Expert Review of Clinical Immunology

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Autoimmune hemolytic anemia: causes and

consequences

B Fattizzo & W Barcellini

To cite this article: B Fattizzo & W Barcellini (2022) Autoimmune hemolytic anemia:
causes and consequences, Expert Review of Clinical Immunology, 18:7, 731-745, DOI:
10.1080/1744666X.2022.2089115

To link to this article: https://doi.org/10.1080/1744666X.2022.2089115

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EXPERT REVIEW OF CLINICAL IMMUNOLOGY
2022, VOL. 18, NO. 7, 731–745
https://doi.org/10.1080/1744666X.2022.2089115

REVIEW

Autoimmune hemolytic anemia: causes and consequences

a,b a
B Fattizzo and W Barcellini
a
Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; bDepartment of Oncology and Hemato-oncology,
University of Milan, Milan, Italy

ABSTRACT ARTICLE HISTORY

Introduction: Autoimmune hemolytic anemia (AIHA) is classified according to the direct antiglobulin Received 29 January 2022
test (DAT) and thermal characteristics of the autoantibody into warm and cold forms, and in primary Accepted 09 June 2022
versus secondary depending on the presence of associated conditions. KEYWORDS
Areas covered: AIHA displays a multifactorial pathogenesis, including genetic (association with con­ Warm autoimmune
genital conditions and certain mutations), environmental (drugs, infections, including SARS-CoV-2, hemolytic anemia; cold
pollution, etc.), and miscellaneous factors (solid/hematologic neoplasms, systemic autoimmune dis­ agglutinin disease;
eases, etc.) contributing to tolerance breakdown. Several mechanisms, such as autoantibody produc­ rituximab; direct
tion, complement activation, monocyte/macrophage phagocytosis, and bone marrow compensation antiglobulin test;
are implicated in extra-/intravascular hemolysis. Treatment should be differentiated and sequenced complement system;
according to AIHA type (i.e. steroids followed by rituximab for warm, rituximab alone or in association monocyte/macrophage
system; thrombosis;
with bendamustine or fludarabine for cold forms). Several new drugs targeting B-cells/plasma cells,
infection
complement, and phagocytosis are in clinical trials. Finally, thrombosis and infections may complicate
disease course burdening quality of life and increasing mortality.
Expert opinion: Beyond warm and cold AIHA, a gray-zone still exists including mixed and DAT negative
forms representing an unmet need. AIHA management is rapidly changing through an increasing
knowledge of the pathogenic mechanisms, the refinement of diagnostic tools, and the development
of novel targeted and combination therapies.

1. Introduction erythrocytes at 0–4°C), but a huge gray zone of mixed and

Autoimmune hemolytic anemia (AIHA) is a rare disease with
an incidence of 0.8 to 3/100,000 people per year and is caused
by an autoimmune attack against erythrocyte antigens [1,2].
While accumulated knowledge exists about disease pathogen­
esis, the etiology of AIHA is still largely obscure and consid­
ered ‘multifactorial.’ Similar to other autoimmune conditions,
several risk factors for AIHA development have been described
including endogenous and exogenous noxae such as drugs,
infections, systemic autoimmune diseases, hematologic and
solid neoplasms, and congenital syndromes [3]. Additionally,
emotional stressors and environmental pollution may possibly
represent under-investigated risk factors for autoimmunity
that may deserve dedicated studies in the future. All these
elements may contribute to the derangement of immune
tolerance and favor the emergence of anti-red cell autoanti­
bodies with different abilities to cause clinically relevant
hemolysis. The severity of the latter, the underlying condition,
and the therapies used to treat AIHA may all lead to several
complications, including infections, thrombosis, and mortality,
but also result in an increasingly recognized burden for
patients’ quality of life. Recently, treatment of AIHA has been
more clearly defined for ‘warm’ (wAIHA, where antibodies are
mainly IgG with highest affinity to the antigen at 37°C) and
‘cold’ forms, where cold agglutinins are IgM binding to
overlapping conditions still exists and deserves separate con­
sideration from both a pathogenic and diagnostic point of
view [4]. Additionally, the advent of novel therapies targeting
specific pathogenic steps, further advocates for better disease
classification. In this review, we will address AIHA pathogen­
esis and treatment and try to shed light on the current and
future understanding of the ‘causes and consequences’ of
anti-red cell autoimmunity.
2. Pathogenic mechanisms
Autoimmunity can arise through several mechanisms, includ­
ing a cross-reaction between antigens of infectious agents and
self-molecules (named ‘molecular mimicry’), a drug-induced
modification of erythrocyte membrane by adsorption or bind­
ing of drugs, and the production of autoantibodies by the so-
called ‘forbidden clones’ during B lymphoproliferative syn­
dromes [1,5]. Additionally, the breakage of ‘self-tolerance’
may be favored by the presence of germinal mutations impair­
ing immune system maturation, such as those of congenital
immunodeficiencies, or by the continuous exposure of ery­
throcyte self-antigens as happens in patients with ineffective
erythropoiesis (i.e. congenital anemias and myelodysplastic
syndromes) [6]. Overall, AIHA pathogenesis is complex

CONTACT B Fattizzo bruno.fattizzo@unimi.it Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, via Francesco Sforza 35, Milan
20100, Italy
This article has been republished with minor changes. These changes do not impact the academic content of the article.
© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
732 B. FATTIZZO AND W. BARCELLINI
Article highlights
● AIHA is a rare and heterogeneous disease with an incidence of 0.8-3/
100,000 people year.
● AIHA recognizes several pathogenic mechanisms including autoanti­
body production, complement activation, monocyte/macrophage
phagocytosis, and bone marrow compensation.
● Standard therapies should be differentiated between warm AIHA
(steroids followed by rituximab) and cold AIHA (rituximab alone or
in association with bendamustine or fludarabine as the first line).
● Several new drugs targeting B-cells/plasma cells (B-cell receptor
inhibitors, anti-CD38 monoclonal antibodies), complement (anti-C1s,
C1q, C3 monoclonal antibodies), and phagocytosis (spleen tyrosine
kinase inhibitor and anti-neonatal Fc receptor monoclonal antibodies)
are under study.
● Several complications, including thrombosis and infections, may
worsen disease course burdening quality of life and increasing
mortality.
(Figure 1) and mainly involves antibody production by (a) the
B-cell compartment and through (b) the alteration of
T-lymphocytes homeostasis, and final erythrocyte destruction
through (c) the mononuclear phagocytic system and (d) the
activation of the complement cascade. Additionally, (e) the
efficacy of bone marrow compensatory response is a major
determinant of AIHA pathogenesis and severity [7].
(a) Anti-erythrocyte autoantibodies are mainly produced by
B lymphocytes at different stages of maturation.
Interestingly, warm IgG autoantibodies are mainly poly­
clonal and produced by self-reactive nonmalignant lym­
phocytes residing in bone marrow and spleen,
while IgM from CAD is more often monoclonal and
sustained by a CD20+ CD5-lymphoid bone marrow infil­
trate. This also happens in AIHA secondary to lympho­
proliferative disorders, such as chronic lymphocytic
leukemia (CLL), where autoantibodies are mainly poly­
clonal IgG (90%) produced by non-CLL lymphocytes and
rarely IgM directly released by the malignant cells [8].
Similar to this mechanism is polyclonal B-cell activation,
occurring in different conditions such as infections with
EBV, CMV, hepatotropic virus, and HIV.
(b) T-cell compartment is also involved through an imbal­
ance among T helper (Th)1, Th2, Th17, and regulatory
T-cells (Tregs), and the production of several cytokines
that favor immune tolerance breakage [5]. Results from
various studies are sometimes conflicting, highlighting
the heterogeneity of immune response in AIHA
patients, often tested at different disease stages
(onset, remission, and relapse) and under various thera­
pies. Specifically, Th2 response appears overactive, with
a consistent increase of interleukin (IL)-4, IL-6, and IL-10
in AIHA patients versus healthy controls [9,10], and
a decrease of Interferon (IFN)-γ, promoting humoral
autoimmunity. Additionally, IL-10 has anti-
inflammatory and regulatory properties and its dysre­
gulation likely contributes to loss of self-tolerance to
RBC autoantigens and autoantibody production.
Cellular immunity is also involved, with elevated activity
of cytotoxic CD8 + T lymphocytes, natural killer cells,
activated macrophages, and increased levels of IL-2 and
IL-12 that promote Th1 differentiation [7]. Transforming
growth factor (TGF)-β is another regulatory cytokine
whose levels were found to be elevated in AIHA. TGF-
β favors the differentiation of Th17, which produce IL-
17 that amplifies the pro-inflammatory and autoim­
mune responses and decreases T-regs [11]. Finally, folli­
cular T-helper cells, which contribute to generate
memory B cells and long-lived plasma cells, have also
been implied in autoantibodies production in AIHA [7].
(c) Hemolysis mediated by the monocytic/macrophage sys­
tem is typical of IgG wAIHA and mainly occurs extra-
vascularly in the spleen [12]. The mechanism includes
the recognition of the Fc fragment of IgG by cells of the
monocyte-macrophage system with consequent ery­
throcyte phagocytosis and antibody-dependent cellular
cytotoxicity (ADCC). Additionally, other immune cells
such as natural killer (NK) and neutrophils that express
receptors for the IgG Fc fragment may also mediate an
ADCC. Notably, the spleen is also a lymphatic organ able
to produce autoantibodies. The monocytic/macrophage
system is also involved in complement-mediated (C3b)
extravascular hemolysis, mainly occurring in lymphoid
organs and liver [5,6].
(d) Complement system is activated through the classical
pathway [13], mainly by pentameric IgM (mainly cold
reactive, rarely warm), but also by abundant monomeric
IgG, also depending on the subclasses (i.e. IgG3 more
than IgG1; IgG2 is a weak activator; no evidence for
IgG4). This results in the phagocytosis of C3b-coated
cells (extravascular hemolysis) or, to a lesser extent, in
the terminal complement activation, with formation of
the membrane attack complex and intravascular
hemolysis.
(e) Hematopoietic bone marrow compensates anemia by
increasing erythropoiesis as mirrored by the increased
number of reticulocytes (immature RBCs with nuclear
residue) in the peripheral blood. Reticulocytosis indi­
cates an efficient bone marrow compensation,
while reticulocytopenia, present in up to 20% of
adults and 40% of children, correlates with greater
clinical severity and slower recovery [14,15] and may
be due to autoimmunity against bone marrow pre­
cursors [7].
Finally, it should be noted that anti-RBC autoantibodies are
not necessarily pathogenic since they can be detected in
healthy individuals (including blood donors); the lack of
pathogenicity may be due to different Ig subclasses, avidity,
ability to fix complement, and thermal amplitude (i.e. cold
autoantibodies reacting at 4°C are less likely to induce clini­
cally significant hemolysis) [1,2].
3. Diagnosis and classification of the different types
of AIHA
The first step is a thorough medical history and physical
examination for signs and symptoms of acute (symptomatic
anemia possibly accompanied by abdominal pain and
 EXPERT REVIEW OF CLINICAL IMMUNOLOGY 733

Figure 1. Pathogenic mechanisms involved in autoimmune hemolytic anemia (AIHA). Several predisposing conditions are involved in AIHA development including genetic
background, presence of underlying immunodeficiencies, infections, solid and hematologic neoplasms. The main immune mechanisms responsible for tolerance breakdown are
molecular mimicry, emergence of forbidden clones, and polyclonal B-cell activation. Additionally, drugs and hematologic treatments, such as checkpoint inhibitors (CPI) and
hematopoietic stem cell transplant (HSCT) may induce immune dysregulation or generation of neoantigens. The resulting immunologic activation involves antigen presenting
cells (APC), T- and B-cells, and cytokines and leads to the production of autoantibodies of several classes. B-cells interact with T helper (Th) cells via CD40 and its ligands but also
present cognate antigens, acting as professional APC. This interaction also contributes to generate class-switched autoantibodies. Concerning cytokines, interleukin (IL)-4, IL-6,
and IL-10 levels are increased and interferon gamma (IFN-γ) decreased favoring Th2 response and boosting humoral autoimmunity; the IL-2 and IL-12 levels are also increased
contributing to Th1 differentiation and cellular autoimmunity. Transforming growth factor beta (TGF-β) is elevated favoring Th17 differentiation and production of IL-17 that
decrease T-regulatory (Treg) levels. Once produced, IgG autoantibodies may weakly activate the complement cascade, and mainly destroy erythrocytes via antibody dependent
cellular cytotoxicity and phagocytosis primarily in the spleen. IgM autoantibodies strongly activate complement system and cause erythrocyte destruction via C3b opsonization
and phagocytosis primarily in the liver. These mechanisms are both called extravascular hemolysis. IgM may lead to terminal complement activation until the formation of the
membrane attack complex (MAC) with consequent intravascular hemolysis.

hemoglobinuria) or chronic hemolysis (milder fatigue, jaun­
dice, gallstones, splenomegaly, peripheral cold-induced cya­
nosis, etc.); furthermore, recent infections, drug exposure,
chronic diseases, tumors, and transplant should be evaluated.
Laboratory workup shows anemia of various degrees with
elevated reticulocytes, increased indirect bilirubin and LDH
(more typical of intravascular hemolysis), and decreased hap­
toglobin [1,2,16]. The direct antiglobulin test (DAT) is the
mainstay of AIHA diagnosis and should be performed with
monospecific antisera (anti-IgG, -IgA, -IgM, anti-complement,
-C), allowing the classification of AIHA into wAIHA (DAT posi­
tive with IgG or IgG+C at low titer, <64), and cold forms (DAT
positive with anti-C only and presence of cold agglutinins
usually of high titer). The latter are further classified in cold
agglutinin diseases (CAD), where monoclonal IgM is sustained
by a clonal lymphoproliferative bone marrow disease similar
to low-grade non-Hodgkin lymphoma, termed ‘CA-associated
lymphoproliferative bone marrow disorder’ [17–19], and cold
734 B. FATTIZZO AND W. BARCELLINI
agglutinin syndrome (CAS), occurring secondary to infections,
such as Mycoplasma pneumoniae, EBV, CMV, and COVID-19, or
malignancies (aggressive B-cell lymphoma). Rarer forms
include mixed AIHA [20], where DAT is positive for IgG and
C and cold agglutinins with high thermal amplitude are
detected. Additionally, some Authors suggest that the titer
of cold agglutinins, along with positive autoagglutination at
20°C, may be helpful to distinguish mixed AIHA from IgG+C
wAIHA [2,4]. The very rare paroxysmal cold hemoglobinuria
(PCH) should also be considered, the latter is sustained by
a biphasic IgG hemolysin called Donath-Landsteiner antibody
[4] that binds to erythrocytes at low temperatures but acti­
vates complement at 37°C resulting in intravascular hemolysis.
The disease is nowadays mainly temporary, self-limiting, and
occurring in children after viral infections [4]. Other atypical
cases include warm IgA driven AIHA, the very severe and
potentially fatal warm IgM cases, characterized by huge intra­
vascular hemolysis due to massive complement activation at
body temperature, and DAT negative AIHA [1,21]. DAT may be
falsely negative due to a low number of erythrocyte-bound
autoantibodies or to low-affinity ones. In these cases, more
sensitive second-level tests may be performed in reference
centers. However, DAT remains negative in up to 10% of
AIHAs and the diagnosis is based on the exclusion of other
hemolytic conditions. Specifically, other acquired forms should
be ruled out, including mechanical and toxic noxae (i.e. ana­
mnesis for intravascular devices, prosthetic valves, angiomas,
radiation, and drugs), paroxysmal nocturnal hemoglobinuria
(high-resolution cytofluorimetry), thrombotic microangiopa­
thies (platelets values, coagulation parameters, blood smear
for schistocytes), and infections (i.e. malaria); subsequently,
congenital hemolytic anemias (i.e. RBC membrane and
enzyme defects) should be considered through the evaluation
of family history, blood smear, osmotic fragility tests, and
enzyme activities [3,7]. Thereafter, response to steroid therapy
allows the ex juvantibus diagnosis of DAT negative AIHA.
In all patients, the clinical evaluation is completed by the
investigation of underlying alloantibodies in patient’s serum
(indirect agglutinin test, IAT), reported in about one-third of
AIHA patients [22]. The latter may cause severe hemolytic
reactions in the case of transfusion. In complex cases, allo-
and autoantibody may be distinguished by immune-
absorbance techniques and extended RBC genotyping [3].
Additionally, autoantibodies may be eluted from the washed
patient’ RBCs to determine the class, specificity, titer, and
thermal range. As regards specificity, in wAIHA autoantibodies
are mainly directed against RBC membrane proteins including
Band 3, glycophorin A, and Rh system, while in CAD the
polysaccharide regions of glycoproteins, such as I/i or Pr anti­
gen, are the most common target.
Bone marrow evaluation is useful to evaluate compensa­
tory erythroid hyperplasia, features suggestive of underlying
bone marrow failure or lymphoma, and the presence and type
of lymphoid infiltrate that may be differently targeted with
available therapy. It is advised in CAD at diagnosis, in
relapsed/refractory cases of wAIHA, and in cases of suspected
underlying hematologic diseases [2,4]. Similarly, whole-body
CT scan, serology for infections and autoimmune diseases, and
testing of anti-phospholipid antibodies are recommended
upon specific clinical suspects. Finally, the determination of
endogenous erythropoietin (EPO) levels is suggested in
patients with inadequate reticulocytosis, particularly if relap­
sing and heavily treated [7].
4. Conditions associated with AIHA development
and secondary forms
Slightly more than 50% of AIHA cases are secondary to asso­
ciated conditions, i.e. chronic lymphocytic leukemia, systemic
lupus erythematosus (SLE), common variable immune deficiency,
or other immunologic or lymphoproliferative disorders [8,23,24].
Furthermore, several endogenous and exogenous noxae may
increase the risk of autoimmunity as detailed thereafter.
4.1. Endogenous noxae associated with AIHA
development
4.1.1. Genetics and congenital conditions associated with
AIHA
Several studies highlighted the association of AIHA with altera­
tions of genes involved in antigen recognition, T- and B-cell
maturation and proliferation, and cytokine homeostasis. The
formers include specific HLA loci (HLA-B8, BW6, DR, and DQ)
[25,26] and stereotyped rearrangements of genes encoding the
variable region of the immunoglobulin heavy and light chains
(IGHV4-34, IGHV3, and IGKV3-20) [27–30]. Additionally, the
G polymorphism of cytotoxic T-lymphocyte-associated protein
4 (CTLA-4, a negative regulator of T-cell responses) and the AG
configuration of lymphotoxin-α (LT-α) gene showed a higher
frequency among patients with AIHA [31,32]. A stronger genetic
predisposition was recently demonstrated in pediatric patients
with Evans syndrome, where AIHA occurs concomitantly with
autoimmune thrombocytopenia and/or neutropenia. By next-
generation sequencing, a genetic lesion was detected in 65%
of cases, mainly involving genes related to primary immunode­
ficiencies (TNFRSF6, CTLA4, STAT3, PIK3CD, CBL, ADAR1, LRBA,
RAG1, and KRAS). These pathogenic hints have prognostic and
therapeutic importance, since mutated patients diagnosed with
primary immunodeficiencies are more prone to catastrophic
infections after immunosuppression [33] and may be candidates
for new biological drugs used in these conditions. Mutations
typical of primary immunodeficiencies include those of KMT2D
and CARD11, which have been described in 69% and 31% of CAD
patients, respectively [34], and are the hallmark of Kabuki syn­
drome. The latter is a congenital disorder characterized by mal­
formations, immune-deficiency, and autoimmune phenomena,
including AIHA in 4% of cases. Other primary immunod
eficiencies include common variable immunodeficiency (CVID)
[35], IgA deficiency [36], and autoimmune lymphoproliferative
syndromes (ALPS) [37]. Finally, AIHA may develop in patients
with congenital anemia, including beta-thalassemia (up to 6%),
sickle cell disease, and hereditary spherocytosis, where erythro­
poiesis is overstimulated and largely ineffective [38]. With more
sensitive methods, such as mitogen-stimulated DAT (MS-DAT),
anti-erythrocyte autoantibodies were demonstrated in up to
61% of patients with hereditary spherocytosis, although with
doubtful pathogenic significance [39].

4.1.2. AIHA in the context of connective tissue diseases
and other autoimmune conditions
AIHA belongs to the criteria for the diagnosis of SLE and may
represent a mild manifestation of the disease or be its leading
feature, with overt hemolysis and treatment requirements. The
frequency of AIHA was about 14% in children and 3% in adults
with SLE, again underlining the likely contribution of a more
immature immune system in the former [40]. Other associated
autoimmune diseases include inflammatory bowel diseases
(complicated by AIHA in about 4% of cases), Sjogren's syn­
drome (in about 2.8% of cases), autoimmune liver disorders (in
1.4% of cases), and case reports of systemic sclerosis, sclero­
derma, CREST syndrome, and thyroid autoimmune disorders.
Although such associations do not necessarily imply
a causative relationship, AIHA severity is correlated with the
degree of organ involvement and disease activity of the
underlying autoimmune condition [3].
4.1.3. AIHA associated with neoplasms
Concerning solid neoplasms, AIHA is highly associated with
ovarian teratoma and thymoma (up to 9% of cases), mainly
occurs during disease progression, and tends to ameliorate
after tumor resection, thymectomy, and/or chemotherapy [41].
An association has also been reported with carcinomas [42].
Very recently, a Japanese group described ectopic band 3
expression and erythrocyte membrane-bound IgG (by flow cyto­
metry) in 97% of 50 colorectal cancer patients, with a DAT
positivity in 10 of them. The presence of anti-band three auto­
antibodies was confirmed in patients’ sera and mouse experi­
ments suggested that ectopic band 3 expression occurs in
tumor cells under hypoxic conditions [43]. Regarding hematolo­
gical diseases, AIHA is frequently associated with lymphoproli­
ferative disorders, such as CLL (5–10% of cases), non-Hodgkin
lymphoma (NHL, 2–3%), and rarely HL (0.2%), which may pre­
cede or follow the hemolytic disease [3,8]. AIHA prevalence
increases in specific NHL subtypes, being maximal in angioim­
munoblastic T-cell lymphoma [44] and in marginal zone lym­
phoma [45]. Notably, CAD is associated with the above-
mentioned CAD-associated lymphoproliferative disease that
may be difficult to distinguish from other NHLs but usually
presents with a 10–15% isolated bone marrow infiltrate [18].
Similar to solid cancers, CLL/NHL treatment usually induces
AIHA recovery. As described for congenital anemias, the positiv­
ity of anti-erythrocyte autoantibodies by MS-DAT is frequent in
CLL [46], but even in myelodysplastic syndromes, and in primary
myelofibrosis [47,48], again with only a few cases developing
clinical hemolysis. Finally, AIHA may rarely complicate multi­
centric Castleman disease and large granular lymphocyte (LGL)
lymphoproliferative disorders and should be included in the
differential diagnosis of anemia in this setting [49].
4.2. Exogenous noxae associated with AIHA
development
4.2.1. Infections associated with AIHA and the role of
microbiome
AIHA may follow several infections, particularly Parvovirus B19,
in up to 20% of cases, hepatotropic virus infections, including
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 735
HCV, HBV, and HAV, and more rarely HIV [3]. In particular, HCV-
related AIHA was mainly sustained by cold agglutinins (CAS)
and had a mortality rate as high as 56% in a large study [50].
The risk of AIHA in HCV patients consistently increased from
2.8- to 11.6-fold in cases treated with interferon [51].
Additionally, AIHA may develop in up to 3% of patients with
infectious mononucleosis, within 1–2 weeks of onset, and
more frequently as a CAS [3]. Regarding bacteria, CAS may
follow Mycoplasma pneumoniae infection [52], as well as
tuberculosis, and usually recovers after anti-tuberculosis treat­
ment [53]. More rare associations include AIHA complicating
brucellosis or undulant fever, and the above mentioned PCH
classically preceded by syphilis and virus infections [3].
Finally, alterations of the microbiome (i.e. the ensemble of
microorganisms colonizing various districts of the body) are
emergingly described through next-generation sequencing
(NGS) techniques and have been associated with the develop­
ment of autoimmune diseases, including autoimmune cytope­
nias [54]. A disrupted microbiome composition may lead to
autoimmunity through molecular mimicry, chronic inflamma­
tion, alteration of epithelial barriers, and dissequestration of
self-antigens. On the other hand, the microbiome perturba­
tions may also be an effect of the autoimmune disease and its
treatment. For instance, in immune thrombocytopenia and
chronic idiopathic neutropenia, a peculiar microbiota compo­
sition has been identified, possibly predictive of response to
therapy, and future studies will possibly clarify its role in other
conditions including AIHA.
4.2.2. Drugs associated with AIHA
Drug-induced AIHAs [1,23,55–57] are generally categorized
according to the pathogenic mechanism in forms due to (1)
drug-dependent antibodies that activate an immune response
only while the drug is present and (2) drug-independent anti­
bodies. The first encompasses hapten-mediated antibodies,
recognizing a mixed epitope composed of erythrocyte parts
and drug non-covalently bound to RBC, and includes penicillin
(DAT positive for IgG only) and ceftriaxone (DAT positive for
IgG, IgG + C, or C only). Drug-independent antibodies induce
AIHA via adsorption, immune dysregulation, or unknown
mechanisms and may occur after cladribine, methyldopa,
and fludarabine [2,3]. The latter, a purine analogue used in
the CLL treatment, further induces an imbalance between
Th17 and T-regs [7], that is restored by the association of
cyclophosphamide and rituximab that kill autoreactive T-cells
and markedly reduce AIHA [58]. Concerning novel oral B-cell
receptor inhibitors, increased incidence of autoimmune hepa­
titis, colitis, and pneumonitis has been reported after the PI3K
inhibitor idelalisib, and a single case report of AIHA post- the
BCL-2 inhibitor venetoclax has been published. Contrarily,
Bruton’s tyrosine kinase inhibitor ibrutinib seems safe and
favored the resolution of primary and secondary AIHAs, pos­
sibly through the inhibition of autoantibodies producing
B-cells and restoration of T-cell homeostasis [59–61]. Finally,
novel immune-activating anti-cancer drugs whose therapeutic
effect is based on enhancing host immune response against
tumor cells may favor AIHA development. Immune checkpoint
inhibitors (CPIs) that reactivate T-cell mediated
736 B. FATTIZZO AND W. BARCELLINI
immunosurveillance are the main example. They inhibit pro­
grammed death receptor 1 and its ligand (PD1 and PDL1) or
CTLA-4 and may evoke severe autoimmune reactions, includ­
ing fulminant AIHA [62,63]. Recent studies reported a total of
82 cases mainly occurring after nivolumab, followed by pem­
brolizumab, ipilimumab, and atezolizumab. All cases displayed
severe hemolysis, with transfusion requirement (80%), high
prevalence of DAT negativity (38%), relapse in about 50% of
patients, and mortality as high as 17% [62,63]. A last mention
deserves the occurrence of cytopenias in about 30% of
patients with acute lymphoblastic leukemia and NHL treated
with chimeric antigen T-cell infusions; these cases may be life-
threatening and steroid-refractory and are likely to be immune
mediated [64].
4.2.3. Transplant associated AIHA
AIHA complicates 5–10% of liver and/or intestinal transplanta­
tion and 2.5% of pancreas transplants [2]. In this context, it is
worth mentioning the passenger lymphocyte syndrome that is
sustained by immune competent lymphocytes transferred
with the donor organ [65]. Onset is between 3- and 24-days
post-transplant, the risk is proportional to the burden of
transplanted lymphocytes, ranging from 9 to 70% of cases
(kidney < liver < heart-lung transplants) [66], and hemolysis
is generally transient. Regarding hematopoietic stem cell
transplant (HSCT), 2–4% of patients may experience AIHA
after 3–10 months, and mortality increases with infections
[67,68]. Some risk factors for AIHA developing post HSCT
include unrelated donor, HLA-mismatch, graft-versus-host dis­
ease (GVHD), cord blood as HSC source, age <15 years, CMV
reactivation, alemtuzumab use, and nonmalignant diagnosis
pre-HSCT. The passenger lymphocyte syndrome may also
occur after HSCT and has been linked to HLA mismatches,
use of peripheral blood as stem cell source, cyclosporine
alone for GVHD prophylaxis, reduced-intensity conditioning,
and female donors, while cord blood use appears protective.
Careful transfusion procedures are warranted in transplanted
patients, particularly in mismatched cases [69].
4.2.4. Possible effect of emotional stress on the
development of autoimmunity
Several hematologic patients, including those with autoim­
mune conditions, report that their diagnosis had been pre­
ceded by a variety of traumas/stressors either physical
(accidents, infections, etc.) or psychological (bereavement, dis­
missal, divorce, etc.). Their contribution to disease develop­
ment is difficult to establish, but it has been speculated that
they may interact with the immune system by altering the
adrenergic/glucocorticoid axis thus interfering with disease
course [70]. Some evidence come from pediatric studies eval­
uating the long-term effects of childhood traumatic stress,
including increased prevalence of altered inflammatory mar­
kers and higher risk of ischemic heart disease [71,72]. A recent
analysis of 15,357 adults enrolled in the Adverse Childhood
Experiences (ACEs) Study from 1995 to 1997 in California, and
followed-up until 2005, evaluated the effect of physical, emo­
tional, or sexual abuse on the frequency of hospitalizations for
autoimmune diseases. The Authors found that 64% of patients
reported at least one ACE and that the occurrence of 2 or
more events conferred a 70% increased risk for hospitalization
for autoimmune diseases [70]. Although only nine AIHAs were
reported in this study, this remains an open field for further
investigation.
4.2.5. Possible association of pollution with autoimmunity
Several epidemiology studies linked air pollution (a heteroge­
neous mixture including carbon monoxide, nitrates, sulfur
dioxide, ozone, lead, toxic by-product of tobacco smoke and
particulate matter) to the occurrence of autoimmune diseases
through various mechanisms including systemic inflammation,
oxidative stress, epigenetic modifications, and chronic airway
damage [73]. In this view, air pollutants have been shown to
bind to the aryl hydrocarbon receptor (AHR) in the lung to
regulate Th17 and Tregs balance. Furthermore, pollutants trig­
ger inducible bronchus associated with lymphoid tissue,
resulting in the production of pro-inflammatory cytokines.
The latter stimulates B- and dendritic cells thus favoring the
production of antibodies and self-reactive T lymphocytes.
Clinical examples are the effect of smoking and occupational
exposure to silica on rheumatoid arthritis and SLE develop­
ment, and the association of inhalation of chemical solvents,
herbicides, and silica with scleroderma and ANCA associated
vasculitis [74]. Although a direct effect on AIHA development
has not been reported yet, air pollutants have been recently
shown to affect erythropoiesis, and particulate matter (PM2.5)
and nitrogen dioxide (NO2) levels have been associated with
lower hemoglobin in older Americans and were also related to
higher C-reactive protein levels as markers of systemic inflam­
mation [75].
5. Standard therapy
Based on the above-mentioned differences in physiopathol­
ogy, the treatment of warm and cold AIHA forms differs in the
choice of the type and sequence of immunosuppressive
agents [1; 2,4,6]. In both conditions, the utility of supportive
measures, including transfusions, nutrient supplementation,
erythropoiesis stimulating agents, and anticoagulant and anti-
infective prophylaxis should be considered. Finally, secondary
forms may necessitate treatment for the underlying disease,
such as chemo-immunotherapy for lymphomas and solid
tumors, intravenous immunoglobulin (IVIG) in primary immu­
nodeficiencies, cytotoxic immunosuppressants in systemic
autoimmune diseases (Table 1).
5.1. Primary warm-AIHA
Predniso(lo)ne 60–100 mg or 1 mg/kg body weight per day for
2–3 weeks is the recommended first-line therapy, followed by
a slow tapering in 3–6 months. About 80% respond initially,
but only 30–40% experience sustained remission after 1 year
[76]. Rituximab addition in the first line, which prolonged
response duration in two prospective studies [77], should be
considered in severe settings (i.e. Hb<8 g/dL, presence of
atypical AIHA or Evans syndrome). In steroid refractory
patients, second-line rituximab is the preferred choice, after
re-assessment for secondary AIHA forms and bone marrow
evaluation, with response rates of 70–80% in about 3–
 EXPERT REVIEW OF CLINICAL IMMUNOLOGY 737

Table 1. Standard therapies for warm AIHA (wAIHA) and cold agglutinin disease (CAD).
Response
Treatment Route and dose rates % Comments
wAIHA
1st line
Steroids Oral or IVPrednisone or 75–80 Curative in 20–30% only; short-term side effects (mood swings, psychosis); long-term side
methylprednisolone 1 mg/Kg/ effects (diabetes, hypertension, infections, osteoporosis, cushingoid syndrome)
day
2nd line
Rituximab IV 375 mg/m2 or 100 mg/week 80–90 Effective even at low-dose; re-treatment equally effective; well-established safety profile
x 4 weeks
Further lines
Splenectomy / 80 Potentially curative; unsuitable for elderly; surgical complications; thrombotic risk; life-long
immune suppression; infection prophylaxis required
Azathioprine Oral 50–100 mg/day 40–60 Steroid-sparing agent; myelosuppression; infections; secondary malignancy; hepatic
toxicity
Cyclophospamide Oral 50 mg day 40–60 Steroid-sparing; myelosuppression; infections; urotoxicity; secondary malignancy; fertility
problems/teratogenic
Cyclosporin A Oral 3–5 mg/Kg/day 40–60 Steroid-sparing; possible dose adjustment; hypertension; arrhythmia; myelosuppression;
infections; nephrotoxicity
Mycophenolate Oral 1–1.5 g/day 80–100 Steroid-sparing; particularly effective in children; good safety profile; mild
Mofetil myelosuppression
Danazol Oral 200–600 mg/day 40 Steroid-sparing agent; low effectiveness in refractory cases; long-term hepatotoxicity
High dose IV boluses 800 mg/m2 70 Response in small series; high toxicity (myelosuppression; infections; urotoxicity; secondary
cyclophosphamide malignancy; infertility)
Additional
therapies
Intravenous IV 0.4 g/Kg x 5 days or 1 g/Kg 40 Effective in AIHA secondary to infections; particularly effective in pediatric settings (60%);
immunoglobulin x 2 days low toxicity
Recombinant Subcutaneous 40,000 IU/week 50–70 Effective in multi-refractory AIHA particularly with reticulocytopenia; useful in very severe
erythropoietin presentation
CAD
Watch and wait - - keeping patient and room warm and transfusion of pre-warmed to 37°C blood
First line
Steroids Oral or IV prednisone or 15–30 Effective only at high dose; short-term side effects (mood swings, psychosis); long-term
methylprednisolone 1 mg/Kg/ side effects (diabetes, hypertension, infections, osteoporosis, cushingoid syndrome)
day
Rituximab IV 375 mg/m2/week x 4 weeks 50 Re-treatment equally effective; well-established safety profile
Further lines
Rituximab/ Oral 40 mg/m2 days 1–5, 29–34, 75 Sustained remissions (median response duration 6.5 years); hematologic and infectious
fludarabine 57–61 and 85–89 toxicity mostly in older and frail patients
Rituximab/ IV 90 mg/m2 days 1–2 for 4 70 Sustained remissions; infectious complications
Bendamustine cycles of 28 days
Bortezomib IV or subctuaneous 1.3 mg/m2 30 Side effects (neuropathy, neutropenia, thrombocytopenia, diarrhea, fatigue and rash)
days 1,4, 8,11
Additional
therapies
Recombinant Subcutaneous 40,000 IU/week 50–70 Effective in multi-refractory cases particularly with reticulocytopenia
erythropoietin

6 weeks. The conventional dose is 375 mg/m2 weekly for 4
weeks, but a fixed low dose of 100 mg weekly for 4 weeks
yielded equal efficacy [78]. Splenectomy is currently recom­
mended in patients unresponsive to/relapsing after rituximab,
with responses of about 70%. The risk of severe infection and
thrombosis should be taken into account and prevented (see
the dedicated section) [79]. Further options include azathiopr­
ine, cyclophosphamide, cyclosporine, mycophenolate mofetil,
or bortezomib, basing mostly on small retrospective series and
case reports. For emergencies, high-dose intravenous methyl­
prednisolone and IVIG may be appropriate, and some reports
or retrospective series of plasma exchange, emergency sple­
nectomy, or partial splenic embolization have been published.
Finally, limited evidence regards the use of bortezomib plus
dexamethasone, high-dose cyclophosphamide, or stem cell
transplantation in ultra-refractory cases [2].
5.2. Cold agglutinin disease
In a proportion of patients with mild anemia or compensated
hemolysis without circulatory symptoms and fatigue, non-
pharmacologic management with thermal protection is
advised [2,4,80]. In symptomatic cases, glucocorticoids are
effective only at unacceptably high doses, so that rituximab
has become the preferred first-line therapy with response
rates of 45–60%, mainly partial. A majority of responders
relapse within 12–15 months, and repeated rituximab courses
have a fair chance to succeed. The addition of fludarabine
738 B. FATTIZZO AND W. BARCELLINI
has yielded higher responses, but with more toxicity,
while the association with bendamustine seems safer and
even more effective. The overall response exceeded 70%
and deepened overtime, with an estimated median response
duration of >88 months [81]. For refractory patients, borte­
zomib monotherapy (1 cycle of 1.3 mg/sm iv for total 4
biweekly doses) may be considered, with responses in 32%
in a small prospective study [82]. Finally, a recent case series
of 10 CAD patients (primary or secondary) treated with ibru­
tinib, described responses in all of them, suggesting the need
of future investigation [83]. Complement inhibitors represent
a promising approach that will be addressed in the following
paragraph.
5.3. Other AIHAs
In mixed AIHA cases [20], which are generally more severe and
refractory/relapsing, clinical-laboratory features (monospecific
DAT, cold agglutinin titers, autoagglutination, presence of cold
agglutinin symptoms) should be evaluated at each relapse to
assess the prevailing form (warm versus cold). Glucocorticoids
should be given generously, and rituximab considered as an
early second line, particularly in the presence of CAD features,
while splenectomy is discouraged [4].
The very rare PCH is usually self-remitting and may be
handled with steroids. Recently, the efficacy of a single dose
of the complement C5 inhibitor eculizumab in a child was
reported [84]. Treatment for drug-induced hemolytic anemia
mainly consists of discontinuation of suspected medications,
and transfusions and steroids were required in severe cases
such as those occurring post-CPIs. For the latter, early ritux­
imab may be required given the high rate of steroid-refractory
cases.
5.4. Supportive measures
Transfusions must not be denied in cases of life-threatening
anemia (i.e. Hb<6 g/dL or conditions of increased O2
requests), but over-transfusion should be avoided. If time
permits, an extended phenotyping should be performed
with respect to Rh subgroups, Kell, MNS, Kidd, S/s, and Duffy
antigens to avoid alloimmunization that may occur in up to
one-third of cases [2,7,22]. Genotyping can be considered but
will often be too time-consuming. In CAD, compatible erythro­
cytes by crossmatching at 37°C are usually available and may
be safely transfused provided adequate warming of the
patient and the extremity chosen for infusion. Vitamin (B12,
folic acid, vitamin D) and iron supplementation should be
tailored on specific deficiencies/need of osteoporosis preven­
tion. Anticoagulant and anti-infective prophylaxis should also
be considered. Finally, recombinant erythropoietin (i.e. epoe­
tin alpha 40,000 UI/week subcutaneously) may improve hemo­
globin in w-AIHA as well as CAD and can be used as
a supportive strategy in patients with inadequate reticulocy­
tosis [85]. Plasma exchange (with the use of albumin substitu­
tion instead of plasma to avoid complement supplementation)
may be successfully used in refractory life-threatening cases,
while specific immunosuppressive therapy is instituted [2,6].
6. New drugs
Novel treatments mainly target autoantibody production by
the B-cell/plasma-cell compartment or the final erythrocyte
breakdown by either complement or mononuclear phagocyte
system (Table 2) [6]. The formers include monoclonal antibo­
dies mainly used in secondary AIHA, such as ofatumumab
(anti-CD20), alemtuzumab (anti-CD52), and daratumumab
(anti-CD38). The latter targets long-lived plasma cells that do
not express CD20 and may cause rituximab refractoriness.
Isatuximab, another anti-CD38 MoAb is under investigation
in wAIHA in phase 1 study [NCT04661033]. Oral B-cell receptor
inhibitors parsaclisib [NCT03538041 and NCT05073458], ibru­
tinib [NCT03827603], and rilzabrutinib [NCT05002777] are also
being studied in clinical trials in wAIHA and CAD with promis­
ing results.
Complement modulation is the most promising drug under
study for CAD: sutimlimab, a monoclonal antibody against
complement protein C1s, demonstrated short time to
response, rapid normalization of hemolysis, and good safety
profile in a phase 3 trial [86]; pegcetacoplan, a pegylated
peptide that inhibits C3 [6] also showed good activity in
CAD and wAIHA with IgG+C DAT positivity. In the latter,
a novel C1q inhibitor ANX005 is also being developed
[NCT04691570]. Notably, complement inhibitors do not elim­
inate antibody production and will probably have to continue
indefinitely. However, the very short time to response may be
particularly helpful in severely anemic patients and acute
crises. The reticuloendothelial system may be targeted by
inhibiting the spleen tyrosine kinase with fostamatinib,
which also inhibits the B-cell receptor downstream pathway
and is now in phase 2 studies in wAIHA [NCT02612558].
Finally, the safety/efficacy of several inhibitors of the neonatal
Fc receptor (FcRn) such as intravenous nipocalimab
[NCT03075878] and subcutaneous RVT-1401 [NCT04253236],
are under investigation. The FcRn, which is homologous to
the MHC Class I receptor family, is widely expressed, and
mediates IgG recycling conferring to the antibodies their
long half-life. Blocking FcRn increases IgG clearance including
that of pathogenic IgG autoantibodies.
7. Consequences of AIHA and its treatment
The detrimental events that may complicate AIHA clinical
course include disease relapse, the above-mentioned associa­
tion with other autoimmune cytopenias, the occurrence of
thrombosis and infections, and the rare evolution into other
lymphoid or myeloid hematologic conditions. All these may
have an impact on patients’ mortality and overall quality of life
and are discussed thereafter [3].
7.1. Risk of AIHA relapse
The severity of anemia at onset constituted the major determi­
nant of disease relapse in two recent large multicenter studies
[15,87]. Relapse risk augmented with Hb decrease, by about 7%
increased risk per each gram of reduction of Hb. Concerning
AIHA type, CAD, mixed and atypical forms were at higher risk of
relapse and required multi-treatment, while wAIHA had a more
 EXPERT REVIEW OF CLINICAL IMMUNOLOGY 739

Table 2. New drugs in warm AIHA (wAIHA) and cold agglutinin disease (CAD). [92], and the close interplay among autoimmunity/autoinflam­
Route of mation, complement aactivation,and the coagulation cascade.
Mechanism Administration Study Phase Setting Moreover, the presence of underlying diseases, such as anti-
B-cell directed therapies phospholipid syndrome, active infections, and neoplasms may
Ofatumumab Anti-CD20 IV Case report Secondary
AIHA favor the occurrence of thrombotic complications [91,93]. On
Alemtuzumab Anti-CD52 SC Case reports Secondary the whole, the use of anticoagulant prophylaxis should be
AIHA considered in hyper-hemolytic, infected AIHA patients, pro­
Daratumumab Anti-CD38 IV Case reports wAIHA/CAD/
Secondary vided safe platelet counts. A comprehensive evaluation of all
AIHA risk factors is advised, including thrombophilia screening (par­
Isatuximab Anti-CD38 SC Phase 1 wAIHA ticularly lupus anticoagulant, LAC) in selected cases and before
Ibrutinib BTKi Oral Case reports, Secondary
Phase 2 plus AIHA splenectomy [2,88,91]. Finally, an ongoing prospective rando­
rituximab mized study will evaluate the efficacy of prolonged oral antic­
Rilzabrutinib BTKi Oral Phase 1 wAIHA oagulation with apixaban in preventing thrombosis during the
Venetoclax Bcl2i Oral Case reports Secondary
AIHA 12-weeks after AIHA diagnosis [NCT05089227].
Parsaclisib PI3Kδi Oral Phase 2, Phase wAIHA/CAD
3
Complement inhibitors
Eculizumab Anti-C5 IV Case reports CAD/Mixed 7.4. Risk of infections
and Phase 2 AIHA
Sutimlimab Anti-C1s IV Phase 1b and 3 CAD Infections may be both a trigger for AIHA development and
Pegcetacoplan Anti-C3 SC Phase 1/2 and CAD/wAIHA
/C3b Phase 3 relapse and be caused by immunosuppressive treatment in
ANX005 Anti-C1q IV Phase 1 wAIHA IgG+C a vicious circle. They complicate about 20% of AIHA cases,
IgG-mediated with a frequency increase along with the number of therapy
phagocytosis inhibitors
Fostamatinib * Syki Oral Phase 2 wAIHA lines. Interestingly, beyond splenectomy, which is associated
Orilanolimab Anti-FcRn IV Phase 1b wAIHA with a known risk of sepsis by capsulated bacteria, repeated
Nipocalimab Anti-FcRn IV Phase 2 wAIHA steroid therapy at each relapse seemed to have a prominent
RVT-1401 Anti-FcRn SC Phase 2 wAIHA
role in recent studies [87,88,94]. In thrombosis, underlying
*Fostamatinib is also a B-cell receptor inhibitor; BTKi: Bruton tyrosine kinase
inhibitor; Bcl2: B-cell lymphoma 2; δPI3Ki: phosphatidylinositol 3-kinase-δ diseases such as primary immunodeficiencies and malignan­
inhibitor; MoAb: monoclonal antibody; mTORi: mammalian target of cies and their therapies may lead to increased infectious risk
Rapamycin inhibitor; Syki: Spleen tyrosine kinase inhibitor; FcRn: neonatal Fc deserving special attention. On the whole, testing HBV/HCV
receptor
status, quantiferon for tuberculosis, is highly advised in patient
candidates to immunosuppression with high-dose steroids,
rituximab, or cytotoxic drugs to prompt adequate prophylaxis.
benign course. Overall, complement activation seems to be an Cotrimoxazole is advisable in patients receiving >25 mg day
additional risk factor for relapse, particularly after rituximab. prednisone (or equivalent) for >4 weeks, anti-viral agents (i.e.
lamivudine) and surveillance in those at risk for HBV reactiva­
tion, referral to an infective disease specialist for anti-
7.2. Evans syndrome (ES)
tubercular treatment if tuberculosis is suspected. HCV positive
The association of immune thrombocytopenia and/or neutro­ subjects should be referred for possible eradication.
penia to AIHA has a significant impact on prognosis. Vaccinations against capsulated bacteria (Meningococcus,
Particularly, relapse rate exceeded 70% in these patients, and Haemophilus, and Pneumococcus) are mandatory before sple­
more than 50% required ≥3 therapy lines in a recent analysis nectomy [95].
[88]. If cumulated with Hb < 8 g/dL or forms other than
WAIHA, the presence of ES was associated wit 3-/4-folds
higher than other cases [87]. ES patients had also a higher
7.5. Steroid induced side effects
incidence of infections (33%) and thrombotic complications
(21%) as compared with primary AIHA cases, correlating with In a recent survey, it has been calculated that AIHA patients
the number of therapy lines and resulting in a mortality rate received a cumulative dose of 8.2 ± 2.1 kg (mean± standard
approaching that of low-grade hematologic malignancies, error) in a mean follow-up of 12 ± 3.5 months [94]. This may
despite young age. be associated with well known but sometimes under recog­
nized side effects including arterial hypertension, diabetes,
gastrointestinal dysfunction, neurological disorders, and
7.3. Risk of thrombosis
osteoporosis. Concerning the latter, assessment of bone den­
Thrombotic events occur in about 15–20% of patients sity scan and supplementation with calcium and vitamin
[17,89,90] and were mainly observed in patients with very D may aid prompt recognition/prevention of harmful sponta­
severe anemia (i.e. Hb levels <6 g/dL) at onset, intravascular neous fractures. Additionally, vitamin D levels were found
hemolysis (LDH > 1.5 x upper limit of normality ULN), previous reduced in AIHA patients versus controls, particularly in
splenectomy, and presence of ES [87,91]. The pathogenesis is those who had received more therapy lines; furthermore, the
still unclear and likely includes the contribution of nitric oxide addition of vitamin D exerted a dose-dependent inhibition on
depletion by free Hb, the release of erythrocyte microparticles in vitro production of anti-erythrocyte antibodies [96]. These
740 B. FATTIZZO AND W. BARCELLINI
findings further suggest preventive administration of vitamin
D in patients with AIHA.
7.6. AIHA evolving into other hematologic diseases
Various reports described the possible evolution of AIHA into
clonal hematologic diseases and bone marrow failure. In par­
ticular, lymphoproliferative diseases may become clinically
overt in patients followed for AIHA, more frequently as the
evolution of an underlying misdiagnosed lymphoid indolent
infiltrate into aggressive forms, particularly in CAD [17].
Concerning myeloid diseases (from idiopathic cytopenia/dys­
plasia of unknown significance to myelodysplastic syndromes),
we previously described the presence of hypercellularity, dys­
erythropoiesis, and increased reticulin fibrosis in about one-
third of AIHA cases, associated with inadequate reticulocyto­
penia and chronic/relapsing course. Furthermore, we reported
two cases of AIHA and ES with anti-erythroblast antibodies,
which showed a clear-cut bone marrow erythrocyte precursor
hyperplasia at diagnosis but evolved into idiopathic cytope­
nia/dysplasia of uncertain significance (ICUS/IDUS) and aplas­
tic anemia (AA) after several years [97]. It may be hypothesized
that refractory/relapsing AIHAs may shift from a predominant
‘peripheral’ pattern toward a ‘central’ autoimmunity over time,
with a likely contribution of chronic inflammation, aging, and
accumulation of somatic mutations [6]. In this sense, small
clones of paroxysmal nocturnal hemoglobinuria have been
described in AIHA patients, possibly evolving to clinically
overt disease [98].
7.7. Mortality of AIHA
In a large retrospective series, mortality of AIHA patients was
around 10–20% and mainly associated with very severe ane­
mia. Patients presenting with Hb <6 g/dL had an overall
mortality of 24% versus 18% of those with Hb >6 g/dL. Age,
multi-treatment, comorbidities, and the occurrence of compli­
cations, further impacted patients outcome [99,100].
Specifically, mortality risk was predicted by ES, acute renal
failure, and infections (hazard risk, HR 8, 95% CI 2.5–26; HR
6.3, 1.4–29; HR 4.8, 1.5–15, respectively) [15,87]. In a series of
13 very severe multitreated primary AIHA mortality reached
57%, despite transfusions, steroid boli, IVIG, rituximab, EPO,
and plasma-exchange [101]. More recently, Lafarge et al.
described a mortality rate of up to 30% in 44 AIHA patients
admitted to the intensive care unit for severe anemia [102]. In
CAD, a recent large multicenter study reported a disease-
related mortality of 11% and a median estimated survival of
16 years from diagnosis (5-year survival 83%) [17]. Finally,
secondary AIHAs may also impact on disease severity and
prognosis and higher mortality was observed in AIHA second­
ary to lymphoma and cancer [2].
7.8. Quality of life in AIHA
Clinicians are used to evaluate patients’ quality of life (QoL)
by simply asking the patients to communicate the percep­
tion of health status and disease impact on daily living in
their own words. However, communication skills of both the
patient and the caregiver are highly heterogeneous and
impede an objective evaluation of quality of life, and the
use of structured surveys is helpful. The UK group recently
administered a semi-structured interview to 12 adult
patients with AIHA and found that the disease had
a significant impact on QoL for most patients [103]. On
a 0–10 scale – where 10 was the ‘worst I’ve ever felt’ –
77% of subjects scored 5–10, and 31% scored 9 or 10.
Patients described that the worst aspects of AIHA included
the impact of investigation or treatment, symptoms (mainly
fatigue), and anxiety over outcome. Multiple QoL domains
were affected, including daily living, working, leisure, and
even sleep. Finally, patients and their partners’ emotional
wellbeing were adversely affected. Available questionnaires
are being actively used in the afore mentioned clinical trials
to assess the beneficial impact of novel drugs on QoL [86].
However, an effort to design disease-specific surveys will be
expected in the next future.
8. COVID-19 infection and AIHA
8.1. AIHA associated with SARS-CoV-2 infection
In the last two years, SARS-CoV-2 has become one of the
major triggers for the development and exacerbations of
autoimmune cytopenias, including AIHA and Evans syndrome
[104–107]. The latter may in turn increase the need of immu­
nosuppressive treatment and, consequently, the frailty of this
patient population. Pathogenically, molecular mimicry among
SARS-CoV-2 antigens and red cells’ epitopes seems the promi­
nent mechanism [108], followed by the over-inflammation
triggered by the virus that may result in cross activation of
the many arms of the immune system (humoral and cellular
immunity, complement, coagulation cascade, etc.) against self-
antigens in an ‘innocent bystander’ fashion. The latter may also
favor the oxidative stress of erythrocytes and platelets with
exposure of phosphatidyl serine and consequent clearance by
the mononuclear phagocyte system [109,110]. COVID-19-
associated AIHA also displays some epidemiologic peculiari­
ties. In fact, a higher incidence of ES was noted, while it usually
complicates only one-fifth of AIHAs [15], and mainly in adults
that are more frequently infected by the virus. Additionally,
a higher prevalence of cold-reactive complement-activating
autoantibodies (up to 50%) was found as compared to primary
AIHA [111]. From a therapeutic point of view, SARS-CoV-2
associated AIHA and ES were mainly handled with
a combination of steroids and intravenous immunoglobulins,
irrespective of AIHA type. Rituximab was administered in only
one case displaying cold reactive autoantibodies [104]. This
drug represents a concern during septic states, including
SARS-CoV-2 infection, and has been associated with a more
severe COVID19 in rheumatic disorders [112]. Supportive treat­
ments, including rEPO and IVIG may be also considered.
Finally, the thrombotic risk of AIHA adds to that of COVID19
thus advocating for anticoagulant prophylaxis with low mole­
cular weight heparin in these patients, provided a safe platelet
count [113].

8.2. AIHA associated with SARS-CoV-2 vaccine
Several reports highlighted the possible impaired immune
response to SARS-CoV-2 vaccines in patients requiring immu­
nosuppression, including autoimmune cytopenias. Specifically,
recent reports showed that hematological patients treated
with monoclonal antibodies (such as rituximab, daratumumab,
and the anti-B cell early maturation antigen belantamab) do
not mount humoral response to vaccines. Additionally, it has
been shown that steroids reduce both humoral and T cell
response to COVID19 vaccine in hematologic patients and in
those with autoimmune diseases [114]. On the other hand,
SARS-CoV-2 vaccine may trigger autoimmune phenomena by
activating ADCC as well as the classical complement cascade.
In the literature, various AIHA, both warm and cold, ITP, and
Evans syndromes developing/reactivating after SARS-CoV-2
vaccine have been reported [115–118], irrespective of vaccine
type and dose. All patients have been successfully managed
with steroids, adjustment of ongoing treatment, or supportive
therapy [118]. Interestingly, two large cohort studies showed
that post-vaccine ITP has an incidence inferior to that
expected for primary ITP in the general population (0.8 versus
3.3 cases per 100,000 adults per year) [119,120]. Overall, it is
suggested to perform vaccinations as far as possible from
rituximab treatment and at the lowest steroid dose and to
strictly monitor Hb and hemolytic markers for possible exacer­
bations since they are manageable if promptly recognized.
9. Conclusions
In conclusion, this review highlights the multifaceted nature of
AIHA including a multifactorial etiology, encompassing genetic
predispositions and environmental exposures leading to toler­
ance breakage. The distinction between cold and warm forms
by DAT with monospecific antisera is pivotal since they show
diverse hemolytic patterns (i.e. IgG mediated extravascular
hemolysis in the spleen versus complement mediated hemolysis
in the liver or intravascularly) and should be treated differently.
Furthermore, a prompt recognition/exclusion of the associated
conditions (infections, drugs, tumors, hematologic conditions,
systemic autoimmune diseases, congenital immunodeficiencies,
etc.) is fundamental, since they may complicate AIHA diagnosis,
and may require specific management (i.e. CT scan, bone mar­
row evaluation, genetic testing). Treatment of wAIHA mainly
relies on steroids as the first line, followed by rituximab in
refractory/relapsing cases. Splenectomy is generally deferred to
the three line in young low-comorbid patients. Asymptomatic
CAD patients may be handled with cold temperatures avoid­
ance, while those subjects displaying moderate-to-severe ane­
mia or disabling peripheral symptoms require frontline
rituximab (since steroids are effective only at high unacceptable
doses). For the rarer mixed and atypical AIHA forms, which may
be more severe and difficult to diagnose, there is great uncer­
tainty and a close interaction with the laboratory physicians is
pivotal. The role of bone marrow compensation and the efficacy
of recombinant erythropoietin in patients with inadequate ery­
thropoiesis should be always considered. Several novel drugs
are being studied in clinical trials and will target the different
pathogenic steps of AIHA that may dynamically change during
EXPERT REVIEW OF CLINICAL IMMUNOLOGY 741
the clinical course. The latter may be affected by several com­
plications including the occurrence of thrombosis and infec­
tions, and the rare evolution into other lymphoid or myeloid
hematologic conditions. Finally, SARS-CoV-2 and its vaccines
represent novel triggers of AIHA development/exacerbations,
including warm and cold forms, as well as cases of Evans syn­
drome with several diagnostic and therapeutic pitfalls.
10. Expert opinion
Anti-erythrocyte autoimmunity has long been recognized as the
most suitable scholastic example to teach autoimmunity, since it
involves different classes of antibodies with variable thermal
characteristics, complement, cytokines, and cellular immune
effectors, such as T-lymphocytes and monocyte-macrophages.
Additionally, AIHA is unusual among autoimmune diseases in
that the target autoantigens are well characterized (i.e. band 3,
glycophorin A, and Rh system in wAIHA and I/i system in CAD).
Notwithstanding the simplicity of the cell targeted by the
immune attack, and the easiness of monitoring the degree of
the immune activation (as compared to other more complex
systemic autoimmune diseases), anti-erythrocyte autoimmunity
highlights the complex interaction of several immune effectors
and the existence of gray zones that elude classification. For
example, complement activation is the well-established key
mechanisms in CAD; however, the disease is undoubtedly due
to an antibody (mainly IgM, usually monoclonal). Thus, the ther­
apeutic strategy would better benefit from a combination of
B-cell/plasma cell-directed drugs and complement inhibition.
Additionally, thermal characteristics are not trivial: while an IgM
reacting only at cold temperature will result in minimal/negligible
clinical consequences, the very rare IgM with thermal range close
to body temperature may be the most severe and potentially
fatal forms. Paradoxically, these cases have not been included in
CAD trials, even if they would probably benefit from early com­
plement inhibition. On the other hand, in wAIHA, it is assumed
that complement plays a secondary role, and most clinical trials
are focused on targeting B-cells/plasma cells, inhibiting ADCC, or
removing pathologic antibodies via the Fc-Rn. This strategy may
exclude the severe wAIHA with DAT positivity for IgG and C,
where complement inhibition may have a definite reason to be
performed. Finally, the strict and scholastic distinction between
wAIHA and CAD leaves an unmet need for mixed forms, which
are generally more severe and relapsing than other AIHAs, and
have no therapeutic options after steroids and rituximab failure.
Altogether, these considerations underline the need to identify
not only the main mechanism involved in erythrocyte destruction
but also all the complex interactions of the different immune
effectors. This is also pivotal for a disregarded mechanism, i.e.
bone marrow compensation, which may be optimal in early
disease, but progressively decreasing with patient’s age, disease
duration/relapses, and presence of anti-erythroblast autoimmu­
nity. Furthermore, in clinical practice, strict classifications may be
difficult to apply, and tests’ positivities may vary overtime and
according to the clinical setting (host immunity, previous treat­
ments, presence of triggers). For instance, a high dose steroid
treatment received for the first AIHA episode may abate/mask the
‘warm’ part of a mixed AIHA at relapse. Additionally, during
polyclonal activation, as that occurring during infections, B-cell
742 B. FATTIZZO AND W. BARCELLINI
populations may release autoantibodies of different Ig subclasses.
This may also occur during primary IgM immune responses that
may be immature and less specific. Finally, the presence of
a complement-activating trigger, such as SARS-CoV-2, may favor
complement deposition on red blood cells and further interfere
with DAT findings.
Beyond the diagnostic issues, the study of the so-called
risk factors and predisposing conditions is gaining impor­
tance. The latter include modifiable environmental triggers
that affect the general population, such as air pollutants and
other stressors, that might allow the establishment of pre­
ventive measures in the future. In this view, a fascinating
field is the analysis of the microbiome and its interactions
with disease development and treatment. Dysbiosis may in
fact represent both a modifiable risk factor for AIHA onset
and relapses, as well as a trigger for infectious complications
following immunosuppression. In the near future, the avail­
ability of NGS techniques will improve our ability to analyze
these microorganisms with a likely impact on how we man­
age several diseases, including AIHA. Broader, molecular
techniques will allow the definition of the genomic land­
scape of this disease, such as the presence of germinal and
somatic mutations that may predispose to autoimmunity
through an altered antigen recognition, an imbalance of B/
T populations and of cytokine milieu, and the emergence of
clonal entities. The latter may include ‘clonal myelopoiesis/
lymphopoiesis of indeterminate potential’ that may be the
herald of autoimmunity in hematology. The same techniques
will possibly help to clarify why some patients are healed,
some relapse and become chronic, and some other evolve
into neoplastic conditions or bone marrow failure.
Therefore, the management of AIHA will significantly
change through a deepening knowledge of the various patho­
genic mechanisms, to the refinement of diagnostic tools and
disease classification, and to the development of novel tar­
geted and combination therapies.
Declaration of interest
B Fattizzo received consultancy for Amgen, Alexion, Annexon, Apellis,
Momenta, Novartis, and Sobi. W Barcellini received consultancy for Agios,
Alexion, Annexon, Sanofi, Novartis, and Sobi. 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 subject matter or
materials discussed in the manuscript apart from those disclosed.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other
relationships to disclose.
Author contributions
B Fattizzo and W Barcellini wrote the manuscript and revised it for
important intellectual content. All authors agree for the final version of
the manuscript to be published.
Funding
This study was funded by Italian Ministry of Health - Current research IRCCS.
ORCID
B Fattizzo http://orcid.org/0000-0003-0857-8379
W Barcellini http://orcid.org/0000-0003-1428-9944
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