International Journal of

Molecular Sciences

Review
Autoimmune Hemolytic Anemias: Classifications,
Pathophysiology, Diagnoses and Management
Melika Loriamini 1,2 , Christine Cserti-Gazdewich 1,3,4 and Donald R. Branch 1,2,5, *

1 Department of Laboratory Medicine and Pathobiology, University of Toronto,

Toronto, ON M5B 1W8, Canada; [email protected] (M.L.); [email protected] (C.C.-G.)
2 Centre for Innovation, Canadian Blood Services, Keenan Research Centre, Room 420, 30 Bond Street,
Toronto, ON M5B 1W8, Canada
3 Laboratory Medicine Program, Blood Transfusion Laboratory, University Health Network,
Toronto, ON M5G 2C4, Canada
4 Blood Disorders Program, Department of Medical Oncology and Hematology, University Health Network,
Toronto, ON M5G 2C4, Canada
5 Department of Medicine, University of Toronto, Toronto, ON M5B 1W8, Canada
* Correspondence: [email protected]

Abstract: Autoimmune hemolytic anemias (AIHAs) are conditions involving the production of
antibodies against one’s own red blood cells (RBCs). These can be primary with unknown cause or
secondary (by association with diseases or infections). There are several different categories of AIHAs
recognized according to their features in the direct antiglobulin test (DAT). (1) Warm-antibody AIHA
(wAIHA) exhibits a pan-reactive IgG autoantibody recognizing a portion of band 3 (wherein the
DAT may be positive with IgG, C3d or both). Treatment involves glucocorticoids and steroid-sparing
agents and may consider IVIG or monoclonal antibodies to CD20, CD38 or C1q. (2) Cold-antibody
AIHA due to IgMs range from cold agglutinin syndrome (CAS) to cold agglutin disease (CAD). These
are typically specific to the Ii blood group system, with the former (CAS) being polyclonal and the
latter (CAD) being a more severe and monoclonal entity. The DAT in either case is positive only
with C3d. Foundationally, the patient is kept warm, though treatment for significant complement-
related outcomes may, therefore, capitalize on monoclonal options against C1q or C5. (3) Mixed
AIHA, also called combined cold and warm AIHA, has a DAT positive for both IgG and C3d, with
Citation: Loriamini, M.; Cserti-
treatment approaches inclusive of those appropriate for wAIHA and cold AIHA. (4) Paroxysmal cold
Gazdewich, C.; Branch, D.R.
hemoglobinuria (PCH), also termed Donath–Landsteiner test-positive AIHA, has a DAT positive only
Autoimmune Hemolytic Anemias:
for C3d, driven upstream by a biphasic cold-reactive IgG antibody recruiting complement. Although
Classifications, Pathophysiology,
Diagnoses and Management. Int. J.
usually self-remitting, management may consider monoclonal antibodies to C1q or C5. (5) Direct
Mol. Sci. 2024, 25, 4296. https:// antiglobulin test-negative AIHA (DAT-neg AIHA), due to IgG antibody below detection thresholds in
doi.org/10.3390/ijms25084296 the DAT, or by non-detected IgM or IgA antibodies, is managed as wAIHA. (6) Drug-induced immune
hemolytic anemia (DIIHA) appears as wAIHA with DAT IgG and/or C3d. Some cases may resolve
Academic Editor: Balik Dzhambazov
after ceasing the instigating drug. (7) Passenger lymphocyte syndrome, found after transplantation, is
Received: 11 March 2024 caused by B-cells transferred from an antigen-negative donor whose antibodies react with a recipient
Revised: 1 April 2024 who produces antigen-positive RBCs. This comprehensive review will discuss in detail each of these
Accepted: 3 April 2024 AIHAs and provide information on diagnosis, pathophysiology and treatment modalities.
Published: 12 April 2024

Keywords: autoimmune hemolytic anemias; AIHA; warm-antibody AIHA; cold-antibody AIHA;

PCH; direct antiglobulin test; DAT

Copyright: © 2024 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article
1. Introduction
distributed under the terms and
conditions of the Creative Commons Autoimmune hemolytic anemia (AIHA) is defined by the destruction of an individ-
Attribution (CC BY) license (https:// ual’s own red blood cells (RBCs), caused by the existence of autoantibodies that target
creativecommons.org/licenses/by/ them [1–8]. This process leads to fewer circulating RBCs. In extreme instances, the lifespan
4.0/). of red blood cells (RBCs) is reduced, dropping from the usual range of 100–120 days to

Int. J. Mol. Sci. 2024, 25, 4296. https://doi.org/10.3390/ijms25084296 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024, 25, 4296 2 of 18

only a few days [1–4]. The internal components of red blood cells are released into the
circulation and surrounding tissues, producing specific symptoms related to the condi-
tion [3]. Autoantibodies usually target antigens found on virtually all RBCs and a direct
antiglobulin test (DAT) is useful in the diagnosis [3]. Because patients with AIHA often
require transfusion to address their anemia, finding compatible blood is challenging [3,9,10].
Fortunately, transfused blood survives, as does the patient’s own, so the transfusion of
antigen-selected units despite incompatibility should not be withheld [3,10]; in so doing, it
is imperative to rule out underlying alloantibodies that could differentially interact with
the transfused donor blood [8,9].

1.1. Acquired Hemolytic Anemias

Importantly, a positive DAT does not automatically indicate AIHA. In one study, 51%
of DAT-positive patients did not have hemolysis [11]. These were predominately patients
with tuberculosis and hepatitis C.
Before one explores whether an anemic patient has AIHA, other causes of anemia
must be ruled out, and evidence of a hemolytic anemia must be confirmed [3,12]. Bleed-
ing must be ruled out as well as metabolic conditions, e.g., iron deficiency anemia or
vitamin B12 deficiency. Congenital reasons for the anemia need to be ruled out, from
enzymopathies (e.g., glucose 6-diphosphate (G6PD)-deficiency) to hemoglobinopathies
(e.g., sickling disorders, thalassemia), and other inherited defects in structure and function
(e.g., McLeod syndrome).
Kinetic and biochemical mimics include liver cirrhosis with active gastrointestinal
bleeding or resorbing large hematomas. If these are excluded, then hemolytic anemia
is inferred by increases in the LDH, reticulocyte count, and (indirect) bilirubin, with the
converse reduction (or complete quenching) in haptoglobin [3]. Once active, acquired
hemolysis is established, the DAT is the most useful way to classify the suite of potentially
explanatory AIHAs.

1.2. Classification and Categories of AIHA

AIHA is classified as either primary or secondary. The disease may manifest either
as a primary ailment or as a secondary condition stemming from an underlying sickness.
Primary AIHA accounts for more than 60% of patients [3]. Secondary AIHA may occur
due to many underlying medical disorders, including autoimmune illnesses such as lu-
pus, chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma (NHL), and other
blood malignancies, as well as infections caused by Epstein–Barr virus, cytomegalovirus,
Mycoplasma pneumonia, hepatitis, and HIV [3,8,13,14]. Recently, AIHA has been associated
with COVID-19 [15] AIHA is further classified according to the temperature at which red
blood cells experience opsonization and destruction.
AIHA comprises seven distinct types that have been categorized mainly based on
serological findings [3,12]. The seven types of AIHA are as follows: warm-antibody au-
toimmune hemolytic anemia (wAIHA); cold-antibody AIHA (includes cold agglutinin
syndrome (CAS) and cold agglutinin disease (CAD)); mixed AIHA (also called combined
cold and warm AIHA); paroxysmal cold hemoglobinuria (PCH), also called Donath–
Landsteiner antibody test-positive AIHA; direct antiglobulin test-negative AIHA (DAT-
negative AIHA); drug-induced autoimmune hemolytic anemia (DIAIHA); and passenger
lymphocyte syndrome (PLS) (related to transplantation, and presenting as AIHA with a
specificity) [2,3,5,15–25] (see Table 1).
Int. J. Mol. Sci. 2024, 25, 4296 3 of 18

Table 1. Categories of Autoimmune Hemolytic Anemias.

Warm-antibody autoimmune hemolytic anemia (wAIHA)

Cold-antibody autoimmune hemolytic anemia
Cold agglutinin syndrome (CAS)
Cold agglutinin disease (CAD)
Mixed autoimmune hemolytic anemia (mixed AIHA)
Paroxsymal cold hemoglobinuria (PCH)
Direct antiglobulin test-negative autoimmune hemolytic anemia (DAT-neg AIHA)
Drug-induced autoimmune hemolytic anemia (DIAIHA)
Passenger lymphocyte syndrome (PLS)

AIHA should be suspected if the patient shows signs and symptoms of anemia and
other causes of the anemia, such as bleeding, underproduction, paroxysmal nocturnal
hemoglobinuria (PNH), and other potential causes of anemia have been ruled out [3],
see Section 1.1 above. The most important serological test for the diagnosis of AIHAs
is the DAT using polyspecific and monospecific anti-human globulins (AHG) [3,12,22].
The initial testing should be with a DAT using a polyspecific AHG that contains anti-
IgG and anti-C3d. If positive, then the DAT should be performed with monospecific
AHG, one containing anti-IgG and one containing anti-C3d. Table 2 shows the expected
results for the DAT depending on the particular type of AIHA. The antibody may only
react by an indirect antiglobulin test (IAT) in wAIHA and mixed AIHA. In cold-antibody
AIHA, the antibody is an IgM cold agglutinin that reacts better in colder temperatures
(i.e., ≤room temperature) and involves (recruits and activates) complement so that the
DAT will be positive only with polyspecific and monospecific anti-C3d AHG. In mixed
AIHA, the IgG antibody reacts in the IAT while the IgM antibody is an agglutinating
antibody having low titer but high thermal activity (≤37 ◦ C). PCH will present with a
positive DAT due to anti-C3d only; however, the serum autoantibody is an IgG bi-phasic
hemolysin that is detected using the Donath–Landsteiner (D-L) test and recognizes the
P-blood group system. Thus, it is non-reactive with p RBCs. Using standard “blood bank
techniques for DAT and elution”, the DAT, eluate and serum antibody are negative in
DAT-neg AIHA; however, the DAT can be shown to be positive with IgG using specialized
tests such as Western immunoblotting [24]. Drug-induced AIHA serologically presents as
indistinguishable from wAIHA. Diagnosis in drug-dependent forms is established when
stopping the suspected drug resolves the destruction, though some drugs may trigger true
wAIHA via iatrogenic immune dysregulation [3,26]. Passenger lymphocyte syndrome is
diagnosed when following a solid organ or bone marrow transplant, the patient develops
an antibody to the recipient’s RBCs. While presenting serologically as a wAIHA, with or
without specificity, it is owing to the donor’s B-cells producing an antibody to the recipient’s
residual RBCs [17,25].
Int. J. Mol. Sci. 2024, 25, 4296 4 of 18

Table 2. Serological Diagnosis of Autoimmune Hemolytic Anemias.

Category DAT # Eluate Serum Antibody

IgG * 67%
wAIHA IgG + C3d 20% Pos (IgG) IgG panagglutinin
C3d only 13%
Polyclonal
CAS C3d only Neg
agglutinating IgM
Monoclona
CAD C3d only Neg
agglutinating IgM +
IgG panagglutinin +
Mixed AIHA IgG + C3d Pos (IgG) agglutinating IgM with low titer but high therm
amplitude (≤37 ◦ C)
Cold-reactive IgG
PCH C3d only Neg
biphasic hemolysin **
DAT-Neg AIHA Neg *** Neg Neg
Drug-induced AIHA IgG only Pos (IgG) ˆ Pos (IgG)
Passenger lymphocyte Syndrome IgG only Pos (igG) IgG &
wAIHA, warm-antibody autoimmune hemolytic anemia; CAS, cold agglutinin syndrome; CAD, cold agglutinin
disease; PCH, paroxysmal cold hemoglobinuria. # Direct antiglobulin test; * Rarely due to IgM or IgA antibodies;
+ Agglutination titer can be high at colder temperatures (≤RT), may react up to ≤30 ◦ C, usually anti-I/i specificity;

** Diagnosis made using Donath-Landsteiner (D-L) Test, usually anti-GLOB, fails to react with p RBCs; *** IgG
can be found in the DAT using special tests but not with standard serological tests or in eluates using standard
methods; ˆ Can be positive with C3d; & Usually has specificity within ABO (anti-A or anti-B) or Rh (anti-D) blood
group system.

1.3. Incidence
Based on current calculations, the yearly occurrence of AIHA is 1–2 cases per
100,000 persons [18]. Out of all the instances, wAIHA is the most common type, account-
ing for two-thirds of all cases [3,18,19]. CAS/CAD is the second most common AIHA,
accounting for 15–20% of cases [3,20]. Mixed AIHA is the third most common AIHA, as
a co-presentation of both wAIHA and cold-antibody AIHA [2,3]. PCH is a rare illness
disproportionately seen in children and associated with infections [3,21]. DAT-negative
AIHA is rare [3,22] and difficult to diagnose [24]. Passenger lymphocyte syndrome (PLS)
occurs after transplantation and is related to the immune cells from the donor, transferred
via the graft, subsequently producing host-specific anti-RBC antibodies, thereby mimicking
AIHA [17,25]. In the future, the incidence of AIHA may increase due to the use of stem cell
transplantation and checkpoint inhibitors in the treatment of cancers [27,28].

1.4. Pathophysiology of AIHA

AIHA occurs mainly due to IgG and IgM antibodies, although, rarely, IgA antibodies
may be causative [1–5,12,13,29]. Furthermore, complement activation is seen in all patients
with CAS/CAD and the majority of patients with PCH [3,12,20]. The pathophysiology
will differ based on the individual components implicated. IgG has a limited ability
to activate complement and has a high affinity for the Fcγ receptor (FcγR) found on
phagocytic cells [3,30]. AIHA that includes IgG can be identified by the phagocytosis of
RBCs [3,31]. IgM demonstrates robust activation via the conventional complement route [3].
Therefore, AIHA characterized by IgM usually involves the destruction of red blood cells
via processes involving the complement system [3]. While monocyte macrophages (MΦ) do
exhibit receptors for monomeric IgM Fc, the IgG-mediated phagocytosis of red blood cells
dominates, while pentameric IgM antibodies are more likely to induce phagocytosis by
complement activation [3,32]. This phenomenon arises due to the presence of receptors on
phagocytic cells that are specifically designed to bind to complement proteins C3b and C4b,
which are detectable using a DAT with anti-complement reagents, primarily anti-C3d [3,12].
In mixed AIHA, both IgG and C3d are found on the patient’s RBCs. This is due to an
Int. J. Mol. Sci. 2024, 25, 4296 5 of 18

agglutinating IgM cold agglutinin having a thermal amplitude to 37 ◦ C where it is able to

activate complement and a warm-reactive IgG antibody that is reactive at 37 ◦ C causing
a positive IAT [3]. In PCH, a “cold-reactive” IgG antibody binds to the patient’s RBCs at
cooler temperatures (below 37 ◦ C) and can activate complement when the temperature
warms to 37 ◦ C, though the patient’s RBCs show only residual complement (C3d) in the
DAT [3]. The removal of the opsonized RBCs by MΦ are by either FcγRs if IgG has been
activated or C3b/C4b receptors if complement has been activated. Usually, the removal of
opsonized RBCs in AIHA with IgG antibodies takes place in the spleen, while IgG plus
complement or complement alone on the RBCs occurs in the liver, specifically in Kupffer
cells [3]. In rare cases, IgA may induce AIHA [3,29,33,34].
The symptoms of AIHA vary depending on the type. The symptoms may include
dyspnea, fatigue, headache, muscular weakness, pallor and/or jaundice [3,8]. The devel-
opment of acrocyanosis and Raynaud phenomenon in CAS/CAD may culminate rarely
with gangrene [3,20]. Spherocytes are often seen in cases of wAIHA when there is the
inadequate phagocytosis of antibody-coated RBCs by MΦ [3,34]. The biochemical signs
of hemolysis identified in immune-mediated hemolysis include decreased hemoglobin
levels, alterations in cell markers linked to hemolysis such as higher lactate dehydrogenase
(LDH), reduced haptoglobin and increased unconjugated bilirubin [3,8,35]. In addition,
compensatory reticulocytosis may occur in those with unsuppressed, feedback-responsive
marrow reserves [3]. Some cases of AIHA occur with reticulocytopenia and the synergy
of underproduction with destruction may result in severe anemia with life-threatening
consequences [36,37].

2. Warm-Antibody Autoimmune Hemolytic Anemia (wAIHA)

wAIHA is commonly acknowledged as the primary occurrence of autoimmune-
mediated hemolytic anemia [2,3,5,12,18]. Roughly half of the instances have an unknown
cause, while the other half may be linked to either an existing underlying illness or the
use of certain drugs [3,14,38]. Unlike cold autoimmune hemolytic anemia conditions like
CAS/CAD and PCH, which are exacerbated in cold temperatures between 28 ◦ C to 31 ◦ C,
wAIHA occurs at normal body temperature [3]. The primary antibody isotype involved
with wAIHA is IgG, but IgA and IgM may be infrequently seen [3,12,29,32–34]. IgG anti-
bodies have a strong attraction to red blood cells when the temperature is at the normal
level of the human body (37 ◦ C). Cold-antibody-induced hemolytic anemia, in contrast, is
distinguished by antibodies that preferentially attach to red blood cells at lower temper-
atures, usually between 28 ◦ C and 31 ◦ C. This category includes CAS/CAD, PCH, and
mixed AIHA. However, in mixed AIHA, the IgM cold-reactive antibody can react to 37 ◦ C
by agglutination [39].
The pathophysiology of the hemolysis in wAIHA is classically extravascular, medi-
ated by the MΦ phagocytosis of antibody- and/or complement-opsonized autologous
RBCs [3,26,31,35]. However, natural killer cell-mediated antibody-dependent cellular cyto-
toxicity (ADCC) may also be active in some cases of wAIHA [40].
Furthermore, MΦ have the capacity to selectively remove portions of the red blood cell
membrane, resembling a biting action, in addition to phagocytosis [3]. This mechanism is
similar to trogocytosis [41]. In wAIHA, this leads to the degradation of the RBC membrane,
causing transformation into spherocytes [3,34]. Spherocytes possess less pliancy compared
to regular erythrocytes and are specifically singled out for removal in the red pulp of
the spleen and other constituents of the mononuclear phagocyte system. The observed
expansion of the spleen, splenomegaly, is related to the trapping of spherocytes and
autoantibody-opsonized RBCs in the red pulp of this organ [3].

2.1. Autoantibody Specificity

The specificity of autoantibodies in wAIHA has historically been tracked with the Rh
blood group system, because autoantibodies that reacted with all normal RBCs would fail to
react with rare RBCs lacking certain or all Rh antigens, from Rh dash-D-dash (-D-) to Rhnull
Int. J. Mol. Sci. 2024, 25, 4296 6 of 18

RBCs. For example, warm autoantibodies that reacted with all normal RBCs but not with
-D- and Rhnull RBCs were termed anti-nl (non-deleted or normal), while autoantibodies
that would react with all normal RBCs and with -D- but not Rhnull were termed anti-pdl
(partially deleted). Finally, autoantibodies that would react with normal, as well as with
both -D- and Rhnull , were termed anti-dl (fully deleted) autoantibodies [3]. Subsequently,
other specificities were reported to exist; for example, anti-Jka , anti-Gerbich, anti-LW and
anti-Kell [3,42,43]. Other scientists found autoantibodies reacting with components of band
3 on the RBCs [3,42]. As investigators dealt with the various specificities reported and
were using Rhnull and -D- RBCs to determine the anti-nl, anti-pdl or anti-dl specificity of
these autoantibodies, two groups, independently, found that in the majority of patients
with wAHIA, autoantibodies reacted preferentially with older, reticulocyte-depleted RBCs
and reacted much less or not at all with younger, reticulocyte-enriched RBCs [44,45]. Both
groups found that the frequency of reactivity was 80% of patients with wAIHA for the
former and 20% for the latter. Thus, warm autoantibodies in 80% of the cases showed a
tropism to “old RBCs” compared to “young RBCs”. One group termed this specificity as
Type I wAIHA versus Type II wAIHA (20% of patients) with wAIHA showing no RBC age
preference [45]. These investigators suggested that Type I wAIHA could be an exacerbation
of the normal clearance mechanism in RBC senescence, whereby natural autoantibodies
deplete old RBCs by targeting band 3 [45–48].
More recently, teams have shown that most warm autoantibodies do, in fact, target
band 3 [42,49,50]. In a recent publication, the authors confirm Type I and Type II wAIHA
and suggest that Type I wAIHA autoantibodies target band 3 and that the hemolytic
anemia may conversely involve patient RBCs that are also aging faster than normal [48].
Type I patients’ RBCs, by aging faster, expose the immune system to more senescent RBC
antigen, which in turn stimulates more autoantibody to senescent band 3, producing a
functional autoimmune disease [42]. In contrast, patients with Type II wAIHA showed a
lack of RBC aging, suggesting rapid and equal-opportunity opsonisation and the removal of
RBCs [49], consistent with a previous report of Type II wAIHA being more severe than Type
I wAIHA [44]. Additional investigations show that the natural autoantibody that binds
to aged RBCs, indeed, recognizes band 3 [49,50]. Taken together, these findings generate
the hypothesis that most wAIHA patients, at 80%, produce more of the senescent RBC
autoantibody that reacts with patients’ accelerated aging RBCs, for Type I wAIHA [42,49].
Another implication from this work is that the specificity of the autoantibody in Type
II wAIHA may be to other RBC antigens (non-band 3) [3,45]. These hypotheses require
further verification.

2.2. Biomarkers
Identifying biomarkers specific to particular disease states is an ongoing quest by
biomedical researchers. Although in AIHAs, the DAT is a true biomarker with classifica-
tion power (Table 3), signature cytokines may also qualify as biomarkers, given specific
associations in wAIHA [51–54]. In the most comprehensive report [51], 54 patients having
DAT+ wAIHA were compared with 36 healthy controls for 38 cytokines, chemokines or
growth factors using a multiplex approach and Luminex technology. This found that
TNFα and interleukin 10 (IL-10) are the most increased in wAIHA, with IL-8/CXCL8 and
IP10/CXCL10 being elevated and, hence, potentially eligible as biomarkers too. Taken
together, wAIHA patients have four possible cytokine/chemokine biomarkers, with TNFα,
IL-10, IL-8/CXCL8 and IP10/CXCL10, and perhaps IL-6 [53]. Future studies may validate
this repertoire.
Int. J. Mol. Sci. 2024, 25, 4296 7 of 18

Table 3. Summary of Pathophysiology and Treatments.

Category Pathophysiology Primary Target Antigen DAT as Biomarker First-Line Treatments &
Extravascular hemolysis Glucocorticoids,
wAIHA Band 3 IgG ± C3d
* steroid-sparing agents #
Intravascular hemolysis
CAS I/i antigens C3d only Keep warm
**
Intravascular hemolysis
CAD I/i antigens C3d only Anti-C1q/-C5
**
Intra- and wAIHA, CAS,
Mixed AIHA Band3; I/i antigens IgG + C3d
extravascular hemolysis CAD options
PCH Intravascular hemolysis GLOB (formally P antigen) C3d Anti-C1q/-C5, Rituximab
DAT-neg AIHA Extravascular hemolysis Unk negative wAIHA options
Intra- and
DIAIHA Unk ˆ IgG and/or C3d Discontinue drug
extravascular hemolysis
antigen-negative
transfusion support;
PLS Extravascular hemolysis ABO/Rh IgG
may adjust
anti-rejection regimen
DAT, direct antiglobulin test; wAIHA, warm-antibody autoimmune hemolytic anemia; CAS, cold agglutinin
syndrome; CAD, cold agglutinin disease; PCH, paroxysmal hemoglobinuria; DIAIHA, drug-induced autoim-
mune hemolytic anemia; PLS, passenger lymphocyte syndrome. & Transfusion if necessary; do not withhold
transfusion if physiologically required; incompatible transfused blood (emulating the recipient antigen profile) is
expected to have survival similar to autologous blood; caveats relate to underlying alloantibodies, which may
be detected in adsorption studies or anticipated by recipient antigen profiling (genotyping, or where feasible,
phenotyping). Depths of matching (mirroring what the patient is antigen-negative for, at a minimum in the
Weiner-Kell system, and ideally beyond this in the Kidd, Duffy, and S systems) are key to avoiding missed
antibodies, preventing the formation of new specificities, and helping to reduce the workload of adsorption
studies assessing for residual/emerging alloantibodies permitted by historic match gaps. * Mediated by monocyte-
macrophage phagocytosis of IgG and/or complement opsonized RBCs. Possibly intravascular, mediated by
antibody-dependent cellular cytotoxicity (ADCC). # First choice treatment; other treatments are available such as
rituximab, daratumumab and kinase inhibitors (see Text). ** Mediated by complement activation. ˆ Target antigen
requires the presence of the drug (in vivo and/orin vitro).

2.3. Treatment
wAIHA is usually initially treated with glucocorticoids [3,55–59]. While glucocor-
ticoids often achieve a partial or complete remission, difficult or dependent cases may
command other treatments such as off-label rituximab (anti-CD20), IVIG, or daratumumab
(anti-CD38). Other investigational agents such as FcγRn inhibitors (nipocalimab), Syk ki-
nase inhibitors (fostamatinib), BTK kinase inhibitors (rilzabrutinib), or PI3 kinase inhibitors
are being explored [57–64]. If complement-mediated hemolysis is suspected, treatment
may include inhibitors of the complement pathway such as sutimlimab (anti-C1s) or
eculizumab (anti-C5) [58,60,63,65]. There continues to be active research on new therapies
for wAIHA [58,63–68]. Although splenectomy was used for years with immense success,
it is rarely used today [3,60]. In a recent cohort study of 1824 patients, splenectomy was
found to be an effective treatment, though with surgical complications in 12% [69].

3. Cold-Antibody Autoimmune Hemolytic Anemia

Cold-antibody autoimmune hemolytic anemia, referred to as cold agglutinin syn-
drome (CAS) or cold agglutinin disease (CAD), is a rare autoimmune condition char-
acterized by the presence of high levels of cold-sensitive autoantibodies in the blood,
primarily IgM, as well as autoantibodies that remain active at temperatures below 30 ◦ C
(86 ◦ F) [3,12,20,70–76]. The antibodies have a particular affinity for red blood cells, often
the Ii RBC antigens [3,12,77,78], causing them to aggregate (agglutination), activate the
complement system, and undergo intra- and/or extravascular hemolysis [3,12,20,67–77].
Historically, CAD included both CAD and CAS; however, these two conditions are now
recognized as different and have been separated into distinct entities.
Int. J. Mol. Sci. 2024, 25, 4296 8 of 18

3.1. Cold Agglutinin Syndrome

Cold agglutinin syndrome (CAS) is a transient condition that is secondary to a bac-
terial or viral infection, such as Mycoplasma pneumoniae or Epstein–Barr virus causing
mononucleosis [2,3,70,77]. CAS can also occur following certain malignancies and other
autoimmune disorders [70,73–75]. CAS is also known as secondary cold agglutinin disease.
CAS is self-remitting, and the condition usually resolves when the underlying infection
clears [3].

3.2. Cold Agglutinin Disease

Cold agglutinin disease (CAD) or primary CAD is a clonal disease whereby the lym-
phoproliferation of a B-cell clone produces a cold-reactive (≤30 ◦ C) monoclonal IgM autoan-
tibody having specificity for the I or i RBC antigens [78]. The monoclonal IgM can activate
complement and cause intravascular hemolysis. CAD is a chronic condition [73–76,78].
Both CAS and CAD have similar findings for diagnosis. Both are caused by an IgM
autoantibody that targets either I or i RBC antigens. The IgM autoantibodies are naturally
occurring, polyclonal anti-I or anti-i with CAS; and, in CAD there is an abnormal production
of a monoclonal autoantibody, associated with a B-cell clone. The IgM autoantibody in
both conditions activates complement and can cause severe intravascular hemolysis [79].
Thus, both will have a positive DAT due to only complement (C3d) on their RBCs, and
both will have a cold agglutinin titer at 4 ◦ C ≥ 64, with a thermal range ≤ 30 ◦ C [3,12].

3.3. Treatment
Treatment for CAS usually involves keeping the patient warm until the condition
resolves with convalescence from the underlying disease. Maneuvers include blanketing
or maintaining higher ambient room temperatures at 37 ◦ C–40 ◦ C, and if transfusion is
required, using a blood warmer. In severe cases of CAS, one may use sutimlimab (anti-
C1s) [79–86].
CAD is a more difficult-to-treat AIHA, as the antibody is usually associated with a
chronic disease and the monoclonal antibody produced can cause more severe anemia than
in CAS [79–86]. Therefore, treatment can consist of rituximab or anti-complement thera-
pies such as sutimlimab to block the classical complement activation pathway [58,65,79–82]
or eculizumab to block the membrane attack complex of complement activation
pathway [58,78,83,84]. To limit the production of the causal antibody and to diminish
the cellular biomass of the driving condition, rituximab with or without strong immuno-
suppressive drugs such as fludarabine or bendamustine may be prescribed [58,78,83,84].
Recently, the use of daratumumab (anti-CD38) has been shown to be effective in the
treatment of relapsing CAD [64,83,84].

4. Mixed AIHA (Combined Cold and Warm AIHA)

Mixed AHIA or combined cold and warm AIHA is characterized by the simultaneous
presence of an IgG warm autoantibody and a cold-reactive IgM antibody with a low titer
but a broad temperature range in the blood circulation, reacting up to 37 ◦ C [2,3,39,87–93].
The syndrome is marked by significant hemolysis, resulting from both intravascular and
extravascular hemolysis. The illness may be severe but shows a favorable response to
steroid therapy [39]. However, mixed AIHA often follows a chronic trajectory with inter-
mittent periods of deterioration [2,3,89,91]. The condition accounts for 5–8% of all AIHA;
of these, 50% of cases are idiopathic while 25–42% of cases are associated with systemic
lupus erythematosus (SLE) [3,4].

Treatment
Treatment usually involves glucocorticoids with patients having a good and rapid
response [39]. However, again, rituximab and/or complement inhibitors may be consid-
ered [60,91]; see treatments under wAIHA and CAD above.
Int. J. Mol. Sci. 2024, 25, 4296 9 of 18

5. Paroxysmal Cold Hemoglobinuria or Donath–Landsteiner Test-Positive

Hemolytic Anemia
Paroxysmal cold hemoglobinuria (PCH) is a rare autoimmune hemolytic anemia that
is defined by the destruction of red blood cells in the blood vessels due to the activation of
the complement system [2,3,94–104]. This destruction happens when the body is exposed
to cold temperatures [3,12,100]. PCH is characterized by the presence of a cold-reactive
IgG autoantibody. This antibody attaches to RBCs at temperatures below 37 ◦ C. When the
RBCs are warmed to body temperature, the antibody activates complement, and the IgG
antibody subsequently dissociates from the RBCs [3,12,100]. As a result, only complement
is detected on the cells.
PCH was first documented in 1904 by Julius Donath and Karl Landsteiner, establishing
it as one of the earliest recognized kinds of AIHA [3]. The illness may present either as a
sudden, non-recurring post-infectious event in children [3,12,21,94,96–102] or as recurring
occurrences in adults with blood cancers or advanced syphilis [3,20,95,96]. Historically,
PCH manifested primarily in patients having syphilis, and exposure to cold resulted in
paroxysms of hemoglobinuria. Today, PCH is almost always encountered as an acute
transient syndrome in young children with a recent history of a viral illness [3,94–96], so
that paroxysms resulting from cold exposure are rarely seen. Thus, it has been suggested
that a better term for this condition would be Donath–Landsteiner test-positive hemolytic
anemia. Another interesting finding in PCH is erythrophagocytosis by neutrophils seen in
the peripheral blood [3]. Although not diagnostic, it should cue to the use of the D–L test.

5.1. Donath–Landsteiner Test

The diagnosis is made in the laboratory using the Donath–Landsteiner test (D–L
test) [3,12,100]. This test utilizes the findings of anti-GLOB (formerly anti-P) as the biphasic
IgG autoantibody, acting against RBCs positive for the GLOB antigen (formerly the P-
antigen) and not RBCs lacking the GLOB antigen, GLOB-null or, formerly, p RBCs. RBCs
incubate with the patient’s serum and a fresh source of complement (AB serum freshly
isolated and stored frozen). The test system moves from room temperature to 37 ◦ C. If
the GLOB + RBCs are hemolyzed and the GLOB-null RBCs are not, then the D–L test is
positive and the diagnosis of PCH is confirmed.

5.2. Treatment
PCH in children is usually self-remitting. In severe cases requiring transfusion, ap-
proaches that have been successfully described include plasma exchange, rituximab, and
complement inhibitors such as eculizumab [3,97,101–104].

6. DAT-Neg AIHA
Approximately 1% to 10% of people diagnosed with AIHA have a negative
DAT [3,103,104]. The DAT is a diagnostic technique that has a sensitivity range from
92% to 97% for detecting AIHA. The diagnosis of DAT-neg AIHA mostly relies on the
method of excluding other possible causes [3,12,105–107]. Several key variables contribute
to the development of DAT-neg AIHA. These factors include the following: (a) the exis-
tence of red blood cell-bound IgG at levels that are too low to be detected using standard
methods [3,24,105]; (b) the relatively weak binding strength of IgG [3]; (c) the lack of a
positive eluate using standard methods; and (c) the presence of RBC-bound IgA where
AHG contains only antibodies to IgG [3,73] or, in rare instances, IgA or IgM [3,22,29,32,108].
In the early 1970s, Gilliland and colleagues [3,105] were the first to emphasize that
the amount of antibodies found on RBCs in patients with AIHA may be insufficient to
detect by standard DAT reagents. They proposed that the distribution of IgG antibodies on
circulating RBCs was non-uniform. RBCs accumulate IgG as they age, leading to a larger
ratio of IgG to RBCs in older RBCs compared to younger ones [44,45,49,50,105].
In some instances of DAT-neg AIHA, less often encountered immunoglobulins, namely
IgA and IgM, may be identified on the outside of RBCs using a DAT with special antibod-
Int. J. Mol. Sci. 2024, 25, 4296 10 of 18

ies [3,22,29,32,33,108]. IgA AIHA has a clinical picture that closely matches that of IgG
wAIHA [106]. While the standard DAT may yield negative results, the use of special DAT
reagents that can detect IgM and IgA may be useful [3]. Western immunoblotting may
detect IgG on RBCs below the detection level of a DAT and could be useful to diagnose
DAT-neg AIHA [24]. This technique could also be adjusted to monitor IgA and IgM levels
on RBCs in DAT-neg AIHA. Most instances of IgM AIHA are caused by the presence of a
pathological cold autoantibody that has a broad temperature range and may respond at
temperatures ranging from 30 ◦ C to 37 ◦ C (see Sections 3 and 4, above). Diagnosing warm
IgM AIHA might be difficult since there are no significant serological findings. Antibody
detection tests may exhibit little reactivity. Both pathogenic IgM cold autoantibodies and
warm IgM autoantibodies exhibit the presence of complement coating on the patient’s
RBCs. As a result of this feature, some people with warm IgM AIHA may be misdiagnosed
with CAD or PCH [3,32].

Treatment
Treatment for severe cases of DAT-neg AIHA would be similar as to those used for the
treatment of wAIHA (see Section 2 above).

7. Drug-Induced Immune Hemolytic Anemia

Drug-induced immune hemolytic anemia (DIIHA) is a rare condition primarily caused
by the existence of drug-induced antibodies, which may be classified as either drug-
dependent or drug-independent [16,38,109–111]. Patients with DIIHA may have signs
of the rapid destruction of red blood cells inside blood vessels quickly after receiving
the medicine. This may be seen, for example, in babies who develop hemolytic anemia
because of ceftriaxone therapy [110,111]. In contrast, some people may have less severe
signs of extravascular hemolysis, which might occur many months after therapy, as shown
in instances of methyldopa-induced hemolytic anemia [26,38].

7.1. Drug-Dependent Antibodies

Drug-dependent antibodies may be detected by analyzing RBCs that have been treated
with drugs, termed the “hapten-specific mechanism”, or untreated RBCs in the presence of
a drug solution, termed the “neoantigen-dependent mechanism” [38]. Both mechanisms
require the drug to somehow interact with proteins on the RBCs, forming a hapten (drug)-
carrier (RBC) complex which can elicit an antibody response to the drug (hapten). Examples
include penicillin and cephalosporins [38,109,110]. Alternatively, a drug may complex with
the RBC in vivo forming a compound antigen, a neoantigen, that requires the drug to be in
the testing system to detect its activity [38]. Examples of this mechanism include second-
and third-generation cephalosporins such as cefotetan and ceftriaxone [38,110,111].
In some instances, the antibody that is detected appears to be an autoantibody re-
sembling autoantibodies found in wAIHA [16,26,38,111]. In this case, the DAT is pos-
itive for IgG without apparent drug-dependence and with IgG antibody detectable in
the patient’s plasma [16]. The prototypical drug that causes this type of DIIHA is alpha-
methyldopa [26,38]. This phenomenon has been termed the “cross-reactive autoantibody
mechanism” [38]. Finally, there is an unusual mechanism whereby following certain drug
therapies, the patient’s RBCs are able to take up proteins from their surroundings, including
immunoglobulins, and give a positive DAT. With IgG on the patient’s RBCs, this can result
in monocyte–macrophage recognition and extravascular hemolysis. This mechanism is
poorly understood, but it has been suggested that it may be pH dependent [38,109].

7.2. Drug-Induced Autoimmune Hemolytic Anemia (DIAIHA)

There are two types of DIAIHA, cross-reactive autoantibody production and im-
munoglobulin adsorption.
Int. J. Mol. Sci. 2024, 25, 4296 11 of 18

7.2.1. Cross-Reactive Autoantibody Mechanism

The mechanism of cross-reactive autoantibody production results in an IgG warm
autoantibody being made by the patient that is dependent on the drug being administered.
The classic example is that of alpha-methyldopa therapy [16,38]. In these conditions of
drug-induced autoimmune hemolytic anemia (DIAIHA), the patient presents with acquired
hemolytic anemia with a DAT positive for IgG and negative for complement and a serum
antibody that reacts with all unrelated RBCs [16,26,38]. Thus, this condition looks like
classical wAIHA. It has been postulated that the drug interacts with the RBC membrane
to modify it enough that it appears as “non-self” to the patient’s immune system, and
this then results in the patient making an antibody to the RBCs plus the drug, but also a
“crossreactive” antibody that does not require the drug [26,38]. Stopping the administration
of the implicated drug will resolve the anemia; however, it may take some time for the
autoantibody to go away [3,16,26]. It is unknown why only a small percentage of patients
will develop this condition; however, the resulting hemolytic anemia can be severe and
even life-threatening. Methyldopa is rarely used anymore. Currently, drugs that can
result in the production of an IgG autoantibody to the patient’s RBCs include cefotetan
and ceftriaxone [38,111]. These drugs also act by both hapten-specific and neoantigen-
dependent mechanisms, making these two drugs potentially dangerous [110,111].

7.2.2. Immunoglobulin Adsorption Mechanism

The other mechanism that can present as wAIHA is when drugs cause the adsorption
of serum proteins onto the RBCs. This previously was termed the “nonspecific adsorption
mechanism” and results in any serum protein being “adsorbed” onto the RBCs, including
albumin, complement components and immunoglobulins, in particular IgG [3,109]. The
patient can develop hemolytic anemia due to the IgG being on the patient’s RBCs. When
the laboratory investigates the reason for the anemia, the DAT is positive for IgG. However,
the eluate will be negative, providing a clue that this may be drug related. Although the
implicated drug can cause the adsorption of complement components, these would be C2,
C3 and C4 that normally circulate in the blood, but not the complement component that is
assessed in the DAT, which is C3d. Thus, only IgG would be detected using AHG.
Any time a patient with acquired immune hemolytic anemia is encountered with an
IgG-positive DAT and a negative eluate, a drug history should be obtained, as this outcome
may be related to a drug-induced hemolytic anemia. The reason for this phenomenon
of immunoglobulin adsorption has been postulated to be due to the chemistry of the
drugs, with some drugs having chemical structures that allows these drugs to covalently
bind to both RBC proteins and external proteins in the patient’s serum [38,109]. In vitro,
drugs optimally bind to RBCs depending on the pH, with a more basic pH allowing for
hapten-specific drug interactions, such as with penicillin [3,16,38]. It has been shown that
immunoglobulin-adsorption also occurs optimally under alkaline conditions [109]. Patients
might therefore manifest this phenomenon if taking applicable drugs in the context of
metabolic alkalosis.

7.3. Treatment
The treatment for these drug-induced conditions often simply involves stopping the
drug therapy, if possible, or switching to a different, chemically unrelated, drug. When this
is done, the hemolysis may abruptly cease [16,26,38]. If the hemolysis is severe, one can
treat the patient with similar therapies as used for the treatment of wAIHA.

8. Passenger Lymphocyte Syndrome

Passenger lymphocyte syndrome (PLS) is an unusual hemolytic occurrence that can
resemble AIHA. Allogeneic transplantation includes bone marrow transplantation (BMT)
and solid organ transplantation. In BMT, host lymphocytes are transferred into the recipient
from the bone marrow or stem cell material obtained from the donor [17,25,112,113]. In
solid organ transplantation, donor lymphocytes can be “passengers” in the solid organ
Int. J. Mol. Sci. 2024, 25, 4296 12 of 18

obtained from the donor, and these can be transferred into the recipient [114–118]. It is
feasible for donor lymphocytes that have been transferred to produce antibodies that are
specific to RBC antigens in the recipient’s body but not present on the donor’s own red
cells [17,25,112–118]. Indeed, sometimes these passenger lymphocytes in either BMT or
solid organ transplants are obtained from donors who have been sensitized to produce
alloantibodies to antigens for which they lack. Thus, passenger lymphocytes from a blood
group O donor transferred into a recipient of blood group A can produce anti-A that
can react with and hemolyze the residual RBCs in the recipient due to those being blood
group A. Likewise, passenger lymphocytes from a donor who is Rh-negative but who
has been sensitized to produce anti-D can induce a hemolytic anemia in an Rh-positive
recipient, which may manifest as a delayed-type hemolytic reaction. In both instances, the
hemolytic anemia can appear as an AIHA, though with an apparent specificity. Indeed, if
this syndrome occurs, its presentation as immune hemolysis after a transplantation may
be misinterpreted as autoimmune hemolytic anemia. The hemolysis can be severe with
outcomes such as renal failure [114,116,117].

8.1. Role of Cyclosporine

The specific influence of cyclosporine on the promotion of antibody production from
the donor is still not fully understood. Because hemolysis has been most frequently related
to cyclosporine [113], a medication that suppresses the immune system, it is suggested that
donor B lymphocytes proliferated and produced antibodies because of cyclosporine effects
to selectively inhibit T-cell function [117]. Alternatively, previously sensitized lymphocytes
when exposed to an antigen in the presence of cyclosporine can still respond to antigens on
recipient RBCs [119].

8.2. Treatment
It is critical to recognize that the stem cell or solid organ transplant patient who is
hemolyzing may be manifesting PLS. Treatment may require the transfusion of antibody-
evading (donor blood type) RBCs, i.e., blood group O cells if hemolysis is due to ABO
antibodies (in a group O donor to a non-O recipient). If anti-D or other alloantibodies
are identified as the cause of the hemolysis, then antigen-negative donor blood should be
used for transfusion [17,113,115]. If hemolysis is severe, one can treat with changes to the
immunosuppressive regimen [113,118] or combine the regimen with IVIG or plasmaphere-
sis [117].

9. Current Opinion
We believe that hyperhemolysis syndrome (context: sickle cell disease (SCD) or other
diagnoses) deserves its own category within AIHA. Although it is a distinctly transfusion-
triggered event, with the absence or presence of an involved alloantibody cognate to a
triggering unit, once triggered, it appears to be an autoimmune-like phenomenon with
extreme bystander hemolysis [120] (kinetically akin to post-transfusion purpura (PTP)).
The complement cascade appears important (as judged by the response of some cases to
eculizumab), with hyperinflammation occurring (as judged by the greater responsiveness
to IVIG with high-dose steroids, +/− the utility of anti-cytokine storm interventions such
as tocilizumab). In virtually every regard, this is a pathology that is not addressed by
any of the conventional AIHA categories. However, because it manifests in part with an
autologous response, there is an argument for its inclusion in the taxonomy of AIHA, just
as there are arguments for the inclusion of passenger lymphocyte syndrome.

10. Summary and Perspectives

The review herein has addressed the full suite of autoimmune hemolytic anemias
with its seven categories (Table 1). Within each category described, there is a discussion
of clinico-laboratory features, diagnostics and management options. Treatments are tai-
lored for each category and patient in “personalized medicine”. This is especially true
Int. J. Mol. Sci. 2024, 25, 4296 13 of 18

if the AIHA is refractory to current first-line therapies, such as glucocorticoids, used in

most AIHAs except for cold-antibody AIHAs. Existing and future options need further
examination [58]. Monoclonal antibody therapies targeting B-cells using rituximab and
daratubumab are showing efficacy in some cases. Targeting the complement activation
pathway using sutimlimab or eculizumab is increasingly suggested in CAD and mixed
AIHA [62,121]. Older therapies are also still viable in some cases, such as splenectomy and
plasmapheresis. The future anticipates more monoclonals to complement components [58],
the inhibition of the neonatal Fcγ receptor (FcγRn) (nipocalimab) [62], immunosuppressive
combinations (mycophenolate mofetil, rapamycin) [122–124], kinase inhibitors (B-cell tyro-
sine kinase (BTK) [125], spleen tyrosine kinase (SYK) [126], Janus kinase (JAK) [127]), and
proteasome inhibitors [128]. Targeting T-cell interactions with co-stimulatory molecules
such as CD80/CD86 on antigen presenting cells (e.g., abatacept) shows utility [129]. In
summary, despite a number of AIHAs exhibiting differential treatment response profiles,
the future looks promising for mechanism-informed therapeutics.

Author Contributions: Conceptualization, M.L., C.C.-G. and D.R.B.; Writing—original draft prepara-
tion, M.L.; Writing—review and editing, C.C.-G. and D.R.B. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

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