Warm Autoimmune Hemolytic Anemia: Recent Progress in
Understanding the Immunobiology and the Treatment
Melca M.O. Barros, Morris A. Blajchman, and José O. Bordin
Autoimmune hemolytic anemia (AIHA) is defined as a
condition associated with the increased destruction of
red blood cells (RBCs) associated with the presence of
IgG anti-RBC autoantibodies. The etiology underlying
the pathogenesis of such autoantibodies is still un-
certain. In the present article, we will discuss the
postulated mechanisms that produce a breakdown of
immunologic tolerance leading to warm AIHA including
the possible roles of RBC autoantigens and the
complement system, the lack of effective presentation
of autoantigens, functional abnormalities of B and T
cells resulting in polyclonal lymphocyte activation and
alteration of cytokine production, and the role of
immunoregulatory T cells. Because warm AIHA is a
relatively rare clinical entity, current recommended
therapeutic strategies for patients with warm AIHA
are mainly based on results from small cohort studies.
Clinicians must also balance the risk of withholding
RBC transfusions against the possible benefit of
UTOIMMUNE HEMOLYTIC ANEMIA
A (AIHA) is defined as the increased destruc-
tion of red blood cells (RBCs) in the presence of
anti-RBC autoantibodies.1 Immune hemolytic ane-
mias have been classified in different ways, but
typically they are subdivided into 4 major types
including warm AIHA, the cold agglutinin syn-
drome, mixed-type AIHA, and paroxysmal cold
hemoglobinuria. Autoimmune hemolytic anemia is
uncommon with an annual incidence of about 1 to 3
per 100 000 individuals. Autoimmune hemolytic
anemia is often classified also by the optimal
temperature at which the autoantibodies bind to a
patient's RBCs in vivo. Warm AIHA comprises
80% to 90% of all cases. In warm AIHA, the
autoantibodies demonstrable in the serum react
optimally with human RBCs at 37°C. In patients
with the other types of AIHA, the AIHA is
mediated by the presence of cold autoantibodies
that exhibit affinity for RBCs at temperatures lower
than 37°C.2
Cases of warm AIHA have also been subclassi-
fied by the presence or absence of various
pathophysiologically associated underlying condi-
tions or diseases. The term primary or idiopathic
warm AIHA is applied when there is no recogniz-
able underlying or associated condition present.
Secondary warm AIHA can also be seen as a
manifestation of an underlying pathophysiologic
Transfusion Medicine Reviews, Vol 24, No 3 (July), 2010: pp 195-210
ameliorating the hemoglobin level with such transfu-
sions particularly in critically ill patients with warm
AIHA. Glucocorticoids are the first-line treatment for
patients with warm AIHA resulting in an 80% clinical
response after 3 weeks of treatment. The latter,
however, also may cause adverse events such as
excessive weight gain, neuropsychiatric disorders,
endocrine, or cardiovascular events. Splenectomy
should be considered for patients who do not show a
satisfactory response to glucocorticoids and may offer
a success rate of up to 70% in patients with idiopathic
warm AIHA. Rituximab treatment in patients with
refractory warm AIHA has been well tolerated with an
overall median response rate of approximately 60%.
Danazol, intravenous immunoglobulin, alemtuzumab, as
well as other immunosuppressive drugs have also been
successfully used in patients with warm AIHA, refrac-
tory to glucocorticoids, splenectomy, and rituximab.
© 2010 Elsevier Inc. All rights reserved.
disorder. Idiopathic warm AIHA represents ap-
proximately 50% of reported cases of AIHA,
whereas secondary cases present associated with
conditions or agents (microbiological or chemical)
including lymphoproliferative disorders (20%),
connective tissue diseases (20%), solid tumors,
infections, or drugs. Most patients with idiopathic
warm AIHA are women in their fourth or fifth
decade of life.2,3
The diagnosis of warm AIHA requires the
demonstration of clinical and/or laboratory findings
of hemolysis and serologic evidence of an autoan-
tibody (usually IgG) to RBCs.2-4 Typically, such
anemias are macrocytic because of the presence of
marked reticulocytosis, but early in the presenta-
tion, transient reticulocytopenia may occur despite
the presence of a normal or even a hyperplastic
bone marrow. Reticulocytopenia may be seen
because of the in vivo hemolysis of the reticulo-
cytes or related to compromised marrow function.5
From the Universidade Federal São Paulo, São Paulo, Brazil;
and McMaster University, Hamilton, Ontario, Canada (MAB).
Address reprint requests to José O. Bordin, MD, Universidade
Federal de São Paulo Rua Botucatu, 740 São Paulo-SP, Brazil.
E-mail: jobordin@hemato.epm.br
0887-7963/$ - see front matter
© 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.tmrv.2010.03.002
195
196
The blood film often reveals important features
such as marked anisocytosis and polychromasia
reflecting the increased reticulocyte count. Spher-
ocytosis is a usual feature of warm AIHA,
particularly in patients with moderate or severe
warm AIHA. Upon presentation, the hemolysis
may be severe and on occasion, may even be life-
threatening. Typically, patients with AIHA exhibit
a mild leukocytosis with neutrophilia. Occasional-
ly, patients present with leukopenia, neutropenia, or
thrombocytopenia, the latter often due to associated
immune-mediated platelet destruction.
The laboratory parameters in patients with
AIHA, which indicates the presence of hemolysis
include a reduced serum haptoglobin level, an
increase in the indirect bilirubin concentration, and
an elevated lactate dehydrogenase level.2-4
The presence of positive direct antiglobulin tests
(DATs) indicates that RBC autoantibodies and/or
complement proteins, or both, are bound to the
circulating RBCs in vivo. A polyspecific antiglob-
ulin reagent that contains antibodies directed
against human IgG and complement proteins
(usually C3) is used to screen the patient's RBCs.
If positive, monospecific antisera with antibodies to
IgG or complement are used to define the pattern of
the DAT. Monospecific antisera to IgM or IgA may
also be used in selected cases. In most cases of
warm AIHA, no autoantibody specificity is appar-
ent, but in such circumstances, antibodies bound to
the patient's RBCs can be eluted off the RBC
surface and potentially identified in the eluate
obtained. Generally, autoantibodies are antigen
nonspecific and react with all panel RBCs tested.
Occasionally, a broad specificity, usually in the Rh
system, will be seen. On occasion, autoantibody
specificities to other blood group antigen systems
have been reported. These have included the Kell,
Kidd, Duffy, and Diego blood group systems.1,4
The etiology underlying the pathogenesis of
warm autoantibodies is uncertain. Recent studies in
both humans and animals have been designed to
explain not only immunologic tolerance to self-
antigens in warm AIHA but also possible mechan-
isms of the increased RBC destruction. In the
present article, we review the various mechanisms
that produce a breakdown of immunologic toler-
ance and discuss current treatment options, as well
as some still emerging new therapeutic approaches.
It is important to note particularly with regard to the
latter that many recently recommended treatments
BARROS ET AL
for patients with warm AIHA are based on results
from relatively small cohort studies.
THE IMMUNOBIOLOGY AND PATHOGENESIS
OF WARM AIHA
The pathogenesis of warm AIHA is likely a
complex process in which many factors play an
essential role. The mechanisms that have been
proposed to explain the spontaneous appearance of
AIHA include the role of autoantigens, the role of
the complement system, the lack of effective
presentation of autoantigens, and functional abnor-
malities of B and T cells. Immunologic data that
support these pathogenetic theories on autoimmu-
nity derive from experimental studies in both
experimental animal models as well as from
human studies. These postulated mechanisms are
summarized in Table 1 and discussed in further
detail below.
The Role of RBC Autoantigens
Studies in humans have indicated that Rhesus
(Rh) polypeptides are the commonest targets for the
pathogenic autoantibodies in patients with warm
AIHA. Other blood group antigen specificities have
been identified in a minority of patients and include
the glycophorins, Band 3 proteins, and the RBC
anion channel protein. In canine AIHA, the
glycophorins are the dominant RBC autoantigen
targets, whereas in mice, the RBC autoantigens are
located on RBC membrane Band 3.6-9 In man,
several studies have shown that Rh-like proteins are
able to stimulate the in vitro proliferation of
mononuclear cells from peripheral blood or spleen
but only in patients with AIHA.10,11 The mecha-
nism that has most often been proposed to explain
the role of autoantigens as a cause of apparently
spontaneous autoimmune disease is cross-reactivi-
ty, or molecular mimicry, between environmental
antigens and autoantigens. Such cross-reactivity
may offer amplified levels of particular antigenic
sequences, or greater avidity of antigen recognition,
to overcome self-tolerance. This molecular mimicry
may enhance immunogenicity by innate stimuli.10-12
A recent study with New Zealand Black (NZB)
mice demonstrated that the development of AIHA
is not dependent on the expression of the major
autoantigen and that a defect in self-tolerance is
directed to the RBC type.13 On the other hand, the
tendency to RBC autoimmunity in NZB mice may
be focused on Band 3 by weak helper T cell (Th
WARM AIHA: IMMUNOBIOLOGY AND TREATMENT
Table 1. Postulated Mechanisms of Autoimmunity in AIHA
I. The role of RBC autoantigens:
1. Rh-related polypeptides are the commonest targets for the
pathogenic autoantibodies in patients with warm AIHA.
2. In NZB mice, the tendency to RBC autoimmunity may be
focused toward RBC Band 3 by weak cross-reactivity
between the T-helper cell dominant epitope and an
exogenous antigen.
3. CD47 functions as a marker of self on murine RBCs.
4. The interaction SIRP-α and CD47 is not responsible for RBC
clearance in humans.
II. The role of the complement system:
1. Altered levels of expression of CR1 are observed in patients
with autoimmune diseases, AIDS, and lepromatous leprosy.
2. CR1/DAF-deficient murine RBCs are eliminated more rapidly
by complement than by the Fc receptor pathway when the
RBCs are opsonized with autoantibody.
3. Patients with autoimmune cytopenias, including warm
AIHA, may have a deficiency of CD59 expression.
III. Lack of effective presentation of autoantigens:
1. The presence of immature DCs leads to the transient
induction of antigen-specific T cell activation followed by
T cell deletion and unresponsiveness.
2. Many self-epitopes are inefficiently processed and
presented by APCs; thus, self-reactive naive T cells
remain in the peripheral T cell repertoire leading
to autoimmunity.
IV. Functional abnormalities of B and T cells result in:
1. Polyclonal lymphocyte activation
(a) Several virus or parasite infections are followed by the
increased production of autoantibodies, which in turn is
linked to B cell polyclonal activation.
(b) In chronic graft-vs-host disease, activation of host
B cells by donor T cells induces autoimmunity,
including warm AIHA.
2. Alteration of cytokine production
(a) Prevalence of Th1 cytokines in NZB mice.
(b) IL-4 and IL-10 treatment ameliorates the anemia
in NZB mice.
(c) Imbalance between IL-10 and IL-12, in NZB mice and
humans with AIHA.
(d) In nonstimulated PBMC cultures from AIHA patients,
there is an in vitro increase of IL-10 and IL-4 production
with a reduction of IFN-γ and IL-12.
(e) PBMCs from patients with AIHA stimulated in vitro with
RhD protein produce IFN-γ and IL-10 but not IL-4.
3. Treg cells have an important role in the maintenance of
peripheral tolerance
(a) Autoimmune diseases are associated with abnormalities
in Treg cell number and/or function.
(b) Older CD25 knockout mice develop AIHA.
(c) Treg cells from patients with AIHA are capable
of inhibiting the Th1 effector responses in vitro by
secretion of IL-10.
cells) cross-reactivity between the helper dominant
epitope and an exogenous antigen.13
CD47 is a transmembrane glycoprotein (integrin-
associated protein) that has been closely associated
197
with RBC Rh proteins in a multiprotein complex
with many other proteins.14 Experimental studies
have suggested that CD47 functions as a marker of
self on murine RBCs; thus, CD47 null murine
RBCs have been shown to be rapidly eliminated
from the circulation of wild-type mice, by splenic
macrophages. This mechanism is based on the
binding of CD47 RBCs to signal regulatory protein
α (SIRP-α) on macrophages resulting in the
inhibition of macrophage activation and phagocy-
tosis.14,15 The human data available show that the
expression of CD47 on RBCs and SIRP-α,β on the
monocytes of patients with active warm AIHA in
remission are not different from those seen in
healthy individuals.16,17 These data thus suggest
that signaling by CD47 in man may be qualitatively
different from that seen in mice and imply that the
interaction between SIRP-α and CD47 is not
responsible for RBC clearance in humans.
The Role of the Complement System in the
Maintenance of Self-Tolerance
The role of complement in the maintenance of
self-tolerance is supported by several lines of
evidence. Altered levels of the expression of
complement receptor1 (CR1) has been observed
in patients with autoimmune disease such as
systemic lupus erythematosus (SLE), AIDS, and
lepromatous leprosy.18 Thus, patients with AIDS
have a progressive decrease of CR1 on their RBCs,
and genetic studies showed unequivocally that this
defect is acquired.19 Data from SLE patients have
been more difficult to interpret, but genetic studies
indicate an acquired loss of CR1.20 A recent study
in mice indicates that mouse RBCs deficient in CR1
are spontaneously eliminated in vivo by the
complement system.21 These studies demonstrate
that RBCs in CR1/decay-accelerating factor
(DAF)-deficient mice are cleared spontaneously
by the alternative pathway of complement, and
when opsonized with an immunoglobulin IgG2a
autoantibody, CR1/DAF-deficient mouse RBCs
were eliminated more rapidly by complement than
by the Fc receptor pathway. These findings suggest
that CR1 (CD35) and DAF (CD55) influence the
hemolytic pathway in AIHA.22 However, RBC
CD55 expression levels are normal in patients with
primary warm AIHA17 as well as in patients with
warm AIHA secondary to SLE.23 On the other
hand, it has been reported that patients with either
primary or secondary AIHA may have a deficiency
198
of CD59 expression on their RBCs.17,23 CD59
deficiency also occurs in various conditions
characterized by complement activation, such as
SLE-associated anemia and lymphopenia. In addi-
tion, CD3+ lymphocytes from SLE patients with
lymphopenia show a diminished expression of
CD55 and CD59 when compared with lymphocytes
from healthy individuals.24 Relevantly, it has been
reported that 36% of patients with autoimmune
thrombocytopenia have deficient expression of
CD59, whereas 16% present with deficient expres-
sion of CD55 compared to that seen in healthy
individuals. Moreover, CD59 deficiency has been
reported to be associated with severity of thrombo-
cytopenia in such individuals.25
Lack of Effective Presentation of Autoantigens
One of the hypotheses put forward to explain the
development of autoimmune diseases such as warm
AIHA is that the clinical entity might be triggered
by changes in the way autoantigens on target cells
are presented, so that they overcome self-tolerance.
Normally, antigen-presenting cells (APCs), includ-
ing dendritic cells (DCs) and macrophages, capture
self-antigens from other cells and present them to
autoreactive T cells to induce T cell tolerance by
either deletion or anergy.26 There are several
hypotheses to explain the lack of effective presen-
tation of autoantigens. One postulate is that the
APCs remain relatively immature and therefore
tolerate rather than activate self-reactive cells. This
type of maturation of APCs thus occurs as a
consequence of signals received by the other cells
of the innate immune system, with the presence of
pathogens leading to the more effective autoantigen
presentation to T cells to induce an immune
response to self-antigens.26 Using antigens targeted
to immature DCs in vivo, Hawiger et al 27
demonstrated that in the absence of additional
stimuli, DCs lead to the induction transient antigen-
specific T cell activation followed by T cell deletion
and subsequent unresponsiveness. However, if the
antigen is targeted to DCs together with anti-CD40
antibodies, which promote APC maturation, there is
prolonged T cell activation and immunity.27
A second explanation for the lack of the effective
presentation of autoantigens is that many self-epitopes
may be inefficiently processed and presented by
APCs. This would result in minimal cross presenta-
tion and as a result, the self-reactive naive T cells
would remain as part of the peripheral T cell repertoire
BARROS ET AL
escaping thymic deletion.28 These epitopes would
thus be generated as a result of the alteration of
endosomal enzymes with consequent changes in the
cleavage of proteins during antigen processing or
weak binding to accessible major histocompatibility
complex (MHC) molecules that would compete
ineffectively with other peptides for presentation.12,29
Relevantly, Liu et al29 demonstrated in mice with
experimentally induced allergic encephalomyelitis
that naive T cells are potentially autoaggressive and
can cause autoimmunity when stimulated.
Functional Abnormalities of B and T Cells
To identify the immunologic factors contributing
to autoimmune onset and maintenance, several
murine strains have been studied extensively.
Functional abnormalities of B and T cells in such
mice have been investigated, particularly their
lymphocyte activation, cytokine production, and
their role in the onset of their autoimmunity.
Playfair and Marshall-Clarke 30 described one
murine model of AIHA, in which the alloimmuni-
zation of mice with cross-reacting rat RBCs
resulted in the development of RBC autoantibodies
as well as rat-specific alloantibodies, suggesting
that the AIHA may have been provoked by the
exposure to foreign antigens that cross-react with
self-antigens. In NZB mice, treatment with anti-
CD4 monoclonal antibody retarded IgG autoanti-
body production demonstrating that the generation
of IgG autoantibody is T cell dependent. 31
Depletion of T cells from such mice prevents the
induction of AIHA in response to immunization by
rat RBCs.32 In human AIHA, Rh polypeptides,
including glycophorins, and Band 3 proteins cause
T cell activation and their proliferation.8-12
Polyclonal Lymphocyte Activation
One explanation for the autoaggressive damag-
ing tissue responses are the polyclonal activators
such, as superantigens or mitogens, that are
associated with a failure to control lymphoproli-
feration. Studies with NZB/W mice thus have
described excessive B cell proliferation with
increased amounts of IgM-secreting cells, particu-
larly in the early life of such animals.33 Thus,
polyclonal activation of B cells has been suggested
as a possible mechanism by which viruses trigger
autoimmune disease. Several virus or parasite
infections are known to be followed by the
increased production of autoantibodies, which is
WARM AIHA: IMMUNOBIOLOGY AND TREATMENT
linked to B lymphocyte polyclonal activation.
Thus, after the intracerebral inoculation of the
lymphocytic choriomeningitis virus, the CH3cB/
FeJ mouse develops AIHA and hypergammaglo-
bulinemia.34 Similarly, we have reported that the
prevalence of a positive DAT on the RBCs of HIV-
infected persons was 16.7% (40/239) probably
because B cells are polyclonally activated and
secrete large amounts of immunoglobulin that can
appear on the surface of RBCs.35 However, one of
the HIV-positive patients we investigated exhibited
reticulocytosis with a decreased hemoglobin level,
unconjugated hyperbilirubinemia, and sphero-
cytes.35 We have also investigated whether the
RBCs of 67 patients with visceral leishmaniasis
(kala-azar) were coated with IgG and found that the
prevalence of a positive DAT before any treatment
was 32.8%. Interestingly, a positive DAT and
positive enzyme-linked direct antiglobulin test
correlated with the presence of either rheumatoid
factor or circulating immune complexes.36
In human chronic graft-vs-host-disease after
bone marrow transplantation, alloreactive donor T
cells that recognize host histocompatibility antigens
cause, by direct interaction between T cells and B
cells, a selective stimulation of host self-reactive B
cell clones, which produces a wide spectrum of
autoantibodies, including those against RBCs that
can give rise to AIHA.37
Cytokine Production
The T cell population comprises distinct subsets
that are characterized by surface markers of helper
T cells (CD4+, Th) and cytotoxic T cells (CD8+).
Th cells have played an important role in the
development of animal models of autoimmunity.
Th cell clones can be classified into different
functional types on the basis of the cytokines they
secrete. Thus, Th1 cells synthesize interleukin-2
(IL-2), interferon-γ (IFN-γ), and tumor necrosis
factor-β (TNF-β), and promote cell-mediated im-
munity, delayed-type hypersensitivity, macrophage
activation, and opsonizing antibody production. In
contrast, Th2 cells secrete IL-4, IL-5, IL-6, IL-10,
and IL-13 and promote both humoral and allergic
responses.38 The role of the Th1 and Th2 cytokines
in the pathogenesis of autoimmune diseases is a
field of on-going investigation. Thus, T cells from
NZB mice stimulated with the RBC Band 3 protein
spectrin, from the RBC membrane skeleton and
mycobacterial heat-shock protein produce high
199
levels of the Th1 cytokine IFN-γ but little or no
IL-4, IL-5, or IL-10.39 Interleukin-12, a cytokine
produced primarily by APCs, promotes the devel-
opment of Th1 cells in vitro and in vivo and may
inhibit IL-4-induced antibody production. Interfer-
on-γ amplifies the IL-12 activation of Th1 cells and
suppresses Th2 cell expansion and promotes B cell
maturation and pathologic antibody generation.40
Mice treated with IFN-γ develop accelerated SLE,
whereas the administration of TNF-α has the
opposite effect.41 Interleukin-4 and IL-10 inhibit
the development of Th1 cell clones through the
down-regulation of IL-12 production by mono-
cytes.37 In NZB mice, the intranasal administration
of soluble peptide containing the dominant helper
epitope that encourage the autoimmune response
toward a Th2 profile resulted in the amelioration of
disease.42 In support of the hypothesis that Th2
cytokines protect against autoimmune disease, it
has been shown that IL-4 and IL-10 treatment
ameliorates the anemia in NZB mice.43 On the
other hand, minimal IL-10 administration acceler-
ates the onset of the autoimmune syndrome. In
contrast, the continuous administration of anti–IL-
10 antibodies delays the onset of autoimmunity in
NZB/W mice, owing to the augmentation of the
TNF-α level.44
Patients with primary or secondary AIHA not
subjected to immunosuppressive therapy show high
serum levels of IL-1α, IL-2, IL-4, and IL-2R.45
Peripheral blood mononuclear cells (PBMCs) from
patients with SLE have been shown to have a
decreased production of IFN-γ, TNF-α, IL-12, and
high levels of IL-10. Decreased production of Th1
cytokines in SLE may be secondary to the
imbalance between IL-10 and IL-12, whereas the
deficiency in IL-12 production may be the effect of
excessive IL-10 production because recombinant
IL-10 strongly inhibits in vitro production of IL-12
in SLE. Peripheral blood mononuclear cell cultures
from normal controls and anti-IL10 reverse IL-12
deficiency of SLE PBMCs. These data suggest that
IL-10 and IL-12 can potentially build an immuno-
regulatory circuit that can regulate the development
and maintenance of an autoimmune disease.46,47
The AIHA patients have been shown to exhibit
increased basal synthesis of IL-4 and also have
decreased levels of IFN-γ produced by AIHA
PBMCs, compared with control subjects. These
results suggest that there is a basal decrease of Th1
cytokines and an increase of the Th2 cytokines.
200
Unstimulated PBMCs from AIHA patients have
shown a constitutively increase of IL-10 production
in vitro compared to those from normal controls.
After lipopolysaccharide (LPS) stimulation of
PBMCs, an increased IL-10 production in both
AIHA patients and controls was observed, compared
to basal levels. Furthermore, IL-12 basal levels are
higher in AIHA patients than in controls; however, a
significant increase in IL-12 production is seen in
LPS-stimulated PBMC cultures from healthy con-
trols but not from those from AIHA patients. The
latter could be explained as the consequence of an IL-
10 inhibitory effect. These data thus support the
hypothesis that the decreased production of Th1 and
the prevalent Th2-type cytokines leading to auto-
antibodies production in AIHA may be secondary to
the imbalance between IL-10 and IL-12 production
by PBMCs.35,48 These observations contrast with
results from parallel studies of PBMCs from patients
with AIHA stimulated in vitro with a major RBC
autoantigen. The RhD protein was thus found to
cause proliferation of Th1 cells that produces IFN-γ
and the regulatory cytokine IL-10 but not IL-4. An
analysis of all the samples obtained from a AIHA
patient panel revealed that high IL-10 responses were
associated with less severe anemia, supporting the
idea that the balance between autoantigen specific Th
cells subsets secreting different patterns of cytokines
is an important mechanism for regulating autoim-
mune responses because Th1 responses appear to be
pathogenic, whereas Th2 responses might mediate
protection from such disease.49
The Role of Immunoregulatory T Cells
For several decades, it had been postulated that
there exists a subset of lymphocytes capable of
suppressing and “managing” the various immune
responses. Several pieces of evidence have revealed
that active suppressive phenomena, including the
control of immune-mediated pathologic conditions,
are mediated by the recently defined CD4+ T
regulatory (Treg) cells. Treg CD4+ cell populations
are characterized by the expression of very high
levels of the IL-2 receptor (CD25), and relevantly,
these cells have been shown to be CD4+CD25+.
These Treg cells have an important role to play in
the generation and maintenance of peripheral
tolerance.50 T regulatory cells are classified accord-
ing to their surface phenotype and cytokine
secretion profile and also whether they are naturally
occurring or inducible. The naturally occurring
BARROS ET AL
CD4+CD25+ Treg cell subset is distinguished by a
transcription factor known as the forkhead box
protein P3 (FOXP3) and also their expression of
CD25. Naturally occurring Treg cells comprise
approximately 6% of the CD4+ T cell population in
healthy individuals. These cells seem to be anergic
to proliferative responses but have the capacity to
suppress proliferation of effector T cells in
vitro.51,52 The inducible Treg cells include T-helper
3 cells, which originate from naive T cells that are
either CD4+ or CD8+, and type 1 Treg (Treg1) cells
derived from CD4+ precursor cells. These inducible
Treg cells may or may not express FOXP3. These
cells nonetheless can secrete the inhibitory cyto-
kines TGF-β or IL-10.51
Several studies have analyzed the number and/or
function of Treg cells in human autoimmune
diseases providing evidence that autoimmune
diseases are often associated with abnormalities in
Treg cells number or function compared to healthy
controls.51 In this context, it has been shown that
CD25 knockout mice, as they age develop
autoimmune disease, including hemolytic ane-
mia.53 In antibody depletion studies using anti-
CD25 antibody, the incidence of AIHA in C57/Bl6
mice increased from 30% to 90%. Adoptive transfer
of purified Treg but not CD4+CD25− cells from
immunized mice into naive recipients prevented the
induction of the AIHA. These findings thus
establish a critical role for Treg cells for the control
of AIHA.54
The peripheral blood of patients with warm
AIHA has been found to contain Treg cells
specific for the target Rh autoantigens capable of
inhibiting Th1 effector responses in vitro by the
secretion of IL-10.49 Studies to characterize
phenotype, origin, and the regulatory function of
Treg cells in human warm AIHA demonstrated
that Treg cells shared phenotypic characteristics
with both natural and inducible Treg cells and
expressed high levels of CD25 and the lympho-
cyte activation gene-3 when expanding nonspe-
cifically, but the Treg cells expressed FOXP3 after
activation by the recognized autoantigen. In
addition, it has been shown that Treg cells
expressed the Th1 signature transcription factor
T-bet, a transcription factor associated with the
Th1 cells phenotype, suggesting that the Treg
cells derive from Th1 cells. These data suggesting
the existence of such cells specific for autoanti-
gens of pathogenic relevance in human patients
WARM AIHA: IMMUNOBIOLOGY AND TREATMENT
show the importance of studying autoreactive
Treg cells in the context of known specificity.
Analysis of T-bet expression suggests that the
Treg cells derive from Th1 cells and may have
arisen as a result of effector T cell stimulation
associated with the chronic autoimmune response
underlying AIHA.55
TREATMENT OF WARM AIHA
Because warm AIHA is a rare disease, treatment
recommendations for patients with warm AIHA are
often based mainly on results from retrospective
data and/or relatively small cohort studies including
Fig 1. Proposed algorithm for the treatment of warm AIHA.
201
heterogeneous patient samples or from single case
reports. A proposed algorithm for the treatment of
warm AIHA is summarized in Figure 1.
Red Blood Cell Transfusions
The RBC transfusions often may be necessary in
the management of patients with warm AIHA,
especially in symptomatic older patients, particu-
larly in patients with coronary artery disease, taking
into account the potential vital risk of the degree of
anemia to their clinical situation. Such patients
should receive RBC transfusions to maintain their
hemoglobin levels at a clinically acceptable level, at
202
least until other treatments come into play that
curtail the degree of hemolysis.56
Transfusions of RBCs to patients with warm
AIHA presents a particular set of potential
problems. One obvious and often underappreciated
issue is the relative short survival time of the
transfused RBCs. Experience has demonstrated that
when incompatibility is due only to the presence of
an RBC autoantibody, the survival of transfused
RBCs is approximately the same as that of the
patient's autologous RBCs. The RBC transfusions
can thus be thought to be able to provide only some
temporary benefit.2,56,57 Patients with warm AIHA
usually have broadly reactive RBC autoantibodies
that are generally panagglutinins that can react with
the alloantigens on all potential donor RBCs. Such
autoantibodies can often mask the presence of RBC
alloantibodies that may be present. The RBC
alloantibodies can develop as a result of previous
transfusions or pregnancies and are the most
important technical problem faced by the transfu-
sion service regarding the RBC transfusion of
patients with warm AIHA. This concern relates to
the fact that these alloantibodies are capable of
causing serious acute hemolytic transfusion reac-
tions in recipients.56
Reports of RBC alloantibodies in warm AIHA
patients range from 15% to 45% (mean, 32%).58-60
These data thus indicate the need for a method to
identify such antibodies to prevent alloantibody-
induced acute hemolytic transfusion reactions.
Selection of the best unit of RBCs to be transfused
to patients with warm AIHA patients thus usually
involves the need for complex compatibility test
procedures. Numerous approaches are available for
selecting RBC units for transfusion for patients who
have warm autoantibodies. These include (a) the
routine testing of the patient's serum against an
RBC panel; (b) the use of a dilution technique,
which will dilute the patient's serum before doing
RBC compatibility testing; (c) the use of adsorption
procedures (a warm autoadsorption technique or
allogeneic adsorption) that removes the autoanti-
body but not the alloantibodies from the patient's
serum; and (d) extended phenotyping of the
patient's RBCs or molecular analyses for RBC
genotyping the patient and supplying pheno-
typically compatible RBC units.56,60,61 Adequate
testing for alloantibodies may take several hours to
be completed. Thus, in an urgent clinical situation,
the clinician must balance the risk of withholding
BARROS ET AL
transfusion against the possible benefit of correcting
the hemoglobin level in a seriously ill patient.56
Use of Glucocorticoids as First-Line Therapy
First-line treatment of a patient with warm AIHA
usually involves the use of glucocorticoids. Gluco-
corticoids influence hemolysis in patients with warm
AIHA by several mechanisms. Patients who respond
to glucocorticoids show a decreased strength of the
DAT and indirect antiglobulin test, suggesting that
improved RBC survival results from a decrease in the
synthesis of anti-RBC autoantibodies.1,62 On the
other hand, this mechanism does not completely
explain the often seen early improvement in
hemolysis seen within 24 to 72 hours after the
onset of glucocorticoid therapy. This is often well
before any alteration in strength of the DAT is
observed. Glucocorticoids are also able to suppress
sequestration of RBCs by splenic macrophages. This
may be the result of the reduced number of Fcγ
receptors observed on blood monocytes of AIHA
patients during glucocorticoid therapy.1,63
Therapy with glucocorticoids is usually started
with oral prednisone, at an initial daily dose of 1 to
1.5 mg/kg or its equivalent for the first 2 to 4 weeks.
Patients with particularly rapid hemolysis may
require intravenous methylprednisolone at a dose
of 250 to 1000 mg/d for 1 to 3 days. Once the
hematocrit begins to rise, the amount of glucocor-
ticoid can be slowly reduced, and when a dose of 20
to 30 mg daily of prednisone in an adult is achieved,
with a persistent remission, as indicated by the
stable hemoglobin level and decreasing reticulocyte
count, it is recommended that alternate day
prednisone doses be commenced. The advantage
of an alternate-day schedule is that it is associated
with fewer corticosteroid long-term side effects.
Relevantly, the total duration of glucocorticoid
therapy can vary from 3 to 12 months, after
obtaining a remission, is usually required in patients
with warm AIHA.1,2,4
After 3 weeks of treatment with glucocorticoids,
approximately 80% of all warm AIHA patients have
a clear clinical response (ie, hemoglobin level N 10.0
g/dL). Patients who do not have a clinical response
at that time and patients who present with recurrent
hemolysis may require second-line treatment, such
as a splenectomy or immunosuppression.2-4
Adverse events such as excessive weight gain,
skin thinning, neuropsychiatric disorders, sleep
disturbances, increased risk of cardiovascular
WARM AIHA: IMMUNOBIOLOGY AND TREATMENT
events, cataracts, and myopathy may occur in
patients taking high doses of glucocorticoids.
Glucocorticoid-induced osteoporosis is a potential-
ly preventable complication; thus, patients on
prolonged courses of glucocorticoids should also
be given calcium and vitamin D supplementation.
Patients at high risk for glucocorticoid-induced
osteoporosis should also be considered for treatment
with bisphosphonates or other antiosteoporosis
medications, particularly when they are receiving
glucocorticoid therapy. High-dose glucocorticoids
are well-known to also increase the risk of infectious
complications. Other comorbidities that can be
exacerbated with the use of glucocorticoids include
diabetes, hypertension, hyperlipidemia, heart fail-
ure, glaucoma, and peptic ulcer disease.64
It is suggested that low-dose folate also be given
to patients with AIHA as long as the warm AIHA
persists because patients with chronic hemolytic
anemia often have folate deficiency.2 The use of
folic acid will often improve the reticulocyte
response particularly in folate deficient patients.
Use of Splenectomy
A splenectomy should be considered in patients
who have not had a satisfactory response to initial
corticosteroids, who relapse after having
responded, or require the equivalent of more than
10 to 15 mg prednisone per day to maintain an
acceptable hemoglobin level. Although the efficacy
of splenectomy for the treatment of warm AIHA
patients has never been compared to that of other
second-line therapies, splenectomy is probably the
most effective second-line treatment. 1-4,65 The
results of splenectomy are variable, with a reported
success rate estimated at 38% to 70% in patients
with idiopathic warm AIHA.2,66-69 Moreover, there
are no long-term data on remission duration after a
splenectomy. In recent studies on patients with
idiopathic warm AIHA or in patients with warm
AIHA secondary to chronic lymphocytic leukemia
(CLL), approximately 70% of patients were
reported to have a complete or prolonged response
with a follow-up of 3 to 20 years.68,69 Patients with
persistent or recurrent hemolysis after a splenecto-
my often will require lower doses of glucocorti-
coids than they had required previously.1
The beneficial effect of splenectomy may be
related to several factors. First, splenectomy
removes the primary site of RBC sequestration
and also decreases the quantity of RBC-bound
203
autoantibody seen in some patients. Apropos of the
latter, it has been reported that the levels of RBC-
bound IgG necessary to maintain a given rate of
hemolysis are 6 to 10 times higher in splenecto-
mized than in nonsplenectomized subjects.1
In many centers, laparoscopic splenectomy is
the procedure of choice because the surgical risk
of laparoscopic splenectomy is lower than that
seen with conventional surgery, particularly if the
size of the spleen is not extensive. In large series
of open splenectomies, mortality is approximately
6%, whereas in recent series reporting on
laparoscopic splenectomy, the mortality rate is
approximately 1.6%.67-69
The most feared complication after splenectomy
is overwhelming sepsis due to infections with
encapsulated bacteria. This complication is fortu-
nately uncommon but is associated with a high
mortality rate and occurs mainly in the first 2 years
after splenectomy. This serious risk can be
considerably reduced by the preoperative vaccina-
tion for Streptococcus pneumoniae, Haemophilus
influenzae type B, and Neisseria meningitidis
serogroup C.1,70 Such vaccinations ideally should
be done at least 2 weeks before the splenectomy. If
not done before surgery, it should be done
approximately 2 weeks after surgery, with revacci-
nation recommended, particularly for the pneumo-
coccus organism every 5 to 6 years. The role and
efficacy of antibiotic prophylaxis in this regard
remains unclear, and not all investigators recom-
mend it.1,70
Use of Danazol
Danazol is a synthetic derivative of ethinyl
testosterone that has mild androgenic properties.
The mechanism of action of danazol in the
treatment of warm AIHA is not completely
understood. Danazol can be used in association
with prednisone as first-line therapy in patients
with warm AIHA, allowing for a shorter duration
of prednisone therapy. The synergistic effects of
danazol and glucocorticoids have been demon-
strated in 2 studies.71,72 The best results (80%
complete response) were observed in patients
receiving a combination of danazol and glucocor-
ticoids as first-line therapy. The outcomes have
been much less impressive in relapsed or refrac-
tory AIHA patients (43% complete response and
14% partial responses).71,72 A retrospective study,
reported from France, did not observe any
204
reduction in the response rate nor in the reduction
in the duration of the prednisone therapy required
in patients treated with danazol.4
Other Immunotherapy Modalities to Treat Patients
With AIHA
Intravenous immunoglobulin. The treatment of
warm AIHA with intravenous immunoglobulin
(IVIG) alone or in combination with prednisone is
controversial, primarily because its use has mainly
been limited to small case series.73 In a retrospec-
tive study of 73 patients, a response to IVIG was
observed in 39.5% of patients.74 Another study
showed that a good response occurred in 5 patients
with recurrent warm AIHA associated with CLL.
In this study, the recovery of the hemoglobin level
was seen to be faster when prednisone and high-
dose IVIG were combined.75 Published reports of
IVIG use in warm AIHA suggest that it may be
necessary to use a high dose of IVIG similar to that
used for the treatment of patients with immune
thrombocytopenia (400-1000 mg/kg per day for 5
days). Intravenous immunoglobulin is also recom-
mended for use in patients with warm AIHA
refractory to conventional therapy with corticos-
teroids. Intravenous immunoglobulin has been
recommended as a possible temporizing measure
before doing a splenectomy.73-77 The mechanism
of the IVIG effect in AIHA appears to be related to
the saturation of Fc phagocyte receptors in
reticuloendothelial system that is amplified in
AIHA patients.73,77
Rituximab. For the past several years, the
effectiveness of rituximab has been evaluated in
several autoimmune diseases and in particular in
autoimmune hematologic disorders. Rituximab is a
monoclonal antibody directed against the CD20
antigen expressed on B cells in the blood, lymph
nodes, and bone marrow. The mechanism of action
of rituximab in patients with warm AIHA is probably
through the depletion of the B cells that produce the
autoantibodies against the RBCs; however, clinical
experience with rituximab in autoimmune diseases
suggests that autoantibody levels are not always
affected by the rituximab treatment.78 This may be
explained by that the binding of rituximab to B cells
generates immune complexes that may serve as
decoys to attract and bind Fcγ receptors expressed
on monoyctes and macrophages, diminishing the
recruitment of these cells at sites of immune complex
BARROS ET AL
deposition, thus, reducing the degree of inflamma-
tion and tissue damage.79
Rituximab has a very favorable benefit-to-risk
ratio in the treatment of patients with refractory
warm AIHA. Published reports indicate that
rituximab (375 mg/m2 weekly for 2-4 weeks) is
effective in treating warm AIHA with an overall
response rate ranging from 40% to 100% (median,
60%), with patients of all ages responding. Initial
studies in pediatric patients reported particularly
promising results, with rates of complete responses
varying from 87% to 100%. 80,84 In adults,
rituximab shows rates of complete responses
varying from 25% to 100%.85-93 Such results with
rituximab has been observed both in patients with
idiopathic warm AIHA and in patients with warm
AIHA secondary to a range of conditions, including
lymphoproliferative disorders, autoimmune dis-
ease, and also in patients having received bone
marrow transplants. The duration of the remission
achieved has been reported to range from 2 to 95+
months. The rapidity with which warm AIHA
patients reached a response to rituximab varied
considerably, with some patients responding very
quickly and others taking weeks or even months to
achieve their maximum response.80-93 Rituximab
has usually been used in patients with warm AIHA
as monotherapy; however, rituximab has also been
used in combination with corticosteroids, other
immunosuppressive drugs, and IFN-α, particularly
in patients with underlying lymphoproliferative
disorders.81,88-90 Preliminary data suggest that
rituximab retreatment may also be effective in
AIHA relapsed patients.80,81,84 A summary of the
data from the various studies using rituximab for
the treatment of warm AIHA is shown in Table 2.
Rituximab treatment in patients with warm
AIHA has been well tolerated, but some adverse
events have been reported including hypotension,
fever, chills, rigors, hypertension, and broncho-
spasm. The occurrence of such side effects seems
to be reduced with subsequent rituximab infu-
sions. The prolonged impairment of antibody
production also increases the risk of viral and
bacterial infections; thus, some infectious events
have been described related to the use of
rituximab therapy.80-93
The use of alemtuzumab in warm AIHA.
Alemtuzumab is a monoclonal antibody directed
against the CD52 antigen expressed on the cell
membrane of virtually all normal as well as malignant
WARM AIHA: IMMUNOBIOLOGY AND TREATMENT 205

Table 2. Reports on the Use of Rituximab for the Treatment of Warm AIHA
Complete Partial No
References Study n Age (y) Treatment protocol Type of AIHA response (%) response (%) response (%)

Quartier et al80 Prospective 6 0.6-2.9 375 mg/m2 weekly Primary 100 – –

for 4 wk
Gupta et al85 Prospective 8 46-70 375 mg/m2 weekly Secondary to CLL 87.5 12.5 –
for 2-5 wk
Shanafelt et al87 Retrospective – 21-79 375 mg/m2 weekly Primary (3) 40 – 60
for 3-8 wk Secondary (2)
Trapè et al86 Prospective 5 44-66 375 mg/m2 weekly Secondary to 60 40 –
for 4 wk lymphoproliferative
disorder
Zaja et al88 Prospective 4 42-84 375 mg/m2 weekly Secondary to CLL 25 – –
for 4 wk
Zecca et al81 Prospective 14 0.3- 375 mg/m2 weekly Primary (10) 71.4 14.3 14.3
13.8 for 2-4 wk Secondary (4)
Narat et al89 Retrospective 11 18-81 375 mg/m2 weekly Primary (2) 27 36 36
for 4 wk Secondary (9)
D'Arena et al90 Retrospective 14 48-87 375 mg/m2 weekly Secondary to CLL 21 50 39
for 3-4 wk
D'Arena et al91 Retrospective 11 23-81 375 mg/m2 weekly Primary 73 27 –
for 4 wk
Rao et al82 Prospective 6 5-17 375 mg/m2 weekly Primary 67 17 –
for 4 wk
Bussone et al92 Retrospective 27 15-81 375 mg/m2 weekly Primary (17) 30 63 7
for 2-4 wk Secondary (10)
Kaufman et al93 Retrospective 20 – 375 mg/m2 weekly Secondary to CLL 100 – –
for 3 wk
Kumar et al83 Retrospective 4 13-16 350-500 mg/m2 Secondary to SLE 100 – –
weekly for 2-4 wk
Svahn et al84 Prospective 4 0.1-0.9 375 mg/m2 weekly Primary (3) 75 25 –
for 4 wk Secondary (1)

lymphocytes, whether they are B or T cells.
Alemtuzumab has the ability to cause the lysis of
such cells by a host-effector mechanism such as
complement fixation, antibody-dependent cell-medi-
ated cytotoxicity, and the induction of apoptosis.
Alemtuzumab therapy can result in a prolonged and
profound peripheral blood lymphopenia particularly
affecting T cells.94 Alemtuzumab is very active in the
treatment of B-CLL, but the experience with the use
of alemtuzumab in patients with warm AIHA has
been very limited. Preliminary results indicate that
alemtuzumab may induce responses in patients with
severe warm AIHA, who failed to respond to
conventional immunosuppression.95-97 Interestingly,
one patient who was refractory to rituximab
responded to alemtuzumab.97 In small series of
patients with warm AIHA secondary to CLL and
refractory to conventional therapy such as corticos-
teroids, rituximab, or splenectomy, complete or
partial responses were achieved in all treated patients,
suggesting that alemtuzumab may be highly effective
in the treatment of severe, resistant B-CLL secondary
warm AIHA. Alemtuzumab should therefore be
considered even before rituximab, if warm AIHA is
accompanied by progressive CLL in patients also in
need of cytoreductive therapy.98-100
The Use of Other Immunosuppressive Approaches
in Patients With AIHA
Immunosuppressive drugs may also be indicated
mainly in patients refractory to prednisone, sple-
nectomy, and rituximab, or for those patients
resistant to these therapies and who may be unfit
to undergo splenectomy. Cytotoxic drugs such as
azathioprine, cyclophosphamide, cyclosporine,
mycophenolate mofetil (MMF) have been used to
treat refractory cases of warm AIHA in an attempt
to suppress autoantibody synthesis. Although
authentic successes have been reported with the
use of such agents, their relative effectiveness
compared to other treatments is unclear.1-4
Cyclophosphamide and Azathioprine. Many
patients with refractory warm AIHA have been
treated with small oral doses of cyclophosphamide
206
(100 mg/d) or azathioprine (100-150 mg/d). Most of
the reported data indicate an efficacy of 25% to 40%
in such patients. Such treatments should be
continued for up to 6 months before being
considered as failed because the response may be
somewhat slow in coming.1-4,65 In one small series,
high-dose cyclophosphamide (50 mg/kg per day for
4 days) was used in 8 patients with highly refractory
warm AIHA. Five patients achieved a complete
response, and 3 achieved a partial response, not
requiring RBC transfusion support. No relapses
were seen after a median follow-up of 15 months
(range, 4-29 months); however, most of these
patients had prolonged cytopenias requiring granu-
locyte colony-stimulating factor support. The use of
high-dose cyclophosphamide was associated with
nonfatal adverse effects including nausea, vomiting,
transient alopecia, and neutropenic fever.101
Vincristine-Loaded Platelets. One old but not
forgotten treatment modality available for the
treatment of patients with warm AIHA is the
infusion of vincristine-loaded platelets. Recently,
this form of treatment was used in 3 refractory warm
AIHA patients and in 3 subjects with refractory
Evan syndrome. All 6 patients with both immune
thrombocytopenia and AIHA initially achieved a
complete response, but 4 patients subsequently
relapsed. Two other patients achieved remissions
lasting for 8+ and 9+ years, respectively.102
Cyclosporin A. Cyclosporin A (CsA) has been
used effectively in a few patients with refractory
warm AIHA. In a comparative study to test the
effectiveness of CsA in the treatment of warm
AIHA and Evan syndrome, a high response rate
(89%) was seen in 18 patients treated with a
combination consisting of CsA, prednisone, and
danazol. In contrast, in 26 patients treated with only
prednisone and danazol, the complete response rate
was only 57.7%. In addition, the patients who
received CsA had a lower relapse rate compared to
those who did not (3.3% vs 70%, respectively).103
In another report, complete responses were ob-
served in 3 of 3 patients with refractory warm
AIHA treated with CsA.104 A good response was
also reported in 56% of patients with warm AIHA
associated with CLL treated with CsA.105
Mycophenolate mofetil. Mycophenolate mofe-
til is the prodrug of mycophenolic acid that inhibits
inosine monophosphate dehydrogenase, a rate-
limiting enzyme acting on the de novo synthesis
of guanosine nucleotides, especially in T and B
BARROS ET AL
cells. Mycophenolate mofetil potently suppresses
lymphocyte proliferation and has been successfully
used in organ transplantation to prevent graft
rejection. There has been only limited data reported
with the use of MMF in patients with refractory
warm AIHA. Most of the patients receiving MMF
have been reported to achieve partial responses, and
continuous treatment was required to maintain the
response.106-108 Mycophenolate mofetil has been
shown to be particularly effective in the treatment
of the refractory autoimmune cytopenias in children
with the autoimmune lymphoproliferative syn-
drome. In a study of 13 autoimmune lymphopro-
liferative syndrome patients with warm AIHA, it
was shown that 12 responded to MMF with
maintenance of an adequate blood count and/or
reduction, or cessation, of the need for other
immunosuppressive drugs. The median follow-up
time in responders was 49 weeks. The MMF
treatment was well tolerated in all patients.109
Hematopoietic Stem Cell Transplantation in
Warm AIHA
There is very limited information on the use of
hematopoietic stem cell transplantation (HSCT) in
patients with warm AIHA. In 2008, the registry of
the European Group of Blood and Marrow
Transplantation reported data on 65 transplants
(37 autologous and 28 alogeneic) in 59 patients
including subjects with AIHA, Evan syndrome,
idiopathic thrombocytopenic purpura, pure red cell
aplasia, pure white cell aplasia, or thrombotic
thrombocytopenic purpura. A complete response
was achieved in 2 of 2 patients with warm AIHA
subjected to allogeneic HSCT, whereas a partial
response was seen in only 1 of 5 patients treated
with an autologous HSCT.110
Therapeutic Plasma Exchange in AIHA Patients
The role of therapeutic plasma exchange (TPE)
in patients with warm AIHA remains unclear. The
indication for TPE in warm AIHA is considered as
a category III intervention (may be beneficial, but
insufficient evidence exists to establish efficacy) by
the American Society for Apheresis. Therapeutic
plasma exchange has been used sporadically in
patients with severe cold and warm AIHA for many
years in an attempt to remove circulating auto-
antibodies. Most of the published data have
indicated reductions in the autoantibody titer, but
clinical responsiveness has been inconsistent. Some
WARM AIHA: IMMUNOBIOLOGY AND TREATMENT
authors believe that TPE increases the efficiency of
RBC transfusions in patients with fulminant
hemolysis.111 In contrast, a recent retrospective
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