environmental
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factors contribute to disease development (Figure 1). As described for other autoimmune diseases, a
familiar predisposition may be recognized, as underlined by the increased incidence of AIHA in a number of
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congenital conditions including Kabuki syndrome and hemoglobinopathies. A primary dysregulated immune
system, as observed in primary immunedeficiency or in connective tissue diseases, carries a higher
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probability of producing anti-erythrocyte autoantibodies and developing clinically overt AIHA. Molecular
mimicry due to microbial molecules with high similarity to self-antigens, the de-sequestration of naturally
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inaccessible self-molecules, as well as the production of neo-antigens due to the perturbation of host
tissues are some of the postulated mechanisms. These involve mainly external triggers, including infections
and drugs. More recently, other factors have emerged, such as acquired somatic mutations that may lead
to the selection of clonal cells that may directly produce autoantibodies. This is the case of the
lymphoproliferative disorders that are associated with a higher risk of AIHA, as well as of solid cancers.
Finally, some complex and multi-faceted settings are emerging, such as solid and hematopoietic stem cell
transplant, and the novel biologic anti-cancer drugs.

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 Therapy of AIHA is mainly based on expert opinions and few prospective trials [5,7,8]. However, an
increasing number of new targeted drugs are appearing, providing new therapeutic options for R/R and
exciting combination strategies, but also complicating the clinical management and the risk-assessment of
the disease [7-9]. The latter is obviously important to ensure an optimal clinical practice and is pivotal to
design prospective trials with the new drugs. Here we describe the several associated conditions that may
determine and increase the risk of developing AIHA. Moreover, we provide intrinsic AIHA-related risk
factors that may predict the severity and outcome of the disease. Finally, diagnostic procedures will be

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reviewed, focusing on the mandatory and recommended investigations, in light of the increasingly

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recognized secondary forms. The manuscript was written after extensive literature review of Medline
including clinical trials, meta-analyses, guidelines, consensus, and case reports published in the last 30

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years.

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2. Genetics and congenital conditions associated with AIHA
The oldest studies focused on the association of AIHA and specific HLA genes demonstrating a strong

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association with HLA-B locus, particularly HLA-B8 and BW6 [10] (Table 1). Later, analysis of DR and DQ
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frequencies showed that HLA-DQ6 was less frequent in direct antiglobulin test (DAT)-positive individuals
(19% versus 53%), 96% of whom had evidence of clinical hemolysis [11]. Molecular studies from the 90s
focused on the configuration of the variable region of the immunoglobulin heavy and light chains (IGHV and
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IGKV, respectively). The latter encodes AIHA autoantibodies and shows stereotyped rearrangements both
in primary forms and in those secondary to lymphoproliferative disorders. In particular, IGHV4-34, IGHV3,
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and IGKV3-20 genes, responsible for I antigen binding, are the most represented in cold AIHA [12-15].
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Clonal CD8+ T-cells have been reported in about 50% of AIHA cases, representing markers of immunity
dysregulation [16]. Likewise, the G polymorphism of CTLA-4 (a negative regulator of T-cell responses) is
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present at a significantly higher frequency among patients with AIHA, particularly if associated with CLL
[17]. Finally, patients with AIHA had a significant greater frequency of lymphotoxin-α (LT-α) AG phenotype
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compared with healthy controls [18]. All these features may represent a risk factor for primary or
secondary AIHA development. With the advent of modern sequencing techniques, a genetic lesion was
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detected in 65% of pediatric patients with AIHA and immune thrombocytopenia (ITP) (Evans Syndrome, ES);
40% of cases showed mutations in genes implicated in primary immunodeficiencies (TNFRSF6, CTLA4,
STAT3, PIK3CD, CBL, ADAR1, LRBA, RAG1, and KRAS). Interestingly, mutated patients showed more severe
disease with higher treatment requirement (> number of therapy lines) and mortality. This finding
suggested that the higher genomic burden in pediatric cases has therapeutic implications: patients with
primary immunodeficiency may be more prone to persistent hypogammaglobulinemia or catastrophic
infections after immunosuppression [19]. Moreover, some mutations are potentially targetable by new
biologic drugs including rapamycin inhibitors (in ALPS or a PIK3d activation syndrome [20,21], CTLA-4 fusion

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 protein (in CTLA-4- and LRBA deficiency) [22], JAK inhibitors (in patients with JAK1 or JAK2 mutations) [23],
and calcineurin inhibitors (in patients with NFATC1 variants) [19]. Mutations of KMT2D and CARD11 have
been reported in 69% and 31% of cAIHA, respectively [24], possibly helping its distinction from non-Hodgkin
lymphoma. Similar mutations have also been reported in Kabuki syndrome, a congenital disorder
characterized by malformations, immune-deficiency, and development of autoimmune diseases (discussed
later).
Various congenital conditions have been associated with AIHA development, among which thalassemia and

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sickle cell disease, and complex conditions such as Kabuki syndrome (Table 1). The latter is a rare genetic

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disorder characterized by specific facial features, intellectual disability, and both immunodeficiency and
autoimmunity. The disease is caused by mutations in the KDM6A or KMT2D genes, involved in tolerance

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and immune system maturation. Several case reports exist, and data from a registry of 177 individuals

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showed a prevalence of 13.6% of autoimmune diseases, higher in adults (25.6%), most frequently ITP
(7.3%) and AIHA (4.0%) [25,26]. Concerning hemoglobinopathies, about 6% of beta-thalassemia patients
develop AIHA, and up to 23% show a DAT positivity [28]. The risk seems associated with beta-thalassemia

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intermedia, family history of AIHA, AB blood group, prior alloimmunization, and splenectomy (HR of 6,
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95%CI of 2-18) [29]. Case reports of AIHA and sickle cell disease have also been reported, again linked to
prior alloimmunization [30,31]. Finally, a case of ES complicating hereditary spherocytosis was described in
an infant [30]. Interestingly, erythrocyte antibody-bound IgG were demonstrated in up to 61% of patients
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with hereditary spherocytosis by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method
able to magnify latent anti-erythrocyte autoimmunity [31].
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3. AIHA in the context of the immune dysregulation

Several immunodeficiencies and autoimmune diseases have been associated with AIHA (table 2). Along
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with the above described Kabuki syndrome, various case reports of common variable immunedeficiency
(CVID) [32], IgA deficiency [33], and autoimmune lymphoproliferative syndromes (ALPS) [34] developing
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AIHA have been described. Moreover, in a Turkish study, AIHA was one of the most common autoimmune
phenomena (prevalence 30%) together with inflammatory bowel disease (IBD) and ITP [35]. Likewise,
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autoimmunity is observed in about 70% of pediatric cases of ALPS, a disease characterized by a disrupted
programmed cell death that is crucial in the development of tolerance [34].
Regarding systemic autoimmune diseases, AIHA may develop either as an epiphenomenon of the
underlying disease, with mild chronic anemia and DAT positivity, or as the leading feature, with devastating
hemolysis and requirement of AIHA therapy. The greatest association has been observed with systemic
lupus erythematosus: in a retrospective study evaluating 336 children and 1830 adults, the frequency of
AIHA was significantly higher in children (14% versus 3%) and correlated with constitutional symptoms [36].
Several case reports exist for AIHA association with systemic sclerosis, scleroderma, and CREST syndrome

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 [37-39]. A higher risk of AIHA development is also described for patients with thyroid autoimmune
disorders, both Hashimoto thyroiditis [40, 41] and Graves’ disease [42, 43]. Furthermore, primary Sjögren
syndrome may present with or be complicated by AIHA [44] with a prevalence of 2.8% (16 out of 565
patients hospitalized from 2000 to 2013); disease activity did not necessarily concur to AIHA development
as it may present without overt sicca symptoms [45]. Finally, inflammatory bowel diseases and
autoimmune liver disorders display increased frequency of immune hemolysis. Specifically, in a multicentric
study, AIHA incidence was 4.1/100,000 patient-years, correlated with colonic involvement (13/14 patients

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with ulcerative colitis), and with a high frequency of extra-intestinal manifestations (37.5%) [46]. Regarding

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primary biliary cirrhosis and autoimmune hepatitis, AIHA prevalence was 1.4% in a cohort of 71 cases,
among whom 44% had autoimmune complications (many of them concomitant in the same patient) [47].

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Accordingly, the association of multiple autoimmune diseases in the same patient, including AIHA,

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autoimmune hepatitis-primary biliary cirrhosis and Hashimoto's thyroiditis, has been described [48].

4. Infections and drugs associated with AIHA

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Various infections have been associated with an increased incidence of AIHA (Table 3). The clearest
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association has been demonstrated for Parvovirus B19 and for hepatotropic virus infections. The former
was associated to DAT positive hemolysis in up to 20% of cases with symptomatic infection [49, 50].
Concerning hepatitis virus, most studies refer to HCV infection and its treatment, particularly with
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interferon, whilst only case reports are available for HAV and HBV [51]. In 2003 Ramos-Casals et al. [52]
described a large series of HCV-related AIHA not treated with interferon. The majority of them had
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crioagglutinins and a history of liver cirrhosis, and the mortality rate was as high as 56%. A further large
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population study among 120,908 US veterans with HCV infection showed a risk of AIHA increased 2.8 fold
(HR, 95% CI 1.8-4.2) that peak up to 11.6 fold (7-19.3) in cases treated with interferon [53]. Comparing
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these results with the risk of ITP (the same in treated and untreated patients), the Authors concluded that
the development of AIHA seems to be associated with HCV treatment. Concerning minor hepatotropic
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virus, AIHA occurs in up to 3% of patients with infectious mononucleosis, typically within 1–2 weeks of
onset [54]. The autoantibody is usually a cryoagglutinin and most cases are self-limiting. Along with virus-
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induced immunodeficiency, HIV infection has also been associated with the development of autoimmune
diseases, rarely AIHA. In a French series of 5,186 HIV infected patients, AIHA occurred in only 1 case out of
36 autoimmune complications [55]. Regarding bacteria, Mycoplasma pneumoniae infection may be
complicated by severe AIHA, mainly cold but even warm forms [56,57]. cAIHA has been also associated with
tuberculosis infection, alone or associated to Mycoplasma pnemoniae [58,59], and may heal after anti-
tuberculosis treatment. In a large UK registry study of patients hospitalized for autoimmune diseases, the
association of tuberculosis and AIHA was particularly high (relative risk = 5.1, 95% CI 3.4-7.4), so that the
Authors recommended TBC screening in patients with AIHA [60]. A number of case reports exist on AIHA

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 complicating acute brucellosis or undulant fever (the chronic phase of this systemic infection), warranting
this differential diagnosis, particularly in migrants from endemic regions [61]. Finally, PCH is almost
invariably preceded by an infection, including syphilis and virus, particularly in children [62,63].
Several drugs have been associated to anti-erythrocyte autoantibodies development [64], and have been
categorized according to the leading mechanism: 1) drug-dependent antibodies that activate an immune
response only while the drug is present and 2) drug-independent antibodies (Table 3). The first is the most
common and includes hapten-mediated antibodies, recognizing a mixed epitope composed of erythrocyte

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parts and drug non-covalently bound to RBC. The drug-membrane binding may be strong, as for penicillin-

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type AIHA with DAT positive for IgG, or weak, as for ceftriaxone, resulting in immune complexes formation
with a late onset AIHA and DAT positive for complement. Drug-independent antibodies induce an

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autoimmune response in the absence of the offending drug, including cladribine, methyldopa and

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fludarabine that may act via adsorption, immune dysregulation, or unknown mechanisms [65]. In particular,
fludarabine, the backbone of chronic lymphocytic leukemia (CLL) treatment for the past decades, has an
additional pathogenic mechanism, that is the induction of an imbalance between Th17 and T-regs [66,67].

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Consistently, the association of cyclophosphamide and rituximab, by potentiating immune-suppression and
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killing autoreactive T-cells markedly reduced AIHA incidence in this setting [68]. Concerning new small
molecules, including the Bruton’s tyrosine kinase inhibitor ibrutinib, the BCL-2 inhibitor venetoclax, and the
PI3K inhibitor idelalisib, few case reports of treatment-emergent AIHA have been published [69-71].
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Increased incidence of autoimmune complications (hepatitis, colitis and pneumonitis) has been reported
for idelalisib [72,73]. Conversely, resolution of secondary AIHA has been described in various studies of CLL
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patients treated with ibrutinib and venetoclax, possibly due to their ability to inhibit autoantibodies
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producing B-cells but even to act on T-cell homeostasis [70,71,74].

Finally, among external triggers for autoimmunity there is great attention for vaccinations, including onset
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of autoimmune diseases for new vaccines. Klein and colleagues reported an incidence ranging from 0.8 to
more than 50/100,0000 person-years, that was maximal for thyroiditis and minimal for AIHA, by analyzing
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electronic records of members of the Northern California Kaiser Permanente from 1998 to 2004 [75].
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5. Other conditions with increased risk of AIHA

As shown in Table 4, solid and hematologic cancers, and transplants may be associated with AIHA
development.
Regarding solid organ malignancies, the most frequently reported as associated with AIHA are ovarian
teratoma and thymoma [76]. Case reports and retrospective studies show that AIHA tends to resolve after
tumour resection, thymectomy, and/or chemotherapy [77-79]. In a large study evaluating 1,083 unselected
cancer patients, a higher incidence of autoimmune phenomena was observed in prostate and ovarian

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 carcinomas (9.01% and 5.6%). The most frequent were vasculitis, polyneuritis and AIHA, and mainly
occurred during disease progression [80].
Concerning hematological neoplasms, AIHA is frequently reported in patients with lymphoproliferative
disorders, particularly CLL and non Hodgkin lymphoma (NHL). CLL patients show the highest risk with up to
5-10% developing AIHA [81,82]. AIHA prevalence in NHL is of about 2-3% but increases in certain subtypes:
13–19% in angioimmunoblastic T-cell lymphoma [83] and 50% in marginal zone lymphoma, mainly AIHA but
also ITP [84]. AIHA onset may precede the diagnosis of a smoldering indolent lymphoma. As a matter of

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fact, cAIHA is associated with a clonal lymphoid infiltrate distinct from other NHL and without malignant

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behavior [85]. Similarly to what described for solid cancers, therapy of CLL/lymphoma usually induces AIHA
recovery [86]. Generally, it is suggested to start with AIHA specific treatment when it is the only clinical

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manifestation, reserving CLL/lymphoma treatment in case of R/R cases. Although very rare, 0.2% of

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patients with Hodgkin lymphoma may develop AIHA, mainly at the advanced stage, but even preceding
lymphoma diagnosis [87].
Considering more recent molecular studies, in CLL the presence of unmutated IGHV status, sterotyped

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IGHV frames, and unfavourable cytogenetics with chromosome 17p and/or 11q deletions represented a
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clear risk factor for the development of AIHA [88-90]. A more recent study in CLL-associated cytopenias
showed that stereotyped subsets were more frequent in ES (66%) and ITP (50%) than in AIHA (33%, which
is similar to the general CLL population) [91]. In addition, nine down-regulated miRNAs were identified as
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risk factor for AIHA development (miR-19a, miR-20a, miR-29c, miR-146b-5p, miR-186, miR-223, miR-324-
3p, miR-484 and miR-660), of whom two (i.e. miR-20a and miR-146b-5p) known to be involved in
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autoimmune phenomena [92]. Of note, a positive DAT without hemolysis is frequent in CLL [93] but even in
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myelodysplastic syndromes (MDS) [94]. In both conditions, as well as in primary myelofibrosis, the
presence of anti-erythrocyte autoantibodies by MS-DAT has been reported in about one third of patients
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[95,96], again with only a small number of patients developing overt AIHA. Finally, in the rare multicentric
Castelman disease AIHA is a reported complication, may revert after anti-IL6 therapy with tocilizumab, and
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should be taken into account in the differential diagnosis of anemia in this setting [97,98].
As regards transplant, AIHA occurs in 5-10% of patients following liver and/or intestinal transplantation [99]
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and 2.5% following pancreas transplantation [100]. A particular, well characterized picture is the passenger
lymphocyte syndrome that involves mainly group O donors, though few cases have been described in AB
recipients with non-AB donors. The risk of hemolysis is proportional to the burden of transplanted
lymphocytes - lowest in kidney (antibody in 17%, hemolysis in 9%), next higher in liver (antibody in 40%,
hemolysis in 29%), and highest in heart-lung transplants (antibody in 70%, hemolysis in 70%) [1,101]. Onset
is between 3 and 24 days post-transplant and hemolysis is generally transient, since the lymphocytes
transferred with the donor organ do not engraft.

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 Concerning hematopoietic stem cell transplant (HSCT), 2-4% of patients develop AIHA after a median of 3–
10 months. Autoantibodies are produced by the donor immune system against antigens on erythrocytes
produced by the graft itself. Mortality may be quite high and increases with infections [102]. Risk factors for
AIHA post HSCT are: use of unrelated donor and HLA-mismatch; occurrence of graft-versus-host disease
(GVHD); use of cord blood, age < 15 years, CMV reactivation, alemtuzumab use, and non-malignant
condition pre-HSCT. The passenger lymphocyte syndrome may occur also in this setting with the following
predictors: use of cyclosporine alone for GVHD prophylaxis, use of peripheral blood rather than bone

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marrow as source of the graft, use of reduced-intensity conditioning, use of a non-genotypically HLA-

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matched donor; and use of a female donor. Umbilical cord blood as source for stem cells appears
protective. Careful transfusion procedures are warranted in transplanted patients, particularly in

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mismatched cases [101-104].

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6. AIHA developing after novel anti-cancer drugs
Several novel biologic anti-cancer drugs are under development and enter the clinical practice. Generally,

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their therapeutic effect is based on enhancing host immune response against tumor cells. Immune
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checkpoint inhibitors are monoclonal antibodies directed against programmed death receptor 1 and its
ligand (PD1 and PDL1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), widely used in lung cancer,
malignant melanoma, and lymphomas. These drugs act via unblocking the immuno-tolerance induced by
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the tumor through PD1/PDL1 signalling. However, they may evoke severe autoimmune reactions, including
fulminant AIHA [105,106] (Table 4). A recent revision of the database of the Food and Drug Administration
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revealed a total of 68 cases: 43 after nivolumab, 13 with pembrolizumab, 7 with ipilimumab, and 5 with
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atezolizumab. All episodes were severe, and the risk appeared higher with PD-1 or PD-L1 targeting agents
(0.15%-0.25%) than with CTLA-4 inhibitors (0.06%). Mortality was as high as 17%, notwithstanding prompt
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aggressive management [107]. In another recent analysis of 14 patients who developed AIHA after
checkpoint inhibitors, median time to AIHA was 55 days (IQR 22-110 days). Compared to primary AIHA,
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these cases showed a higher proportion of DAT negativity (38%) and of severe anemia (median Hb 6.3 g/dL
(IQR, 6.1-8.0 g/dL). Finally, 50% of cases relapsed after first line and 14% became chronic [108].
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7. Disease-related risk factors

Along with the associated conditions that may predispose to AIHA development, there are also disease
related risk factors that may have an impact on outcome (Figure 2). Two recent large multicentre studies
highlighted the importance of the severity of anemia at onset to predict relapse and mortality in primary
AIHA [2,3]. More in detail, the risk of relapse increased significantly from 1.61 to 1.74 and 1.98, expressed
as hazard ratios, in patients with Hb 8.1-10, 6.1-8, <6 g/dL, respectively; relapse risk increased also
considering Hb level as a continuous variable (7% higher risk per each gram of reduction of Hb). Very severe

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 cases (Hb <6 g/dL) were characterized by a higher overall mortality (24% vs 18% of those with Hb >6 g/dL, p
= 0.04), which is still higher than that historically reported in literature.
Concerning thermal characteristics of the disease, cAIHA, mixed and atypical forms were at higher risk of
relapse and required multi-treatment, whereas wAIHA displayed higher and more durable rate of response
to steroids and rituximab. Complement activation seems to be an additional risk factor for R/R cases,
particularly to rituximab. The concomitant presence of ITP (ES) has been shown to negatively impact
prognosis. In particular, relapse rate was cumulatively higher if ES was associated with Hb <8 g/dL at onset,

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forms other than wAIHA (3-fold higher) or the presence of both (4-fold higher) [3]. Moreover, bone marrow

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features impact on disease severity: an analysis of 47 primary AIHA showed that reticular fibrosis,
dyserithropoiesis, and hypercellularity correlated with shorter relapse-free survival and lower response rate

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to immunosuppressive therapies [6]. Age, multi-treatment, comorbidities, and the occurrence of

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complications, may further complicate the clinical picture, management, and outcome of patients
[109,110]. ES, acute renal failure, and infections have been associated with increased mortality (HR 8, 95%
CI 2.5-26; HR 6.3, 1.4-29; HR 4.8, 1.5-15, respectively) [2,3]. A case series of 13 very severe R/R primary

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AIHA reported a mortality of 57%, despite intensive treatment, including transfusions, steroid boli,
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intravenous immunoglobulins, rituximab, recombinant EPO, and plasma-exchange [111]. These findings are
strengthened in a recent report, including 44 AIHA admitted to intensive care unit for severe anemia [112],
where mortality reached 30%. Thrombotic events, generally proportional to active hemolysis [113], occur in
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about 15-20% of patients. In particular, pulmonary embolism has been reported as fatal event in past series
[114], whereas thrombotic events do not impact survival in more recent studies on primary AIHA. Risk
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factors for thrombotic events are Hb levels <6 g/dL at onset, intravascular hemolysis, and previous
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splenectomy [3]. The presence of allo-antibodies, which occurs more often in AIHA (15-40%) than in
general population, also complicates the disease course, since can increase post-transfusional hemolysis if
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not detected, worsening the degree of anemia [115]. Finally, secondary AIHAs may also impact on disease
severity and prognosis. In lymphoproliferative disorders and systemic lupus erythematosus, AIHA needed
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more therapy lines [116]. Higher mortality was observed in AIHA secondary to lymphoma and cancer
[110,116].
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8. Diagnostic procedures
Given the several associated conditions and the intrinsic disease factors, an accurate diagnostic approach
to AIHA is fundamental for a comprehensive risk assessment. A thorough medical history may reveal signs
of acute hemolysis (usually symptomatic anemia, mainly intravascular, possibly accompanied by abdominal
pain and hemoglobinuria), versus chronic features (milder symptoms of anemia, jaundice, gallstones,
splenomegaly, and iron overload). Drug assumption is of great importance, and acrocyanosis or Raynaud
phenomena upon cold exposure are typical of cAIHA. Table 5 shows basic evaluation and tests

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 recommended in particular settings. Blood counts reveal anemia with a wide range of Hb levels.
Reticulocytes are usually elevated, but reticulocytopenia may be present in up to 20% of adults and 40% of
children, possibly due to autoimmunity against erythroblast precursors. Elevated indirect bilirubin is
particularly indicative of extravascular hemolysis, and increased LDH of more severe intravascular
erythrocyte destruction. Haptoglobin is the most sensitive hemolytic marker being persistently decreased
until full remission [117]. The DAT (or Coombs test) is the cornerstone of AIHA diagnosis and should be
performed with monospecific antisera (anti- IgG, -IgA, -IgM, anti-complement, -C), allowing the distinction

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between wAIHA due to an IgG autoantibody (DAT positive with IgG or IgG+C at low titer), and cAIHA due to

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an IgM antibody (DAT positive with anti-C and presence of high titer of cold agglutinins). This distinction is
fundamental, as warm and cold forms have different responses to therapy, in particular, steroids are poorly

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effective and splenectomy is contraindicated in cAIHA. Mixed forms show a DAT positive for IgG and C and

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the presence of high titer of cold agglutinins. The very rare PCH (due to an IgG that binds to patient RBCs in
the cold but causes severe intravascular hemolysis at 37°C) can be diagnosed with the Donath-Landsteiner
test [7,118]. Notably, complement positivity, as well as reticulocytopenia, characterize more severe cases

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and negatively impact on prognosis. It is worth reminding that the DAT may yield false negative results due
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to the presence of RBC-bound antibodies below the threshold of the test or to low affinity antibodies. In
these cases, excluded other common causes of hemolysis and persisting the clinical suspect of AIHA, it is
recommended to ask for second-level tests in a reference center. Of particular importance is the
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identification of atypical AIHA due to warm IgM, as these autoantibodies are potent activators of
complement, and often escape detection since may detach from the RBC during washing procedures. The
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DAT may appear negative of weakly anti-C positive, causing detrimental delay in diagnosis and therapy of
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these rare forms, usually severe and potentially lethal. Notwithstanding extensive evaluation, about 5-10%
of AIHAs may be DAT negative. In these cases the diagnosis is made after the exclusion of the many
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hemolytic disorders (congenital hemolytic anemias, paroxysmal nocturnal hemoglobinuria, thrombotic

microangiopathies, mechanical and toxic noxae) and on the basis of an ex adiuvantibus therapy with
C

steroids. Diagnosis is completed by the detection of autoantibodies in patient’s serum (indirect

agglutination test, IAT), using a test panel of standardized RBC. The IAT reveals also the presence of
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alloantibodies that have been reported in 1/3 of AIHA patients and may cause severe hemolytic reactions in
case of RBC transfusion. In complex cases, allo- and autoantibody may be distinguished by
immunoabsorbance techniques and extended RBC genotyping [7,118].
Bone marrow evaluation (morphology, cytometry, cytogenetics and biopsy) is increasingly performed in
recent years, and just advised in cAIHA at diagnosis and in wAIHA relapsed after or refractory to first line
therapy with steroids [7]. This recommendation is based on several considerations: 1) the awareness about
the importance of bone marrow erythroid hyperplasia and consequent compensation, 2) the increasing
number of complicated cases with borderline diagnosis of bone marrow failure, MDS or lymphoma, 3) the

Information Classification: General

 possibility to harness therapy based on the type of bone marrow lymphocyte infiltrate (T or B), or generally
on bone marrow characteristics (fibrosis, dyserythropoiesis), avoiding further detrimental
immunesuppression [6], and 4) the availability of targeted therapies (mostly in clinical trials at present).
Further recommended diagnostic workup includes the investigation to identify secondary forms, i.e, whole
body CT scan, serology for infections and autoimmune diseases in case of clinical suspect (Table 5). Of
particular importance is the test of anti-phospholipid antibodies (cardiolipin, beta-2 glycoprotein 1, and
lupus-like anticoagulant), that may add a risk factor to the known thrombotic diathesis of acute/severe

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AIHAs. These tests may also modify therapy, for example suggesting intravenous Ig administration in

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infective-associated cases, advising thromboprophylaxis in anti-phospholipid triple-positive subjects, or
avoiding splenectomy in AIHA secondary to systemic autoimmune diseases or primary immunodeficiencies.

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Of particular relevance is the determination of endogenous EPO levels particularly in R/R and heavily

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treated subjects, since these patients may benefit from recombinant EPO administration [119]. Finally,
molecular analysis and next generation sequencing would help to confirm associated conditions (congenital
anemias, lymphoproliferative disorders, MDS) as already described in previous paragraphs.

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9. Conclusion
AIHA is a heterogeneous disease depending on both disease-related risk factors and associated
predisposing conditions. Among the latter, genetic background, lymphoproliferative neoplasms,
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autoimmune diseases, and immunodeficiency play a pivotal role, underlying that immune dysregulation is a
shared pathogenic mechanism. In addition, various environmental triggers may contribute to elicit the
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autoimmune reaction by different means, mainly by molecular mimicry. The close association between
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immunodeficiency and autoimmunity may be further reinforced by the vicious circle between the two: the
first predisposes to chronic infection that may in turn trigger autoimmunity. Breakdown of immune
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tolerance is fundamental, as highlighted by AIHA following HSCT or new check point inhibitors, both able to
induce an “immunologic storm”. Among disease-associated risk factors, the severity of anemia at onset is
C

the most important and correlates with recurrence of relapses, refractoriness to therapy and fatal
outcome. This is not surprising since the degree of anemia reflects the concomitant presence of the several
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immune mechanisms involved in AIHA, i.e. highly pathogenic antibodies, complement activation, and
failure of bone marrow compensation. Finally, a precise diagnosis is of great importance to distinguish AIHA
types (warm and cold forms) and to appropriately define associated conditions, as therapy and prognosis
may be quite different.

10. Expert opinion

If primary AIHAs are highly heterogeneous, secondary forms are even more complex, since associated
conditions may complicate the clinical picture and management. The few disease-associated risk factors

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 identified in primary forms are particularly useful in the acute setting, to identify patients requiring an
aggressive treatment and a stringent monitoring. However, their value has not been assessed in secondary
forms that are increasingly diagnosed. In this view, new molecular tools will be of great importance, to
properly disclose even subclinical associated conditions, in order to choose the most appropriate therapy.
Currently, several target therapies are accessible or under development, and will soon be available in the
clinical practice. The challenge for the next future will be to establish the best choice, sequence or
association of these target therapies depending on the prevalent immunopathogenic mechanism of the

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disease. Moreover, treatment selection would be ideally guided by disease-related risk factors, and/or

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associated conditions. These factors would in fact suggest a more aggressive and early intervention in cases
with predictable worse prognosis. Other unmet needs deserving further investigation are prevention and

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management of AIHA- and therapy-related complications, particularly severe and life-threatening

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infections. In this view, immunosuppression should preferably target pathogenic mechanisms, ideally
preserving immunocompetence and bone marrow stemness. A risk-adapted approach, taking into account
detrimental factors such as age, comorbidity, marrow fibrosis/dyserythropoiesis, should be therefore

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pursued. Additionally, guidelines for anti-infectious prophylaxis in the different settings need to be defined.
AN
Other harmful complications are thrombotic events, which may characterize severe forms with
intravascular hemolysis. The lack of a specific “thrombotic risk score” for AIHA, makes it difficult to select
which type, timing and dose of prophylactic anticoagulation/antiaggregation would better fit each different
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patient and setting. Finally, there are several new drugs under development for AIHA, such as complement
inhibitors (like sutimlimab for cAIHA), B-cell targeting molecules (i.e. PI3K and BTK inhibitors), and
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proteasome inhibitors (bortezomib). Other drugs are directed at cellular immunity and cytokines, such as
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subcutaneous low-dose IL2, and monoclonal antibodies against the B cell-activating factor of the tumor
necrosis factor family (BAFF) (like belimumab). Additional drugs may act on the serine treonine kinase
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mTOR (mammalian target of rapamycin), part of T cell receptor (such as sirolimus). Targeting IgG-driven
extravascular hemolysis by inhibiting the spleen tyrosine kinase (Syk) is also an attractive approach in
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wAIHA (for example fostamatinib). Finally, inhibition of the neonatal crystallizable fragment receptor (FcRn)
is a new interesting approach: these drugs avoid protection of circulating IgG, including pathogenic
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autoantibodies, from catabolism and regulate innate and adaptive responses initiated by IgG immune
complexes. All these drugs will be available in the next future, providing new therapeutic options to
patients, but also complicating the clinical management of the disease. The identification of risk factors
related to the disease itself, to the type and degree of the immunologic dysregulation, and to the
associated conditions will be mandatory to design good clinical trials. The better selection/stratification of
patients' populations, would reduce clinical heterogeneity, allowing stronger evidence of efficacy in the
different settings. On the whole it is time to start a risk-adapted therapy also in AIHA.

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 Funding
This paper was not funded.

Declaration of interest
W Barcellini received consulting honoraria from Alexion, Incyte, Bioverativ, Momenta, and B Fattizzo
received consulting honoraria Momenta and Apellis. The authors have no other relevant affiliations or
financial involvement with any organization or entity with a financial interest in or financial conflict with the

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subject matter or materials discussed in the manuscript apart from those disclosed.

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Reviewer Disclosures

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Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

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Oncology Clinical Studies Group; NCRI CLL Working Group. The prognostic significance of a positive
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100. Elimelakh M, Dayton V, Park KS, et al. Red cell aplasia and autoimmune hemolytic anemia following
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101. Skeate R, Singh C, Cooley S, et al. Hemolytic anemia due to passenger lymphocyte syndrome in solid
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 102. González-Vicent M, Sanz J, Fuster JL, et al. Autoimmune hemolytic anemia (AIHA) following
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Okawa S, Kayatani H, Fujiwara K, et al. Pembrolizumab-induced Autoimmune Hemolytic Anemia
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106. Algaze SD, Park W, Harrington TJ, et al. Autoimmune haemolytic anaemia in a patient with
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107. Tanios GE, Doley PB, Munker R. Autoimmune hemolytic anemia associated with the use of immune
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* A recent and comprehensive series of AIHA secondary to immune checkpoint inhibitors

109. Barcellini W, Fattizzo B, Cortelezzi A. Autoimmune hemolytic anemia, autoimmune neutropenia and
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 111. Fattizzo B, Zaninoni A, Nesa F, et al. Lessons from very severe, refractory, and fatal primary
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113. Audia S, Bach B, Samson M, et al. Venous thromboembolic events during warm autoimmune

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114. Allgood JW, Chaplin H Jr. Idiopathic acquired autoimmune hemolytic anemia. A review of forty-

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117. Barcellini W, Fattizzo B. Clinical Applications of Hemolytic Markers in the Differential Diagnosis and
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119. Fattizzo B, Michel M, Languille L, et al. Predictors of Response to Erythropoietin in Autoimmune

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Hemolytic Anemia. Blood. 2019,134:3516.

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 Figure legends

Figure 1: genetic, immunologic and environmental factors involved in the pathogenesis of autoimmune
hemolytic anemia (AIHA). CLL: chronic lymphocytic leukemia, NHL: non-Hodgkin lymphoma, PD1/-L:
programmed death 1 and its ligand, CTLA-4: cytotoxic T-lymphocyte-associated protein 4.

Figure 2: Associated conditions predisposing to AIHA and disease related risk factors that may have an

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impact on outcome. Reference for hazard risks of AIHA relapse and AIHA-related death are: (2) Barcellini

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W, et al: Blood. 2014;124(19):2930-2936, and (3) Barcellini W, et al. Am J Hematol. 2018;93(9):E243-E246.

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Figure 1
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Figure 2
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 Table 1: genetic findings and congenital syndromes associated with increased risk of AIHA
development.

Risk factor Frequency Key findings Reference

GENETIC FINDINGS
HLA I and II Case series HLA-B8 and BW6 are strongly Abdel-Khalik, Br Med J
associated to wAIHA. 1980 [10]; Wang-
HLA-DQ6 is less frequent in Rodriguez, Transfusion
DAT-positive individuals 1996 [11]
IGHV and IGKV region >60% of Specific IGVH and IGKV regions Silberstein, Blood 1991

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cAIHA are related to anti- red blood [12]; Potter, J Immunol
cell autoantibodies 2002 [13]; Malecka,

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development Haematologica 2016
[15]
TCRG and TCRB 50% of Pathogenic T-cells are clonally Smirnova,

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tested AIHA restricted in AIHA Autoimmunity 2016
[16]
Cytokine polymorphisms 41% of AIHA AIHA shows higher frequency of D’Abronzo, Int J Lab

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TNF-α, LT-α, IL-10, IL-12, CTLA-4 tested LT-α (+252) AG phenotype Hematol 2012 [18]
CTLA-4 exon 1 73% of CTLA-4 signalling is defective in Pavkovic, Am J
tested AIHA AIHA, particularly in CLL cases Hematol 2003 [17]
Genes involved in PIDs 40% of Hadjadj, Blood 2019
Majority of paediatric ES
TNFRSF6, CTLA4, STAT3, PIK3CD, CBL,
ADAR1, LRBA, RAG1, and KRAS
paediatric
ES
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display somatic mutations
found in immune-deficiencies
[19]
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KMT2D and CARD11 69% and Autoreactive B-cells display Malecka, Br J Haematol wAIHA:
31% of somatic mutations favouring 2018 [24] warm
cAIHA proliferation autoimm
tested une
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hemolyti
CONGENITAL SYNDROMES c
Kabuki syndrome 4-5% AIHA and ITP are the most Margot, Genet Med. anemia,
frequent autoimmune 2019 [25]; Almécija, J cAIHA:
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complications of Kabuki Pediatr Hematol Oncol cold

autoimm
Syndrome 2019 [26]
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une
Hemoglobinopathies DAT positivity is frequent, but Xu, Pediatr Hematol hemolyti
Thalassemia 6% clinically overt AIHA is rarer and Oncol 2012 [27]; c
correlates with alloimunization, Khaled, Blood Cells Mol anemia,
prior splenectomy, beta- Dis 2019 [28] CTLA-4:
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thalassemia intermedia, cytotoxic

familiarity Inaba, Am J Hematol T-
Sickle cell disease Case reports 2005[38]; Chaplin, Arch lymphoc
Intern Med 1981 [29] yte-
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Congenital hemolytic anemias Case reports 61% of cases with hereditary Yoshida, J Hematol associate
spherocytosis had Oncol 2009 [30]; d protein
autoantibodies against Zaninoni, Transfusion 4, ES:
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erythrocytes 2015 [31] Evans

syndrom
e, ITP: immune thrombocytopenia, DAT: direct antiglobulin test, CLL: chronic lymphocytic leukemia, PID: primary
immunodeficiency.

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 Table 2: immunodeficiencies and autoimmune diseases displaying increased risk of AIHA
development.

AIHA: autoimmune haemolytic anemia, ES: Evans syndrome, ITP: immune thrombocytopenia, DAT: direct antiglobulin test, SLE:
Risk factor Frequency Key findings Reference
IMMUNEDEFICIENCIES
Autoimmune lymphoproliferative 70% of cases develop AIHA is the most Oliveira, Curr Opin Pediatr 2013 [34]
syndrome autoimmune frequent
phenomena autoimmune
Common variable immunodeficiency 2-5% complication Feuille, J Clin Immunol 2018 [32]

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IgA deficiency Moderate association together with ITP Odineal, Clin Rev Allergy Immunol
and ES 2019 [33]

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AUTOIMMUNE DISEASES
Systemic lupus erythematosus 3-14% AIHA is higher in Gormezano, Lupus 2017 [36]
pediatric than in

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adult patients with
SLE

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Systemic sclerosis Case reports AIHA is a rarely Katsumata, Mod Rheumatol 2006
association of [37]; Jordana, Med Clin 1990 [38];
systemic sclerosis, Rosenthal, JAMA 1971 [39]
scleroderma and
CREST syndrome
Autoimmune thyroiditis Case reports
U Both Hashimoto
tyroiditis and
Oh, Korean J Hematol 2011 [40];
Moore, Transfus Apher Sci 2017 [41];
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Graves’ disease may Hegazi, J Thyroid Res 2012 [43]
associate with AIHA
Sjogren Syndrome 2.8% Sjogren Syndrome is Qiao, Int J Rheum Dis 2016 [44];
often marked by Wen, Clin Rheumatol 2015 [45]
hematologic
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alterations, however
true DAT positive
AIHA is a rare entity
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Inflammatory bowel diseases 4.1/100,000 patient- Although rare, AIHA Uzzan, Dig Liver Dis 2017 [46]
years may complicate
IBDs, particularly
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ulcerative colitis
Autoimmune hepatitis/Primary biliary 1.4% autoimmune Efe, Eur J Gastroenterol Hepatol
cirrhosis phenomena 2012 [47]
complicate 30% of
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autoimmune
hepatitis, AIHA is
rare
systemic lupus erythematosus, CREST: calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia,
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IBD: inflammatory bowel disease

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 Table 3: environmental factors associated with AIHA occurrence.

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 Risk factor Frequency Key findings Reference
INFECTIONS
Parvovirus B19 20% and case Persistent ParvoB19 infection El Khoury, Blood 2018 [49]; Giovannetti,
reports correlates with AIHA Blood Transf 2013 [50]
development
Hepatitis C 0.7% - 11.4 per Chronic HCV infection Chiao, Arch Int Med 2009 [53]
100 000 correlates with AIHA
person-years development, significantly in
treated cases
Hepatitis A/B Case reports A sporadic association among Khawaja, J Pak Med Assoc 2011 [51]
AIHA and HAV and HBV
infections has been reported

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HIV 0.02% 1 AIHA out of 36 autoimmune Lebrun, AIDS 2017 [55]

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events in a French series of
5186 HIV infected pts
Mycoplasma spp Case reports Cold agglutinin disease but Atta, Am J Hematol 2017 [56]; Stein, JAMA
even warm AIHA may follow 2018 [57]

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atypical Mycoplasma
pneumonia

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Tubercolosis/Babesiosis Case reports Association between AIHA and Morell, Lupus 2006 [58]; Wu, Transfus Med
TBC is not infrequent and 2012 [59]
hospitalized patients with AIHA
are at higher risk for TBC
Brucellosis

Syphilis/Epstein-Barr virus/Respiratory
Case reports

Case reports U
AIHA may complicate the
course of chronic brucellosis
Syphilis, EBV, and RSV may
Eskazan, Intern Med 2014 [61]

Leibrandt, Case Rep Hematol 2018 [62]

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Syncytial Virus complicate with paroxysmal
cold hemoglobinuria
DRUGS
penicillins, cephalosporins, tetracycline, Case reports Hapten and drug absorption Garratty, Expert Opin Drug Saf 2012 [64];
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carbromal, hydrocortisone, oxaliplatin, and reviews mechanisms Arndt, Semin Hematol 2005 [65]
and tolbutamide

Stibophen, metformin, quinine, Immune/ternary complex

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quinidine, cephalosporins, amphotericin mechanisms

b, rifampicin, antazolinc, thiopental,
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tolmetin, probenecid, nomifensine,

cephalosporins, diclofenac and doxepin

cephalosporins, tolmetin, α- Autoantibody mechanism

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methyldopa, L-dopa, mefenamic acid,

teniposide, pentostatin, cladribine,
fludarabine, lenalidomide,
procainamide and diclofenac
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Cephalosporins, carboplatin, cisplatin Non-immunologic protein

and oxaliplatin
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mesantoin, phenacetin, insecticides, Unknown methods of AIHA

chlorpromazine, acetaminophen,
ibuprofen, thiazides, omeprazole,
carboplatin, nalidixic acid,
erythromycin, and streptomycin
Fludarabine
causation
21% Fludarabine induced AIHA, Weiss, J Clin Oncol 1998 [66]
possibly through T-cell
dysregulation may be avoided
by rituximab association.

BTKi 6% Ibrutinib was associated to low Hampel, Br J Haematol 2018 [74]

risk of AIHA development

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 PI3Ki Registrative Idelalisib has been linked to Lampson, Blood 2016 [72]; Furman, N Engl J
trials autoimmune complications but Med 2014 [73]
not to AIHA
VACCINES
Vaccines 0.8/100.000 AIHA was the rarest Klein, Vaccine 2010 [75]
person-years autoimmune complication in a
population study

AIHA: autoimmune hemolytic anemia, BTKi: Bruton tyrosine kinase inhibitor, PI3Ki: phosphoinositide 3-kinase inhibitor

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 Table 4: Other conditions with increased frequency of AIHA.

Risk factor Frequency Key findings Reference

CANCER
Solid cancers 1.29% with Thymoma, prostate and ovarian Nenova, Folia Med 2016 [80]
autoimmune carcinomas have the highest
phenomena association with autoimmunity

Thymoma 30% with Bernard, Autoimmun Rev 2016 [78]

autoimmune
phenomena

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 Ovarian/Prostate 6% / 9% Ugoeke, J Investig Med High Impact Case
Rep 2017 [79]
Others Case reports
Hematologic cancers
Chronic lymphocytic leukemia and NHL 5-20% Autoimmune cytopenias may Visco, Am J Hematol 2014 [82]; Crickx,
frequently complicate chronic Eur J Haematol 2019 [83]; Dasanu, Conn
lymphoproliferative disorders Med 2015 [84]; Lechner, Leuk
and usually correlate with Lymphoma 2010 [87]
advanced disease and high
biologic risk
Castleman disease 6% and case Tajima, Ann Hematol 2013 [97]
reports

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Myelodysplastic syndromes Case reports Sokol, J Clin Pathol 1989 [93]; Zaninoni,

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Myelofibrosis Transfusion 2016 [94]
TRANSPLANT
Solid organ 2,4% AIHA is the most common Li, Transfusion 2012 [98]

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immune cytopenia. Time to
AIHA varies depending on

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transplant: from 3 m (liver) to
74 m (heart).
10-15% AIHA onset correlates with: González-Vicent, Transfus Med Rev
unrelated donor, HLA- 2018 [101]; Kruizinga, Biol Blood Marrow
mismatch, GVHD, Cord blood Transplant 2018 [102]

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use, Age<15, CMV reactivation,
alemtuzumab use, non-
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malignant condition
Passenger lymphocyte syndrome 9-70% by The risk and degree of Skeate, Transfusion 2013 [100]
transplant hemolysis is in proportion to
type the mass of lymphocytes
transplanted: kidney<liver <
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heart-lung
NOVEL ANTICANCER DRUGS
Checkpoint inhibitors 0.15-0.25% AIHA occurred more frequently Okawa, Intern Med 2019 [104]; Tanios,
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with PD-1 or PD-L1 targeting Eur J Haematol 2019 [106]; Leaf, Am J

agents than with CTLA-4 Hematol 2019 [107]
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inhibitors

AIHA: autoimmune hemolytic anemia, NHL: non-Hodgkin lymphoma, GVHD: graft versus host disease, PD1/-L: programmed
death 1 and its ligand, CTLA-4: cytotoxic T-lymphocyte-associated protein 4.
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 Table 5: Diagnostic procedures in AIHA.
Basic evaluation
Blood counts
Hemolytic markers (reticulocytes, LDH, fractionated bilirubin,
haptoglobin)
DAT with monospecific antisera (anti-IgG, -C, -IgA, -IgM)
Cold agglutinin titer
Folic and B12 serum levels, iron status
Consider confounding alterations of LDH, bilirubin and
haptoglobin due to diseases other than AIHA
Consider false positive (intravenous Ig, Rh immune globulins, ATG,
paraproteins or elevated serum globulins) and false negative due
to low sensitivity of the test.
Consider positivity due to alloantibodies in recently transfused
patients, in delayed hemolytic transfusion reactions, and in the
hemolytic disease of the newborn
Essential to exclude deficiency

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Serum electrophoresis, renal and hepatic chemistry Important for the preliminary assessment of secondary forms

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Tests recommended in particular settings
Blood smear To exclude congenital membrane defects, thrombotic
microangiopathies

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Serology for Mycoplasma pneumoniae, EBV, CMV, hepatitis B To assess underlying infections, autoimmune diseases, and
and C, and HIV; Anti-nuclear, -DNA, -extractable nuclear immunodeficiencies in case of clinical suspect
antigens, -cardiolipin, -beta-2 antibodies, and lupus-like

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anticoagulant, IgG, IgA and IgM levels and lymphocyte
subpopulations
Bone marrow evaluation (morphology, cytometry, In cAIHA at diagnosis and in relapsed/refractory wAIHA
cytogenetics and biopsy) and whole body CT scan

ionic strength solutions (LISS) and cold washings, Donath-

Landsteiner biphasic hemolysin U
DAT with more sensitive tests (solid phase, microcolumn, low In case of strong clinical suspect of AIHA and a negative standard
DAT-tube
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Hemoglobinuria and hemosiderinuria Indicative of intravascular hemolysis
Endogenous levels of erythropoietin In case of persistent reticulocytopenia and in heavily treated cases
Molecular analysis To confirm associated conditions (congenital anemias,
lymphoproliferative disorders, myelodysplastic syndromes,
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immunodeficiencies)

AIHA: autoimmune hemolytic anemia, DAT: direct antiglobulin test, ATG: antithymocyte globulins, CT: computed tomography,
cAIHA: cold AIHA; wAIHA: warm AIHA.
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