Autoimmunity Reviews 9 (2010) A350A354

Contents lists available at ScienceDirect

Autoimmunity Reviews
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev

Geoepidemiology of autoimmune hemolytic anemia

Jean-Francois Lambert a, Urs E. Nydegger b,
a
b

Service of Hematology, University Hospitals of Canton Vaud (CHUV), Lausanne, Switzerland

Transfusion Therapy Consultancy TTC, Emeritus Medical Faculty University of Bern, Switzerland

a r t i c l e

i n f o

a b s t r a c t
Autoantibodies against red blood cell antigens are considered the diagnostic hallmark of AIHA: Direct
antiglobulin test (DAT) completed by cytouorometry and specic diagnostic monoclonal antibodies (mAbs)
allow for a better understanding of autoimmune hemolytic anemia (AIHA) triggers. Once B-cell tolerance
checkpoints are bypassed, the patient loses self-tolerance, if the AIHA is not also caused by an possible
variety of secondary pathogenic events such as viral, neoplastic and underlying autoimmune entities, such as
SLE or post-transplantation drawbacks; treatment of underlying diseases in secondary AIHA guides ways to
curative AIHA treatment.
The acute phase of AIHA, often lethal in former times, if readily diagnosed, must be treated using plasma
exchange, extracorporeal immunoadsorption and/or RBC transfusion with donor RBCs devoid of the autoantibody target antigen. Genotyping blood groups (www.bloodgen.com) and narrowing down the blood
type subspecicities with diagnostic mAbs help to dene the triggering autoantigen and to select well
compatible donor RBC concentrates, which thus escape recognition by the autoantibodies.
2009 Elsevier B.V. All rights reserved.

Available online 27 November 2009

Keywords:
Autoimmune hemolytic anemia
Coombs test
Antiglobulin test
Direct antiglobulin test (DAT, sometimes
referred to as Coombs Test
Immunoglobulin
Complement (C)

Contents
1.
2.

Introduction . . . . . . . . .
Pathophysiology . . . . . . .
2.1.
Thermal range and type
2.2.
Drugs . . . . . . . . .
3.
Diagnosis . . . . . . . . . .
4.
Treatment . . . . . . . . . .
Take-home
.
messages . . . . . . .
Acknowledgments . . . . . . . . .
References . . . . . . . . . . . .

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1. Introduction
Autoimmune hemolytic anemia (AIHA) is a result of insufciently
compensated peripheral red cell destruction. Hemolysis involves a
shortening of the half life of red blood cells (RBC), from normally
100 days down to a few days in overt cases. Like in other
autoimmune diseases, AIHA shares the offence of the hosts' immune
system against his/her own tissue, in this case the (RBC). The antibody
(Ab) and complement (C)-offended RBC either lyses or becomes
phagocytosed by Fc- and C-receptor bearing cells. Some authors

Corresponding author. Transfusion Therapy Consultancy, P.O. Box 784, CH-3000

Bern 9, Switzerland. Tel.: +41 79 652 9573; fax: +41 313811083.
E-mail address: urs.nydegger@octapharma.ch (U.E. Nydegger).
1568-9972/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.autrev.2009.11.005

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compare AIHA to aplastic anemia, caused by autoreactive T lymphocytes which suppress or destroy hematopoietic cellsan aggression
against the RBC at an early stage of maturation brought about by early
stages of (auto-) immune responses indeed.
Epidemiology of AIHA is mainly limited to adults. Although
children with primary immunodeciency disease (PID) are commonly
affected, the rarity of this association does not tip the balance to
pediatrics. Normally children show self extinguishing mild courses
often induced by viral infections. Affected adults tend to develop a
more severe anemia requiring medical management. AIHA has no
known predisposition. There is no familial hereditary component,
there is no age preselection, nor have we identied genetic
background making an individual susceptible to develop AIHA.
Different forms of AIHA can be distinguished based on involved
autoimmune process with autoantibodies maximally active at body

J.-F. Lambert, U.E. Nydegger / Autoimmunity Reviews 9 (2010) A350A354

A351

temperatures or at cooler ranges respectively referred to as warmtype AIHA or cold agglutinin disease. An alternate classication of
AIHA is based on the presence/absence of an underlying disease in the
latter case the condition is termed primary or idiopathic AIHA.
Secondary AIHAs may be associated with lymphoproliferative
disorders (e.g. Hodgkins or non Hodgkins lymphomas), with
rheumatic disorders, primarily SLE, with certain infections [1], with
nonlymphoid neoplasma (e.g. ovarian tumors), certain chronic
inammatory diseases (e.g. ulcerative colitis) or with certain drugs
(e.g. -methyldopamin, alemtuzumab, mycophenolate, purine analogue alkylating agents [2]) (Table 1).

2. Pathophysiology
2.1. Thermal range and type of hemolysis
Thermodynamics is the rst factor: complexes of anti-RBC Ab and
their antigen on or adsorbed in the form of previously soluble immune
complexes [3] to RBCs are unable to form strong bridges, requiring the
intervention of rabbit anti-human antibodies to link and agglutinate
the RBCs. While most cold antibodies are IgM class, Donath
Landsteiner (DL, paroxysmal cold hemoglobinuria) antibodies are
IgG: they bring C to the surface at warm temperatures and leave the
RBC b25 C with C left back on the cell surface. To the immunohematologist, b30 C is already cold and for patients suffering from
this type of AIHA, exposure to cold air or food can sufce to trigger an
AIHA crisis. The diagnosis of mixed-type AIHA is based on demonstrating the presence of warm IgG autoantibody and low titre
(b64 2 at 4 C), high thermal amplitude (reacting at/or N30 C)
cold IgM autoantibody. While mixed-type AIHA on the same patient
is uncommon, large groups of AIHA patients classify to different
temperature spectra of anti-RBC antibody reactivity. RBC agglutination on the peripheral blood lm is a common nding in mixed-type
AIHA and can lead, initially, to a misdiagnosis of cold agglutinin
disease. Behind the three major types of autoantibodies, i.e. warm,
cold and DL, a multitude of etiological backgrounds are hiding as
shown in Fig. 1.
Hemolysis mechanisms are different according to the Ab subtype.
In warm Ab reactions, lysis is triggered by xation of Ab to the RBC
followed by Fc recognition on spleen macrophages (Fig. 2A). The
antigen specicity of warm-type antibodies remains most often
elusive.
With cold Ab, the complement pathway is engaged, resulting in
C3b mediated phagocytosis mainly by the liver Kupffer cells while
membrane associated complex (MAC) is a minor mechanism if IgM
titer is low (Fig. 2B).
The blood type I/i, carbohydrates related to the ABO and Lewis
blood groups, is always involved in cold agglutinin disease. Cold
agglutinins specic for i are often polyclonal in children following
viral infections. Specicity for I is classical for Mycoplasma lung
infection with polyclonal antibodies while monoclonal anti-I are
found associated to lymphoproliferative syndromes [4].
Table 1
Classication of immune hemolytic anemia.
Autoantibody

Alloantibody

Primary

Secondary

Idiopathic

Lymphoproliferative
syndrome
Infections
Systemic autoimmune
disease
Drug associated
Other

Hematopoietic stem cell.

Pregnancy
Post-transfusional
Post HSC or solid organ
transplantation

Fig. 1. The triangle of antibody types involved in autoimmune hemolytic anemia upper
left corner: warm reactive autoantibodies are typical for AIDS, cold reactive are typical
for mycoplasma and quite frequently, Treponema pallidum infections become
complicated by paroxysmal cold hemoglobinuria. The center overlap of the gure
mentions etiologic factors involved in multiple antibody subtypes.

Paroxystic cold hemoglobinuria associated to DL Ab has a biphasic

occurrence with cold xation of DL IgG followed by warm
engagement of complement mainly leading to MAC hemolysis
(Fig. 2C). In this setting, the P RBC antigen is involved.

Fig. 2. Mechanistic background of red blood cell hemolysis in autoimmune hemolytic

anemias. A. In warm reactive IgG AIHA, the red blood cell binds the autoantibody to a
membrane expressed cognate autoantigen of elusive specicity. Thereafter (right
panel), the RBC is trapped by spleen macrophages through Fc receptor interaction. B. In
cold reactive IgM AIHA, the I/i antigens on RBCs become covered with IgM class
antibodies simultaneously xing C1q whereupon the IgM leaves the cell at higher
temperature leaving opsonising complement (C3b) and MAC back on the cell.
Thereafter, RBC is trapped by liver macrophages (Kupffer cells) through complement
receptor interaction. C. In paroxysmal cold hemoglobinuria, the biphasic Donath
Landsteiner IgG interacts with P antigen carrying red blood cells at cold temperature,
xes complement, and leaves the cell surface at warm temperature. Thereafter,
complement dependent hemolysis is triggered mainly through membrane attack
complex (MAC) perforation.

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J.-F. Lambert, U.E. Nydegger / Autoimmunity Reviews 9 (2010) A350A354

2.2. Drugs
There are three types of drug-associated Ab: (i) hapten type is due
to the noncovalent binding of the drug to the RBC then targeted by the
antibody in a drug-dependent manner (Fig. 3A); (ii) autoantibodies
specic to the drug also recognize a self-Ag at the RBC surface, thus
triggering a drug-independent hemolysis (Fig. 3B); (iii) drugAb
immune complexes can activate the complement, thus inducing a
non-specic, but drug-dependent RBC hemolysis (Fig. 3C). The drugs
most frequently associated with AIHA are cefotetan (Cefotan) and
cefriaxone (Rocephin) [5].
AIHA has been noted with increased incidence in patients
receiving purine nucleoside analogues for hematologic malignancies.
Oxaliplatin, a drug used for colo-rectal malignancies, may cause nonimmunologic protein adsorption to RBCs, similarly to cisplatin and has
been shown to induce drug-dependent antibody formation [2]. Upon
hematopoietic stem cell transplantation recipients of ABO-mismatched products can receive non-self, becoming self in the
posttransplant life, anti-RBC antibodies, transferred by passenger
lymphocytes from the donor; fortunately, AIHA in these cases is
generally transient, even upon severe hemolysis [6,7]. Venous
thromboembolism is a little-recognized, though likely common,
complication of AIHA and in some instances may be related to
coexistent anti-phospholipid antibodies [8].
Monoclonal antibody therapy of an ever-increasing number of
labeled indications can induce, in rare instances, AIHA [9] due to the
formation of soluble immune complexes involving mAb.
3. Diagnosis
The diagnosis of AIHA must go beyond the clinical triad of anemia,
splenomegalia and jaundice, the latter two signs often being absent
(Table 2). It is the direct antiglobulin test (DAT, Coombs test), which is
the most powerful diagnostic tool after a long train of deductions for
AIHA. If AIHA still remains idiopathic in many patients, currently
many forms come to light as secondary form for different reasons
related to modern diagnostic tools.
A reactive DAT should be sought by all means and, if reactive,
rarely proves to be capable of bearing another interpretation than
AIHA. The distinction between primary and secondary AIHA is more of
a scholarly problem since the former is an exclusion diagnosis and
must remain one during the follow up care of the patient. The
presence of anti-RBC autoantibodies with a reactive DAT are the
mainstays of AIHA. Using agglutination tests and/or ow cytometry,
IgG, proteolytic fragment of complement (mainly C3 and C4) or both
are found on the thus damaged RBCs. The blood lm exhibits
polychromasia and spherocytosis, the latter a blood lm hallmark of

Table 2
Signs and symptoms of autoimmune hemolytic anemia in percentage.

Breath shortage
Pallor
Splenomegaly
Jaundice
Rapid heart rate

Percent cases
with this sign

Circumstance

Reference
number

6070
100
20
60
50

Relates to acute cases

Hb b 80 g/L
Ultrasound diagnosis required
[26]
Upon physical effort

the disease visible not only at microscopic inspection but detectable

also by the CellAVision device [10]. Indirect hyperbilirubinemia,
increased urinary urobilinogen and serum lactate dehydrogenase and
decreased serum haptoglobin are variably present but not necessary
for the diagnosis.
Laboratory tests, if fully detached from clinical pictures must be
reviewed critically (Table 3): the importance of the DAT, while rocksolid, should not be overestimated because DAT negative AIHA exist
[11]. To stretch diagnostic inference too far with DAT may mislead the
physician and patient. Thus the antiglobulin serum used to detect
RBC-bound IgG may fail because it might not be reactive with IgA antiRBC. DAT results may become reactive only, when C protein fragments
cover the RBC surface brought about by C-activating antibodies, albeit
non-C-activating antibodies are inducing AIHA with identical erceness, as recently conrmed with a well-established mouse model [12]
in which erythrophagocytosis by Kupffer cells was also evaluated.
Accordingly, the quality/composition of RBC-reactive immune complexes has been shown as relevant in human medicine [13] IgG2
containing IgMIgG immune complex being innocuous to RBCs.
Evidence is accruing, that the DAT will more frequently become
completed using, instead of reading agglutination patterns,
performing ow cytometry (FC). In 33 Chinese patients with AIHA
who were found to have RBC-bound IgG, both the mean uorescence
intensity (MFI) and percentage of uorescence-activated RBCs were
remarkably increased and results of both were considered positive
[14]. This indicates that modern Chinese medicine is now equipped
with laboratory automation systems and more sophisticated set ups.
The prevalence is the estimated population of people who are
managing AIHA at any given time, and incidence means the annual
diagnosis rate, which in turn depends on clinical pathology laboratory
technology. An attempt to extrapolate the occurrence of AIHA in
different countries on the 5 continents is given on Table 4 and is
dependent on a fair reporting system. In anemic patients with
negative Coombs test the results of FC were always negativethe
incidence of AIHA thus depends on the capability to push diagnosis to
a stage, where conrmation/rejection is possible at the laboratory
level.
FC performed in suspected AIHA cases can be reactive whilst
conventional, agglutination-based Coombs test remained negative
[15]. In this study, there was a poor correlation between strength of

Table 3
Laboratory ndings bearing strong evidence for autoimmune hemolytic anemia.

Fig. 3. Major subtypes of drug-associated hemolytic antibodies. A. Classical, hapten

related autoantibody of the penicillin type depending on a low afnity attachment of
the drug to the RBC surface allowing warm reactive IgG xation with consecutive Fc
opsonisation. B. Drug-independent hemolytic mechanism (e.g. levodopa) associated
with the random occurrence of a drug-specic antibody that crossreacts RBC membrane
components. C. Immune complex autoimmunohemolytic mechanism related to the
non-specic attachment of drugantibody immune complex (e.g. quinine) to the RBC
membrane followed by engagement of the complement system leading to membrane
attack complex (MAC)-dependent lysis.

Percent cases with this

nding% (approx)

Circumstance

Hyperbilirubinemia

80

Increased serum lactate

dehydrogenase,
Reticulocytosis
Mean reticulocyte
hemoglobin content
Coombs test

100

Indirect, prehepatic
bilirubin increased [26]
Iso-form 5

Flow cytometry.

100
Reduced
90% (revealed by
agglutination)

95% revealed by FCa [20]

J.-F. Lambert, U.E. Nydegger / Autoimmunity Reviews 9 (2010) A350A354

Table 4
Geoepidemiology and age distribution of autoimmune hemolytic anemia*.
AIHA
Geography Brazil , China , Europe, India,
USA, Philippines, Russia,
Saudi Arabia,
Age-range 4050 years
Gender
m/f 40/60

Cold agglutinin PNH

disease
Northern
climates
Young adults
50/50

PIG-A gene on x
chromosome, i.e.
non-sex linked

Based, at least in part on: www.cureresearch.com/a/autoimmune_hemolytic_anemia/

stats-country.htm.

Coombs test and mean uorescence intensity by FC. Preliminary

evidence suggests that FC-based Coombs test is a more sensitive test
than the hemagglutination-based and helps in the serological
diagnosis of hemagglutination-based Coombs negative AIHA. In
resource poor settings, the hemagglutination-based DAT remains
state of the art.
The quantity of IgG subclass bound to the surface of RBCs can be
estimated [16]. A mean number of 80,000 to 120,000 molecules of
IgG1/RBC were detected. The mean concentration of IgG2a autoantibodies was lower than 40,000 molecules/RBC. In humans, the
healthy RBC normally carries several thousands of IgG molecules
because in the normal state, RBCs transport physiological immune
complexes [3] but cut offs of both Coombs tests and CF need to be set
such that these normal amounts do not show up as pathological. This
might not be the end of increasing the sensitivity of the DAT principle.
Single-cell analysis which avoids loss of information associated with
ensemble averaging of large cell numbers is now possible: a
microuidic device lets one manipulate, lyse, label, separate and
quantify protein contents of single cells using molecule uorescence
countingif such technology will perhaps not help to improve patient
care, at least it could help us to better understand the development of
AIHA at the molecular level [17]. Other researchers use the activity of
phagocytes to ingest damaged RBCs with the erythrophagocytosis
assay which has the advantage of revealing function (Table 3). Such a
system may help in the identication of AIHA patients with negative
DAT test as well [18].
As with immune thrombocytopenic purpura syndrome, predictive
values for AIHA do exist but since this disease may or may not express
itself in a number of underlying immunopathological disorders their
exchange for corroboration of diagnosis is relatively weaktoo many
(laboratory) criteria must act in concert to boost positive prediction of
laboratory criteria [19] (Table 5).
There exists a susceptibility of AIHA in case of underlying diseases.
Thus, in contrast to the NZLB/W mouse, whose SLE is preceded by
AIHA, in humans, AIHA is a well-known but not an obligatory
complication of overt SLE. However, AIHA remains a rare complication
of any of the cited underlying diseases such that a PPV is way from
reaching signicance. A reactive DAT in individuals without clinical
overt AIHA cannot be used to predict development of AIHA. The
frequency of healthy blood donors with a positive direct DAT was
approximately 1 in 3000 in a recent study [20]. None of the donors
had hemolysis. This makes the DAT reactive donors a reason for
incompatibility results upon RBC donor/recipient compatibility
testing.
The hemolytic anemia that concurs with immune thrombocytopenia and/or neutropenia can be ascribed to a general tolerance defect
underlying all immune cytopenias. Thus, defects at various stages of
peripheral tolerance checkpoints are not lineage-specic [19] suggesting that the proclivity to develop red cell specic antibodies arises
through diverse mechanisms, blurring the distinction between ITP
and Evans syndrome.

A353

Table 5
Serological markers of autoimmune hemolytic anemia.
Antibody property

Particularity

Anti-RBC
Often with Rhesus D
autoantibodies
specicity
Association of auto- with 1547% of cases
alloantibodies
Circulating immune
Defective composition
complexes
Anti-phospholipid
antibodies
Cold agglutinins
DonathLandsteiner
Erythrophagocytosis

Part of anti-phospholipid
syndrome; part of disease
cluster
20% of cases
5% of cases
In the presence/absence of
opsonizing antibodies

Reference number
[27]
(Meta-analysis [28])
Other than IgG2 subclass
in mixed IgMIgG
complexes [13]
[8]

4. Treatment
The range of hemolysis intensity is wide. Thus most AIHA will be
compensated by an increased RBC production. In some instances, the
hemolysis is so active that treatment becomes an emergency. Many
years ago, MuellerEckhardt and his disciples have taught us, that RBC
concentrate transfusions should not be withheld to severely anemic
AIHA patients with reactive DAT. Compatibility testing is the standard
protocol that identies suitable blood for patients requiring transfusion. When autoantibodies or non-clinically signicant alloantibodies
compromise the indirect DAT the supply of RBC transfusions will
delay the supply of blood to the patient. No adverse reactions were
reported in a recent study where suitable blood was provided after a
serologically mismatched DAT testing.
Glucocorticoids are effective in slowing the rate of hemolysis. Since
speculation exists that the spleen is the principal producer of anti-RBC
autoantibodies, splenectomy may become indicated for patients who
require an unacceptable high maintenance of prolonged administration of steroids, but these cases should rst go through other options
than the surgeon's knife. In contrast to immune thrombocytopenic
purpura, where intravenous immunoglobulins (IVIg) are often
curative for thrombocytopenia, IVIg in AIHA may provide shortterm ultimately non-curative control of hemolysis, especially in
children, and immunosuppressive drugs and danazol have been
used with success in refractory adults. Rituximab (anti-CD20), directly
targeting the Ab-producing B-cells can be used in selected cases.
[21,22].
With anti-RBC antibodies of known specicity, novel approaches
in therapy might prove useful for some patients. Thus, anti-A/B
antibodies could also become depleted using an absorber technology
(www.absorber.se). Prior to removing such antibodies by plasma
exchange or absorption, a careful diagnosis now involves such
techniques as Luminex or XM-ONE of AbSorber whose commercially available product is based on beads (magnetic nanoparticles)
carrying synthetic blood group A and B antigens. By adding patient
sera to the beads, the presence of blood group antibodies can be
measured by ow cytometry. The most common measurement
methods today are hemagglutination or ABO-ELISA. Measurement
results are unreliable in that they vary from time to time and from
clinic to clinic.
Recently, the successful use of a mAb targeted against C5,
preventing the formation of the membrane attack complex (eculizumab, Soliris, Alexion Pharmaceutical's), has transformed the management of paroxysmal nocturnal hemoglobinuria. Early experience
in severe AIHA is promising, although sometimes side effects, such as
gall stones, may arise and the prize is considerable [23].
Most vaccines contain adjuvants in addition to the target antigen.
Therefore, before vaccinating subjects prone to develop autoimmune

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J.-F. Lambert, U.E. Nydegger / Autoimmunity Reviews 9 (2010) A350A354

conditions, the prescriber ought to carefully rule out possible

potentiation of preexisting autoimmune reactions [24,25].
Take-home messages
Diagnosis relies on regenerative anemia, hemolysis and positive
direct antiglobulin test.
Generally, warm antibodies trigger an extravascular hemolysis with
microspherocytes while cold antibodies induce intravascular hemolysis through activation of complement.
AIHA is rare in children and frequently associated to drugs in adults.
Treatment include immunosuppression (steroids, azathioprin) and
splenectomy for warm Ab and conservative measures for cold Ab
(cold avoidance).
Transfusion is always difcult, but should not be delayed in severe
anemia.
Acknowledgments
UEN acknowledges the continuous support of Octapharma
(Lachen, Switzerland) and Labormedizinisches Zentrum Dr. Risch
(Schaan, Liechtenstein); as yet, the text has been written without
conict of interest.
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