Pathobiology of Transfusion Reactions

PM10CH04-Zimring ARI 2 December 2014 8:47
ANNUAL
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• Top cited articles James C. Zimring1,2 and Steven L. Spitalnik3
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Puget Sound Blood Center Research Institute, Seattle, Washington 98102;
email: jzimring@psbc.org
2
Departments of Laboratory Medicine and Internal Medicine, Division of Hematology,
University of Washington, Seattle, Washington 98195
3
Department of Pathology and Cell Biology, Columbia University College of Physicians
and Surgeons, New York, NY 10032
Annu. Rev. Pathol. Mech. Dis. 2015. 10:83–110 Keywords

The Annual Review of Pathology: Mechanisms of red blood cells, hemolysis, transfusion reactions
Disease is online at pathol.annualreviews.org
This article’s doi: Abstract

10.1146/annurev-pathol-012414-040318
Antibody-induced hemolytic transfusion reactions were first described over
Copyright c 2015 by Annual Reviews. 300 years ago. Indeed, during its early evolution, transfusion medicine fo-
All rights reserved
cused almost exclusively on issues in immunohematology to prevent such
events. However, despite the best of efforts to avoid them, incompatible
transfusions still occur, through both error and an inability to obtain com-
patible red blood cells for patients who are alloimmunized against multi-
ple antigens. Because transfusing units of incompatible blood is potentially
lethal, studies on human volunteers are not ethical. Thus, understanding of
hemolytic transfusion reactions is generated through clinical cases, animal
models, inference from related human pathologies, or studies using small
volumes of transfused red blood cells. Over the past several decades, sub-
stantial new knowledge has been accumulated regarding the mechanisms
of hemolysis, the metabolism of products of hemolysis, and the effects of
both on recipient biology. Using these data sources, this article traces the
historical generation of this knowledge and describes recent advances.
83
INTRODUCTION
Red blood cell (RBC) transfusions are the most common interventional therapy for hospital-
ized patients; ∼15,000,000 RBC units are transfused into ∼5,000,000 Americans each year. Im-
munohematology services primarily screen for RBC alloantibodies, monitor alloimmunization,
and provide sufficient numbers of units against which the recipient has no antibodies (i.e., com-
patible blood). Nonetheless, hemolytic transfusion reactions (HTRs) still occur, resulting from
errors, limited amounts of compatible blood, and primary alloimmunization. Accelerated RBC
destruction also occurs in other pathological [e.g., autoimmune hemolytic anemia (AIHA)] and iat-
rogenic (e.g., anti-RhD infusion for immune thrombocytopenic purpura) settings. Much biology
underlying antibody-mediated hemolysis has been elucidated, producing mechanistic understand-
ing. However, genetic differences (between donors and recipients) produce broad variations in
HTR pathology. In addition, less is understood regarding how initial hemolysis in HTRs leads to
downstream consequences [e.g., renal dysfunction, disseminated intravascular coagulation (DIC),

shock, and death]. Finally, although seemingly logical therapeutic interventions are attempted,
it is unclear whether these are efficacious. This article focuses on historical and recent advances
in understanding HTR pathophysiology, with an emphasis on evolving understanding and the
potential for translation into clinical care.
These issues may best be illustrated by a real clinical case (Figure 1):
In 2004, two teenage boys with sickle cell disease were scheduled, simultaneously, to receive routine
RBC exchange transfusions in a hemotherapy unit. One patient’s blood type was Group B, the other
Group A. Serological testing was correct, and correctly labeled units were released from the blood bank.
The hemotherapy nurse mistakenly began transfusing one Group B unit into the Group A patient. Vir-
tually immediately, symptoms of his stereotypical sickle cell crisis appeared (i.e., severe headache, chest
pain, palpitations, mild respiratory distress). The nurse recognized the reaction, immediately stopped
the transfusion, and contacted the pathology resident and attending physician. Gross hemoglobinemia
was seen in a posttransfusion sample. Fluids, methylprednisolone, diphenhydramine, and furosemide
a b
Figure 1
Blood sample and urine samples from the patient in the described clinical case. (a) Immediate
posttransfusion blood sample from the patient demonstrating gross hemoglobinemia in the supernatant.
(b) Serial posttransfusion urine samples showing gross hemoglobinuria in the first sample (sample 1), with
rapid and progressive clearing in subsequent samples (samples 2–10).
84 Zimring · Spitalnik
were administered. Renal function was not compromised, and gross hemoglobinuria, noted in initial
posttransfusion urine samples, cleared rapidly. The patient was admitted overnight for observation, had
an uneventful course without long-term sequelae, and continued returning for elective RBC exchange
transfusions.
This adverse event was due to a medical error, the most common cause of an ABO HTR. The
patient’s symptoms occurred immediately and, interestingly, manifested as a sickle cell (i.e., vaso-
occlusive) crisis, and not with the textbook description of back pain, fever, or “a sense of impending
doom.” In addition, significant intravascular hemolysis occurred, evidenced by hemoglobinemia
and hemoglobinuria. Although not measured in this case, one would expect decreased levels of
haptoglobin and increased levels of lactate dehydrogenase and, perhaps, potassium. The patient
was treated according to current standards, some of which may be pathophysiologically relevant
(e.g., fluids and diuretics) and some not (e.g., diphenhydramine). Finally, although his acute pre-
sentation was dramatic, dangerous downstream consequences (i.e., renal dysfunction, DIC, shock,
and death) did not occur.
IMMUNOGLOBULIN DIVERSITY
Antibodies primarily facilitate the destruction, neutralization, and/or elimination of their targets.
Antibodies perform these functions using the complement (C ) cascade and Fc receptor (FcR) lig-
ation, both of which clear antibody-bound targets. In addition, C and FcRs produce inflammatory
and physiological changes coincident with target clearance. Moreover, cross talk between the C
and FcR pathways produces a complex integrated system. In general, with exceptions discussed
below, IgM functions predominantly through C , whereas IgG functions primarily through Fcγ
receptors (FcγRs), involving C in some settings.
Although immunohematology laboratories focus on IgM and IgG RBC-binding antibodies,
human antibody diversity is substantially more complicated. Humans express four IgG subclasses
(IgG1–4), each with different affinities for FcγRs and different abilities for activating C (1).
Additional isotypes include IgA (IgA1 and IgA2), IgE, and IgD, although the latter is not normally
detected in secreted form. IgM, IgG, and rarely IgA can induce HTRs. Although alloantibodies
in some patients may have one dominant, or exclusive, IgG subclass, other antisera are mixtures of
IgG1–4 (2). Thus, a given antibody-coated RBC has a mixture of bound antibodies, yielding a cell
that can simultaneously ligate activating and inhibitory FcγRs, and that fixes C well, poorly, or not
at all. Importantly, FcγR ligation and C fixation are enhanced by IgG density. Thus, the ultimate
effect of alloantisera on incompatible RBCs depends on target antigen density and serum IgG1–4
composition. Moreover, genetic variation in effector pathways and donor RBCs influences the
outcome of incompatible transfusions (see below). Evaluating this complexity is beyond the scope
of current clinical testing, but it may explain why some, but not other, allo- and autoantibodies
cause hemolysis in vivo.
Murine antibody isotypes and subclasses correspond largely with human forms, but with im-
portant differences (1); this is relevant because murine models are useful in studying HTRs. Mouse
B cells secrete IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, and IgE. In some strains, IgG2c replaces
IgG2a (3). IgM primarily exerts its effects through C and IgG through FcγRs, with C contribut-
ing sometimes. Mouse IgG function is as diverse as human IgG1–4; however, precise orthology
between murine and human IgGs is lacking, which is one caveat in inferring human biology from
murine phenomena (1).
www.annualreviews.org • Pathobiology of Transfusion Reactions 85

THE ROLE OF COMPLEMENT IN HEMOLYTIC

TRANSFUSION REACTIONS
The C system is a cascade of proteases, receptors recognizing activated C components, and
regulatory molecules. Although a detailed description is outside the scope of this article, excellent
reviews are available (4). Nevertheless, a synopsis is required to explore mechanisms underlying
HTRs. Although several pathways can activate C , including lectins and bacteria, the most relevant
for this review is activation by RBC-bound antibodies (i.e., the classical pathway). Antibodies have
antigen-binding and Fc domains; the latter perform effector functions. Antibody effects on C
depend on their Fc regions, which vary by isotype and subclass.
IgM is the most potent C activator; one IgM molecule is sufficient (5). However, unbound
IgM does not bind and activate the first C component (C1); otherwise, C would be activated
indiscriminately. Rather, when IgM binds its target, a conformational change exposes C1q-binding
sites (a component of C1), inducing autocatalytic activation of C1s by C1r, followed by C1s-
mediated activation of downstream events. Unlike IgM, one IgG does not bind C1q and induce
C1 activation, even if bound to its target. Rather, at least two bound IgG Fc regions must be
nearby to activate C ; indeed, recent evidence suggests that hexamers of closely spaced IgG are
required (6). Thus, IgM and IgG activate C only if the target antigen is encountered and bound.
C is the main effector of IgM-mediated clearance. Initial C1 activation is amplified by C2 and
C4 activation, producing C3 convertase (C4bC2b), which covalently binds its target through a
thioester bond. C3 convertase then cleaves C3 into C3a and C3b. C3a, a soluble anaphylatoxin,
regulates inflammation and vascular tone and contributes to signs and symptoms of hemolysis. In
contrast, C3b is a potent opsonin that covalently binds its targets through a thioester bond (one C3
convertase induces binding of up to 1,000 C3b molecules). Although C3b can bind the C -fixing
antibody, the target, or other nearby proteins, much C3b binds the antibody itself. C3b is recog-
nized by complement receptors (CRs) CR1 and CRIg (7), which are expressed on macrophages
and activate ingestion of their opsonized targets. Although potent, C3b has a short half-life and is
rapidly converted from C3b to iC3b to C3c to C3dg. Each C3b degradation form is recognized by
different CRs on different cells (7). For example, iC3b binds CR1–4 and CRIg. Because CR3 and
CR4 stimulate macrophage phagocytosis, iC3b is a highly potent opsonin. However, C3dg is rec-
ognized only by CR2, which is expressed on B cells and follicular dendritic cells, not macrophages,
and does not promote phagocytosis. Thus, C3 degradation provides intrinsic negative feedback,
as the final C3 form (i.e., C3dg) is nonopsonizing. Moreover, C3dg masks thioester-binding sites
that could be C3 targets, preventing ongoing C3 fixation. Hence, RBCs surviving C3 binding long
enough for C3 conversion to C3dg should be protected from C3-based opsonization. Similarly,
membrane-attached C4 (as C4b) is recognized by CR1; however, it is unclear how the low levels
of RBC-bound C4b would affect opsonization without amplification through C3.
C3 is also integral to amplification of downstream C components, particularly C5, which splits
into C5a and C5b. C5a is a soluble anaphylatoxin, whereas C5b attaches to the antibody target,
nucleating membrane attack complex (MAC) assembly. In addition to C5b, MACs contain C6–9,
which form a membrane-permeabilizing channel, producing osmotic dysregulation and cell lysis.
Thus, MAC-mediated hemolysis occurs independently of macrophages and can even occur in
vitro.
In summary, C activation induces two RBC destruction pathways: (a) C3 fixation (either
directly or on bound antibody) opsonizes RBCs, promoting CR-dependent phagocytosis, and
(b) MAC formation directly hemolyzes RBCs by disrupting membrane integrity. C3 is central to
these processes: C3b and iC3b are opsonins, and C3b (with C4b and C2a) is the convertase acti-
vating C5 to initiate MAC formation. C5 is also activated by other proteases (notably thrombin) in
C3-deficient animals; however, it is unclear whether C3-independent pathways are important in

RBC antibody-induced MAC formation (8, 9).
FC RECEPTORS AND ANTIBODY EFFECTOR FUNCTIONS

Antibodies also affect physiology through receptors recognizing their Fc domains. The FcγR fam-
ily members vary in binding affinity, IgG subclass preference, structure, signaling pathways, and ef-
fects. Eight human FcγRs have been described (10). FcγRI (CD64), FcγRIIA (CD32A), FcγRIIC
(CD32C), and FcγRIIIA (CD16B) are activating receptors: IgG ligation induces phagocytosis.
These single-pass transmembrane proteins either contain an immunoreceptor tyrosine-based ac-
tivation motif (ITAM; FcγRIIA and FcγRIIC) or are coupled to a common, ITAM-containing
γ-chain (FcγRI and FcγRIIIA). In contrast, FcγRIIB (CD32B) contains an immunoreceptor

tyrosine-based inhibitory motif (ITIM) that inhibits phagocytosis. Because FcγRs have differing
affinity for IgG1–4 and different tissue distributions, the net effect of circulating IgG is a balance
of affinities for activating and inhibiting receptors.
Other IgG receptors include FcγRIIIB (CD16B), FcRn, and TRIM21; however, these are un-
likely to clear antibody-bound RBCs. FcγRIIIB, a glycosylphosphatidyl inositol (GPI)-anchored
protein, binds IgG1 and IgG3, is predominantly expressed on neutrophils, and is not clearly in-
volved in phagocytosis. FcRn, the neonatal FcR, transports maternal IgG. Because TRIM21 is
intracellular, it is unlikely to ingest IgG-opsonized RBCs.
The IgG affinity of particular FcγRs has strongly influenced phagocytosis paradigms. Tradi-
tionally, much is made of FcγRI’s higher affinity for IgG1, IgG3, and IgG4 than other FcγRs
(FcγRI essentially does not bind IgG2). Although FcγRI’s high affinity allows monomeric IgG
binding, other FcγRs have much lower affinities, requiring increased avidity by multiple IgGs
bound to a common target. Thus, FcγRI was regarded as always occupied by polyclonal IgG and
thus not available for clearing immune complexes (ICs) or IgG-coated RBCs; however, this has
recently been challenged (10, 11).
Murine FcγRs have similarities with and differences from human FcγRs (10). Like those of
humans, murine activating FcγRs signal through an ITAM (FcγRI, FcγRIII, and FcγRIV) and an
inhibitory FcγR signals through an ITIM (FcγRII). Mouse FcγRs also have differential affinities
for mouse IgG subclasses. However, unlike humans, mice lack a structural ortholog for FcγRIIIa
and an analog of GPI-linked FcγRIIIB. In addition, mouse FcγRs do not encode ITAMs; activating
mouse FcγRs signal through a common γ-chain. Whether the mouse FcγR system functionally
recapitulates the human system despite differences in sequence-based orthologs has been debated.
Although mouse models predict general themes of human antibody-mediated destruction, the
extent of the similarities is unclear. Mice humanized for FcγRs and IgGs may address potential
limitations (1).
IgM FcRs are not considered important in IgM-mediated HTRs, due to the overwhelming par-
ticipation of C and CRs. Nevertheless, IgM FcRs were described [e.g., pIgR (12), Fcα/μ (13), FcμR
(14)]. However, pIgR is selectively expressed by intestinal epithelial cells (12), and macrophage
FcμR expression is controversial (15–17). Fcα/μ is expressed on activated macrophages and con-
sumes IgM-containing IC; however, its role in clearing IgM-coated RBCs is unclear.
NEGATIVE FEEDBACK LOOPS IN ANTIBODY BINDING

TO RED BLOOD CELLS
FcγR-mediated opsonization and C -mediated opsonization are self-limiting processes con-
taining negative feedback mechanisms, which not only limit but also reverse them, producing

“deopsonization.” Thus, RBCs must become sufficiently opsonized to induce phagocytosis, but if
they avoid consumption long enough, they can revert to RBCs that are not targeted for removal.
The C negative feedback system (described above) rapidly converts RBC-bound C3b to C3dg,
which does not bind phagocytosis-promoting CRs. Thus, even antibody- and C3b-coated RBCs
may circulate normally if C3b is converted to C3dg before clearance. The resulting C3dg also
occupies sites to which new C3b would attach, thereby preventing continuing C3b deposition
(and C4 deposition, because both use thioester linkages). In addition, in patients receiving RBCs
and plasma, the recipient’s C system is influenced by donor plasma C proteins.
Negative feedback in the FcγR system is less understood but appears to exist. First, C3b binds
IgG Fc domains and theoretically masks FcγR-binding sites. Thus, C3b conversion to C3dg may
inhibit FcγR-mediated ingestion if Fc-bound C3b blocks FcγR access. Whether this occurs in
vivo during HTRs is unknown; however, bound C can block binding of IgG Fc domain–specific
secondary antibodies (18).

Another negative feedback pathway involves antigen modulation, also denoted as antigen loss,
weakened antigenicity, and antigen suppression. In this case, antibody binding induces a process by
which antigen and antibody become less detectable or undetectable (19). This occurs in multiple
blood group systems, including Kell, Kidd, Rh, Gerbich, LW, AnWj, and Cromer (19). It is unclear
whether antigen is altered (e.g., by proteolysis or reduction), masked by antibody that is then
undetectable (e.g., by C3dg binding), or removed by trogocytosis of antigen-membrane complexes.
In one patient with Kell system antigen modulation, Kell glycoprotein became undetectable by
Western blotting with either C- or N-terminal-specific antisera (20). Thus, in this case, masking
by altered antibody is excluded and complete Kell glycoprotein removal is probable. In murine
studies of antigen modulation during incompatible RBC transfusions, FcγRs are required for this
process (21, 22). Because IgG binding opsonizes through FcγRs and also induces FcγR-dependent
antigen modulation, the latter provides negative feedback analogous to C3 conversion to C3dg.
In addition, C3 is important for antigen modulation in the Kell system (23) but not in the mHEL
system (22; J.C. Zimring, unpublished observations). When incompatible transfused HOD, M,
and Kell-system RBCs circulate in the long term, each demonstrates antigen modulation on
RBCs that are not cleared. This requires anti-HEL antibodies that simultaneously bind multiple
epitopes but also occurs with monoclonal anti-Fy3; however, little clearance occurs with the
former, whereas substantial clearance occurs with the latter. In both cases, FcγRs, but not C ,
are required for antigen modulation. Monoclonal anti-M and polyclonal anti-Kell similarly cause
antigen modulation, but are less well characterized.
Normal immunobiology also encompasses removing RBC membrane proteins by antibody-
containing ICs. Circulating ICs fix C and bind RBC CR1 (the Knops system) (24). When RBCs
with CR1-bound ICs traverse the liver, Kupffer cell FcγRs ligate the ICs, promoting their inges-
tion. IC phagocytosis releases them from the RBC surface but does not damage these RBCs, which
continue circulating. Thus, patients with disorders that chronically generate ICs can type as Knops
null; however, during remission, they are Knops positive. Several hypotheses suggest how CR1
ICs are removed without RBC damage, including CR1 proteolysis and trogocytosis (24). Overall,
this IC transfer reaction and antigen modulation by RBC antigen-bound antibodies are similar.
INTEGRATED HEMOLYTIC TRANSFUSION REACTION MODEL

INVOLVING COMPLEMENT, COMPLEMENT RECEPTORS,
AND FC RECEPTORS
Classically, HTRs are divided into two categories. Acute HTRs (AHTRs) occur rapidly (within
minutes), from efficient IgM-induced C fixation, with MAC formation producing massive
intravascular hemolysis. In contrast, delayed HTRs (DHTRs) occur over hours to days because
of extravascular hemolysis due to phagocyte ingestion of IgG-opsonized RBCs. For example, the
signs, symptoms, and laboratory findings in AHTRs are described in the clinical case presented
above. Although the distinction between AHTRs and DHTRs is useful conceptually, it is not
always accurate. First, the association of IgM with AHTRs and IgG with DHTRs does not al-
ways reflect C biology. For example, for IgM-mediated C activation to produce MAC-induced
intravascular hemolysis, C3b must be deposited, which also opsonizes RBCs for phagocytosis.
Although MAC assembly may occur so rapidly that intravascular hemolysis occurs before CR-
mediated phagocytosis, this does not explain why C -fixing IgG-bound RBCs do not behave
similarly, as sufficient C3 activation to opsonize RBCs would also be amplified to assemble MACs.
However, low levels of C3 activation could produce sufficient C3b for opsonization, but not for
MAC formation, resulting in extravascular hemolysis. Nonetheless, in patients with incompatible

transfusions and DHTRs, the direct antiglobulin test can demonstrate high levels of RBC-bound
C3b. A kinetic argument could suggest that IgG-dependent C3b deposition is slow and rapid
conversion to C3dg prevents MAC formation; however, given C cascade amplification and the
impression that one IgM molecule can activate enough C3 to induce MAC formation and hemo-
lysis, this argument may not hold. Finally, C -regulatory proteins (e.g., CD55, CD59), which
downmodulate C -mediated responses, may also be involved (25).
Because human anti-RBC antisera often contain IgM and IgG, this simplified model may not
suffice. For example, most human anti-A and/or anti-B (in the ABO system) are both IgM and IgG.
In addition, during primary alloimmunization, IgM is produced first and initially predominates.
After ∼10 days, IgG begins to predominate and IgM diminishes. During primary RBC alloim-
munization, RBCs carrying the offending antigen have shortened survival times, presumably due
to alloantibody. However, rapid clearance is not observed during the IgM-predominant, primary
immune response, even with abundant RBC surface antigens.
Effector Mechanisms in Hemolytic Transfusion Reactions

in Humans and Animal Models
Understanding the mechanisms underlying incompatible transfusions is complicated by human
diversity. For example, in addition to the well-known ABO and Rh blood group systems, there are
dozens of blood group systems and hundreds of antigens and epitopes (26, 27). For known RBC
antigens, incompatible transfusions are virtually never hemolytic for some, seldom hemolytic for
others, and variable for the rest. In addition, RBCs in a given unit are not all identical; some
are cleared rapidly following an incompatible transfusion, and others circulate in the long term.
Therefore, labeling an incompatible transfusion as hemolytic or an anti-RBC antibody as clin-
ically significant is conceptually simplistic. Because incompatible transfusions are avoided, ex-
cept in extreme clinical circumstances and following medical errors, the exact frequency and
extent of hemolysis are unknown. However, even for ABO incompatible transfusions, which, at
their extremes, produce severe intravascular hemolysis and death, ∼50% of these events pro-
duce no signs or symptoms of hemolysis (28). Moreover, in both humans and mice, incompatible
RBCs have a rapid clearance phase that then dissipates, leaving a population of RBCs that sur-
vive in the long term (29). Deductive arguments posited that the surviving RBCs in humans
are not uniquely resistant to clearance (29); however, other human and murine data indicate
that the RBCs surviving early clearance are indeed resistant, in both incompatible transfusions
and cold agglutinin disease (23, 29–37). Thus, while synthesizing existing knowledge into general
paradigms of HTR biology, it is important to acknowledge biological variation and avoid simplistic
generalizations.

Precise mechanistic studies of human HTRs following transfusion of entire incompatible RBC
units are lacking, because incompatible transfusions are given only in error or in patients needing
urgent transfusion when no compatible blood is available. However, insightful studies have been
performed in which small quantities (<5 mL) of radiolabeled RBCs (typically using 51 Cr) are
infused and their circulation is followed. In some cases, incompatible RBCs were transfused into
patients with either naturally occurring alloantibodies or alloantibodies produced following prior
transfusion or pregnancy. In other cases, alloantibody-coated RBCs were infused. Finally, some
recipients were passively immunized with alloantibodies, followed by infusion of incompatible
RBCs. Although significant insights were obtained (see below), there are several caveats. The
results from transfusing small amounts of incompatible RBCs may differ when compared with
intact units (29, 38–40), especially at low titers but less so at high titers. The potential morbid-
ity and mortality of transfusing humans with whole units ethically preclude such experiments.
Moreover, currently, incompatible RBCs are often given from frozen RBC stocks, to decrease the
risk of infecting healthy volunteer recipients with transfusion-transmitted pathogens by allowing
donors time for seroconversion. The extent to which incompatible frozen RBCs reflect the biol-
ogy of typical RBC units is therefore unclear. Finally, very few such studies using very few human
volunteers have ever been performed. Thus, although straightforward conclusions were drawn,
making general inferences from limited data is dangerous, especially given human diversity in the
relevant effector pathways (see above). Nevertheless, there are several conclusions from these few
published studies.
For example, antibody density on RBC surfaces correlates with incompatible RBC clearance
rate (41), suggesting that incompatible RBC antigen density also correlates with clearance rate.
Although perhaps true for donor-to-donor variability for a given antigen, or for RBC-to-RBC
antigen density in a given donor, it is less clear that average RBC antigen density determines
whether alloantibodies induce HTRs (i.e., clinically significant and insignificant alloantibodies).
In addition, antibody titer (especially in the ABO system) correlates with the rate and extent
of transfusion-induced hemolysis and predicts the likelihood of hemolytic disease of the fetus
and newborn (especially the Rh system), although the latter may also relate to the amount of
alloantibody crossing the placenta. In human studies of passive immunization with polyclonal anti-
D followed by transfusion of 51 Cr-labeled RhD+ RBCs, as few as 10 molecules of anti-D per RBC
produced 50% clearance by 100 h (42); this is below the level of detection by RBC agglutination
(42). In addition, IgG alloantigen affinity correlates with clearance; when 51 Cr-labeled RhD+
RBCs were presensitized with anti-D of differing affinities and transfused, higher-affinity anti-
D produced faster clearance (43). Finally, IgG subclass influences hemolysis. In AIHA patients,
the presence of multiple IgG subclasses correlated more with hemolysis than did isolated IgG
subclasses (2, 44–47). In addition, although isolated IgG1, IgG2, or IgG3 was associated with
AIHA, in no case was IgG4 alone hemolytic (2, 48). However, given that the amount of RBC-
bound antibody required for hemolysis is less than that detectable by agglutination (42), it is unclear
whether detecting an isolated IgG subclass precludes the presence of other subclasses. In addition,
because ∼0.1% of healthy blood donors have a positive direct antiglobulin test (i.e., nonhemolytic
RBC-bound autoantibodies) but patients with AIHA can have life-threatening hemolysis with
similar direct antiglobulin test results, the biology of anti-RBC autoantibodies is likely as complex
as that of anti-RBC alloantibodies.
In human studies of isolated IgG subclasses, autologous RhD+ RBCs were presensitized with
either a monoclonal IgG1 or IgG3 anti-D antibody. Following autologous transfusion, RBC
survival was monitored; IgG3 more effectively cleared antibody-coated RBCs than did IgG1 (49).
In vitro data also suggested that IgG1 and IgG3 anti-D cleared RBCs by different mechanisms
(50). However, because these different monoclonal antibodies did not express identical anti-D
variable domains, issues of differing antibody affinity cannot be excluded.
Antibody thermodynamic binding properties also correlate with signs and symptoms of hemo-
lysis; thus, alloantibodies binding RBCs in vitro at room temperature, but not at 37◦ C, are generally
nonhemolytic in vivo; however, this may simply be due to an inability to lyse what cannot be bound.
Considerable evidence supports the importance of C and FcγRs in antibody effector function,
encompassing biochemistry, cellular biology, in vitro phagocyte models, and in vivo animal models.
Even more compelling, several human diseases involve genetic C defects, with pathophysiology
predicted by the role of C in immune function. For example, humans with combined deficiency of
CD55 and CD59 experience paroxysmal nocturnal hemoglobinuria (51), which is ameliorated by
preventing C5 activation with a monoclonal antibody (51). Similarly, much evidence supports the
paradigms in which C and FcγRs together produce incompatible RBC destruction. Thus, some
anti-RBC alloantibodies fix C in vitro, inducing rapid MAC-mediated hemolysis. In addition, C3b
is often found on incompatible RBCs recovered posttransfusion (e.g., by the direct antiglobulin
test). Moreover, in animal models, phagocytosis of opsonized RBCs in vitro is prevented when
FcγRs are removed or inhibited (21).
Murine models also provide robust evidence for the role of C and/or FcγRs in clearing in-
compatible RBCs. Inbred mouse strains have genetically well-defined but otherwise poorly char-
acterized erythrocyte alloantigens (EA1–10). When the mice were alloimmunized by repeated
transfusions, C fixation coincided with destruction of transfused incompatible murine RBCs.
More importantly, C depletion in vivo inhibited incompatible RBC clearance. More recently,
transgenic mice expressing human or chimeric model blood group antigens were used to study
HTRs in vivo. Several groups described incompatible transfusions with mouse RBCs expressing
transgenic antigens from the Duffy (21, 52), MN (33, 34, 53, 54), and Kell systems (21, 23, 55),
in addition to a chimeric antigen consisting of hen egg lysozyme (HEL) and ovalbumin (OVA)
sequences fused to Duffy (i.e., the HEL-OVA-Duffy or HOD mouse) (19, 22). Polyclonal anti-
sera induced clearance of RBCs expressing Duffy(b), M, Kell, or Cellano antigens. Because the
entire transgene is missing from recipient, wild-type mice, these alloantisera bind more epitopes
on donor RBCs than is typical in humans; nonetheless, similar situations arise in patients with
anti-RhD and in rare blood group–null recipients. However, monoclonal antibodies, including
anti-M and anti-Fy3, also cleared incompatible murine RBCs (21, 33, 34).
Although monoclonal IgM anti-M cleared M+ RBCs in wild-type mice by intravascular hemol-
ysis, this clearance was substantially decreased in C3- or C5-deficient recipients. In addition, al-
though monoclonal IgG2a anti-Fy3 cleared HOD RBCs in wild-type mice, which was unchanged
in C3 knockout recipients, there was essentially no clearance in mice lacking the FcγR common
γ-chain (these mice lack activating FcγRs: FcγRI, FcγRIII, FcγRIV) (21). Thus, these results cor-
respond with established paradigms for incompatible human transfusions. Similarly, monoclonal
anti-HEL antibodies bind HOD RBCs in vivo, but cause no detectable clearance, providing the
equivalent of a clinically insignificant alloantibody (56). Interestingly, although monoclonal anti-
HEL and anti-Fy3 bind the same protein on HOD RBCs, there is no clearance with the former
but brisk clearance with the latter (21, 56). The basis for this is unclear but relates to IgG subclass
and/or antigen density, because some synthesized HOD protein lacks HEL sequences and thus
higher levels of Fy3 antigen are present ( J.C. Zimring, unpublished observations). In addition,
polyclonal antisera briskly cleared incompatible Kell RBCs, and this clearance was only slightly
decreased by inhibiting C3 or FcγR function (23); however, clearance was absent when C3 and
FcγR pathways were both eliminated ( J.C. Zimring, unpublished observations), demonstrating
their functional redundancy in vivo.

Interestingly, monoclonal anti-M antibodies directly damage human RBCs in vitro by a C -

and FcγR-independent mechanism (57–59). As determined by calcium flux and other assays, these
bound antibodies directly induced eryptosis (57–59). A similar observation was reported for anti-A
(60). In addition, transfused M+ RBCs were rapidly cleared in wild-type murine recipients pas-
sively immunized with the same monoclonal anti-M, and clearance was not significantly decreased
in C3- or FcγR-knockout recipients (34). Clearance also persisted when FcγRI and FcγRIII were
blocked in C3-knockout mice, suggesting a novel C3- and FcγR-independent clearance pathway
(although FcγRIV was not blocked). Finally, clearance persisted despite liposomal clodronate
treatment, which ablates mononuclear phagocytes. Nonetheless, RBCs reentered the circulation
24 h later in clodronate-treated mice but never reappeared in untreated animals, suggesting
that sequestration made these incompatible RBCs susceptible to phagocytosis. Interestingly,
sequestration was previously identified in a human AIHA case and a murine model, in which ery-
throphagocytosis correlated with an agglutinating autoantibody (61, 62). Although sequestration

and reappearance seem unlikely for eryptotic RBCs, these reports suggest hitherto underappre-
ciated potential pathways of antibody-mediated, C - and FcγR-independent RBC clearance.
Additional Interesting Animal Models

Mouse models are particularly useful, because one can genetically modify inbred strains. This
facilitated the introduction of model blood group antigens and testing of effector function (e.g.,
C and FcγRs). However, mice have clear disadvantages, including difficulties in physiological
monitoring. Moreover, mice lack naturally occurring alloantibodies analogous to the human ABO
system. Accordingly, there is utility in studying various species, ranging from rodents (63) to
nonhuman primates (64, 65). In addition to modeling human HTRs, these studies are relevant
for veterinary transfusion medicine. Although multiple species have been studied, and particularly
important results obtained with guinea pigs (66, 67), this discussion is limited to rabbits, cats, dogs,
and nonhuman primates (see Table 1).
Rabbits have primarily been used to identify blood group antigens and their role in immune-
mediated RBC clearance. For example, almost a century ago, alloantibodies induced by deliberate
immunization caused agglutination in vitro and RBC clearance in vivo (68, 69). Incompatible
RBC transfusions into alloimmunized recipients produced scenarios consistent with IgG-induced
AHTRs and DHTRs but not clinically impressive intravascular hemolysis (70). Indeed, these
approaches were typically exploited to produce models of hemolytic disease of the fetus and
newborn rather than models of HTR (71, 72). Nonetheless, in studies in vivo in which soluble
hemoglobin was infused to mimic intravascular hemolysis, recipient dehydration was required
for hemolysis-induced renal dysfunction (73, 74); this provides some rationale for hydration and
diuresis in human AHTRs.
Cats have naturally occurring alloantibodies to carbohydrate RBC antigens that are somewhat
analogous to the human ABO system. Cats are either Group A, B, or AB (but not Group O), and
nonimmunized, nontransfused cats have naturally occurring antibodies (75–78). Severe AHTRs
can occur with the first therapeutic transfusion, with no prior exposure, and produce dramatic
clinical symptoms, including hemoglobinemia, hemoglobinuria, and death (75, 79). No other cat
blood groups were identified until recently; the Mik system is clinically significant, causes HTRs
due to naturally occurring alloantibodies, and is unrelated to the AB system (80).
Cats have also been used experimentally to study HTRs, by deliberately transfusing incom-
patible RBCs into alloimmunized recipients (75, 77). Group A RBC transfusions into Group
B recipients induced severe AHTR symptoms (including anaphylactoid symptoms), rapid RBC
clearance, intravascular hemolysis, hemoglobinemia, hemoglobinuria, markedly decreased C lev-
els, and DIC (77). In contrast, Group B RBC transfusions into Group A recipients produced
Table 1 Models for the study of HTRs

Blood group
Speciesa genetics Models Advantages Disadvantages
Humans Classical Rh immune globulin infusion in Well controlled; clinically Severe reactions are rare and
immune thrombocytopenic relevant unpredictable
purpura; monoclonal and
polyclonal anti-Rh(D) antibodies
Mice Classical, IgM- and IgG-induced HTRs; Well controlled; No evidence of DIC or renal
transgenic, monoclonal and polyclonal recombinant reagents dysfunction; difficult to
knockout antibodies available (e.g., recombinant study physiology
cytokines and inhibitors)
Rabbits Classical HTRs (useful for hemolytic Renal dysfunction Lack of impressive
disease of the fetus and newborn); intravascular hemolysis
hemoglobin infusion
Cats Classical Naturally occurring and Relevant for veterinary No evidence of renal
deliberately induced HTRs; practice; severe dysfunction
xenotransfusions intravascular hemolysis;
similarities to human ABO
system
Dogs Classical Naturally occurring and Relevant for veterinary Questionable evidence of
deliberately induced HTRs; practice; DIC; excellent renal dysfunction
xenotransfusions methods to study physiology
Nonhuman Classical Deliberately induced HTRs AHTRs and DHTRs; severe AHTRs are IgG-induced;
primates (cynomolgus monkeys); reactions with DIC, renal DHTRs are IgM-induced;
hemoglobin infusions (baboons) failure, and death ethical concerns
a
Although blood groups have been described in many other animals, in many of which immune-mediated hemolysis has been described (e.g., guinea pigs,
sheep, horses, and cows), the discussion in this article is limited to the species described here.
Abbreviations: AHTR, acute hemolytic transfusion reaction; DHTR, delayed hemolytic transfusion reaction; DIC, disseminated intravascular coagulation;
HTR, hemolytic transfusion reaction.
milder signs and symptoms, with some intravascular, but mostly extravascular, hemolysis; the
direct antiglobulin test was positive, incompatible RBCs were cleared over days rather than min-
utes, and C levels decreased slightly. Unfortunately, no AB recipients were examined. To evaluate
differences between incompatible RBC transfusions and incompatible plasma infusions (i.e., major
and minor cross-match-incompatible transfusions), incompatible plasma from Group B cats (i.e.,
containing anti-A) was infused into Group A recipients (77); this had no apparent clinical effect,
suggesting that incompatible RBC transfusions induce more severe reactions than do incompatible
plasma infusions.
The biochemistry of these feline antigens was partially elucidated. Group A RBCs express
a series of sialic acid–containing glycosphingolipids, with GD3 as the major species, that have
N-glycolylneuraminic acid (NeuGc) as the predominant sialic acid and only minor amounts of
N-acetylneuraminic acid (NeuAc). In contrast, glycosphingolipids of Group B RBCs contain
only NeuAc, again with GD3 predominating. Western blots were negative, suggesting that A
and B antigens are only on glycosphingolipids. These results suggested that the difference be-
tween A and B was the presence or absence, respectively, of a NeuAc hydroxylase that converts
NeuAc into NeuGc (81). This was confirmed by identifying mutations in the CMAH hydroxylase
gene, producing an inactive enzyme or an absent protein, in Group B cats; therefore, the B blood
group allele is denoted as b (82). Group AB cats do not produce anti-A or anti-B antibodies and
are universal recipients for RBC transfusions (83, 84). The allele in AB cats (i.e., aab ) remains

mysterious; it is not explained by CMAH hydroxylase mutations. Nonetheless, the three cat AB
system alleles have the following order of genetic dominance: A > aab > b (82, 84, 85). The im-
mune response to carbohydrate antigens is predominantly thymus independent, producing IgM
alloantibodies that efficiently activate C , thereby inducing intravascular hemolysis; this is similar
to the human ABO system.
Using an alternative approach (86, 87), rabbit blood transfusions into feline recipients produced
severe, often fatal, AHTRs. Although the consequences of these xenotransfusions were not shown
to result from immune-mediated RBC clearance, this system, along with alloantibody-induced
HTR models, was exploited for elucidating clinical scenarios, mechanistic pathophysiology, and
therapeutic interventions. Similarities and differences between feline and human HTRs were
described. Cat AHTRs were dramatic and potentially fatal, with signs and symptoms of the in-
travascular hemolysis observed in humans, including DIC (77, 86). However, unlike in humans, no
renal dysfunction was noted (77), although anaphylactoid responses were observed (77). Although
cytokines were not measured, inflammatory sequelae due to prostaglandin-like substances were
identified (87). In the xenotransfusion model, corticosteroids were therapeutically effective (86,
87), and aspirin, indomethacin, and a thromboxane synthetase inhibitor ameliorated AHTR signs
and symptoms, including decreasing DIC and preventing death (86, 87).
Most canine blood groups were identified by investigating clinical HTRs seen during veteri-
nary practice (88–92). However, not all hemolysis seen clinically is immune mediated (93). In
addition, experimental RBC alloantibody production and incompatible RBC transfusions in dogs
began more than 100 years ago (91, 94–98). These experiments further elucidated dog blood group
antigen complexity and HTR mechanisms. Nonetheless, it remains controversial whether incom-
patible transfusions and xenotransfusions induce renal dysfunction in dogs (98–102). In contrast,
DIC induction by HTRs is more clear-cut, although therapeutic interventions were not attempted
(97, 100–102). Although cytokine storm was not evaluated, it is likely, because proinflammatory
cytokines were observed in dogs transfused with compatible older, stored RBCs (103). In addition,
whether incompatible RBC clearance in dogs exacerbates infection is unknown, but this is likely
to be the case given the results with older, stored RBC transfusions (104–106). In general, studies
evaluating therapeutic interventions in dog HTR models are few and without definitive results; for
example, it is unclear whether corticosteroids are beneficial, dangerous, or ineffective (90, 101).
Few reports describe nonhuman primate HTR models, despite the history of the Rh (i.e., rhe-
sus) blood group system (107) and the evolutionary similarity between humans and nonhuman
primates. Most studies are derived from a single group using cynomolgus monkeys. Although
formal blood group antigens were not identified, the investigators deliberately alloimmunized
individual monkeys, thereby allowing incompatible transfusions (108). Complete agglutinins and
hemolysins rapidly cleared incompatible transfused RBCs, accompanied by intravascular hemolysis
(i.e., hemoglobinemia, decreased haptoglobin, hemoglobinuria), renal dysfunction (e.g., anuria),
cardiac dysfunction, tachypnea, DIC, and death (108, 109). Surprisingly, these complete alloan-
tibodies were IgG (110). In contrast, incomplete agglutinins and hemolysins produced predomi-
nantly extravascular hemolysis, and transfused RBCs were cleared over hours (108). Again surpris-
ingly, these incomplete alloantibodies were IgM (110). In addition, decreased circulating levels of
complete, but not incomplete, alloantibodies were seen immediately after incompatible transfusion
(108). Thus, although of great interest, these models do not reflect human AHTRs or DHTRs re-
garding antibody isotype. Nevertheless, because DIC accompanied intravascular hemolysis, these
studies evaluated whether the antigen-antibody complexes, RBC stroma, and/or free hemoglobin
were pathophysiologically relevant (108). Infusions of sonicated RBCs or washed stroma pro-
duced DIC, renal dysfunction, and death, whereas neither antigen-antibody complexes (111) nor
stroma-free hemoglobin (112) was effective. The roles of the kallikrein system and Hageman
factor were also examined (113). Taken together, the data from this group suggested that, al-
though alloantibodies caused intravascular hemolysis, the resulting RBC membrane fragments—
not the released hemoglobin or the antibodies themselves—induced DIC. In baboons, hemolyzed,
stroma-containing blood infusions also induced DIC (114); however, interestingly, intravenous
infusion of dextran, a volume expander, exacerbated DIC (114).
AHTR-induced renal dysfunction was also studied in cynomolgus monkeys (108). RBCs
hemolyzed by sonication, or washed RBC stroma, induced renal dysfunction, and interestingly,
unwashed RBC stroma was worse; however, antigen-antibody complexes were not required (111)
and stroma-free hemoglobin was ineffective (112). Although the authors suggested that DIC might
induce renal dysfunction (110), particularly because of fibrin thrombi in small renal vessels (109),
this was not thoroughly investigated. For example, studies preventing DIC (e.g., using anticoagu-
lants) and then evaluating the effect on renal dysfunction were not performed. Finally, hypotension
and shock, seen in human AHTRs, were also seen in the cynomolgus monkey IgG-induced AHTR
model (113). Although preliminary studies implicated the kallikrein system and Hageman factor
(113), definitive conclusions were not drawn.
Effects of Recipient Genetic Variability

Human RBC clearance variability was identified when RhD+ RBCs, opsonized with anti-D, were
infused in vivo; this assay evaluated reticuloendothelial system Fc receptor function in autoimmune
disorders (115). Clearance variability, seen in healthy controls and patients (116), was attributed
to genetic differences in FcγR function. However, in a subsequent study, 4 of 24 subjects had sub-
stantially prolonged clearance and, although reproducible in volunteers, prolonged clearance was
not observed using RBCs from other donors (117). This finding challenged the original interpre-
tation that antibody-opsonized RBC clearance reflected only FcγR function, and it demonstrated
intrinsic RBC variability in clearance susceptibility. In addition, RBC clearance variability was not
due to antibody density on the opsonized RBCs (117). Nonetheless, subsequent analyses strongly
argued that FcγR genetics was also involved in regulating RBC clearance. Thus, recipient FcγR
polymorphisms correlated with clearance of RhD+ RBCs infused into RhD− volunteers passively
immunized with monoclonal anti-D (118, 119); FcγRIIIa and FcγRIIa polymorphisms corre-
lated with clearance by IgG3 and IgG1 anti-D, respectively. Because these differences diminished
with polyclonal anti-D (118, 119), it is unclear whether these findings extend to typical alloan-
tisera. Nonetheless, these studies provide compelling evidence that donor and recipient genetics
may substantially influence whether a given patient will hemolyze an incompatible unit, to what
extent, and in what fashion; however, this phenomenon remains poorly understood.
DOWNSTREAM CONSEQUENCES OF RED BLOOD CELL HEMOLYSIS

HTR pathophysiology includes more than the cellular and molecular biology of RBC clearance de-
scribed above. To fully understand the mechanisms underlying various downstream consequences
of HTRs (e.g., cytokine storm, renal dysfunction, shock, DIC, and death), it is important to review
how RBCs and their contents are handled during intravascular and extravascular hemolysis.
Responses to Intravascular Hemolysis

Following intravascular hemolysis, haptoglobin rapidly binds hemoglobin with defined stoichiom-
etry (120–122). This binding stabilizes hemoglobin; in this complex its vasoactive and oxidative
effects are diminished (123). Hemoglobin-haptoglobin complexes are recognized by CD163 (124),

found only on monocytes and macrophages (125), and ingested by receptor-mediated endocytosis.
Human haptoglobin polymorphisms exist; the most important are Hp 1-1, Hp 2-1, and Hp 2-2
(126). Interestingly, Hp 1-1 binds hemoglobin with higher affinity than does Hp 2-2 (126) and is
superior at clearing hemoglobin through CD163 (127).
This classical mechanism may have evolved to protect the host from the oxidative effects of
free hemoglobin released by hemolysis (e.g., malaria) (126). However, circulating haptoglobin
is limiting (∼100–200 mg/dL) and rapidly depleted during HTRs. Nonetheless, haptoglobin is
an acute-phase reactant; cytokines (e.g., IL-6) and inflammation rapidly induce its expression,
primarily by hepatocytes (128). Haptoglobin’s importance is illustrated in knockout mice (129), in
which host susceptibility to the damaging effects of free hemoglobin is greatly enhanced. Humans
with genetic anhaptoglobinemia are at risk for anaphylactic transfusion reactions (130); although
they are perhaps predicted to respond poorly to hemolysis, no data are available. Analogously,
haptoglobin infusions might be therapeutically beneficial during hemolysis (131, 132); this is
discussed below.
Although CD163 expression might also be limiting and, thus, important in responding to
hemolysis, studies with CD163-knockout mice have not been informative on this point (133) be-
cause of human/mouse differences in the hemoglobin-haptoglobin-CD163 system. Nonetheless,
CD163 expression is induced by cytokines (e.g., IL-6) and downregulated by lipopolysaccha-
ride (LPS), interferon-γ, and tumor necrosis factor (TNF)-α (125). Glucocorticoids also induce
CD163 expression (125), thereby promoting hemoglobin-haptoglobin complex clearance (134).
Following hemoglobin-haptoglobin complex ingestion, ferritin, ferroportin, antioxidant enzymes
[e.g., heme oxygenase 1 (HO-1)], and heme carrier protein 1 (HCP-1) (135) are induced, thereby
enhancing free hemoglobin catabolism by macrophages (136). Therefore, glucocorticoids may
have therapeutic benefits during HTRs (see below).
Although the hemoglobin-haptoglobin-CD163 pathway is the classical cellular clearance
mechanism, other pathways and hemoglobin-binding proteins exist. For example, CD163, al-
beit with lower affinity, binds and clears free hemoglobin by receptor-mediated endocytosis, even
without haptoglobin (137). In addition, proteolytically shed, soluble CD163 (i.e., sCD163) di-
rectly binds plasma hemoglobin; the latter also binds IgG, and this ternary sCD163-hemoglobin-
IgG complex is cleared by FcγR-mediated endocytosis (138). Although interesting, this novel
pathway needs confirmation. Because FcγR-mediated signal transduction also mediates sCD163
release (139), this is a potential novel pathway during IgG-mediated HTRs involving intravascu-
lar hemolysis. Finally, apolipoprotein A-1 binds free hemoglobin; these complexes are cleared by
macrophage scavenger receptors (33, 140).
Following receptor-mediated endocytosis, hemoglobin is degraded in the endosomal-
lysosomal system (135). After initial degradation in acidic endosomes, HCP-1 transports the
released heme to the cytosol, where it encounters HO-1. If Fe2+ is released in the endosome, it is
transported to the cytosol by divalent metal transporter 1 (DMT1); there, free iron participates in
redox reactions, is incorporated into ferritin, or is exported by ferroportin. Finally, the resulting
globin polypeptides, and any remaining hemoglobin, are degraded in lysosomes, with analogous
cytosolic export of heme and Fe2+ .
If released hemoglobin is not bound by haptoglobin (e.g., haptoglobin depletion by rapid
intravascular hemolysis) or cleared by the other pathways described above, then it circulates as
free hemoglobin, visualized clinically as hemoglobinemia. Most free hemoglobin undergoes renal
clearance (133, 141), producing hemoglobinuria and potentially causing renal dysfunction due, in
part, to NO scavenging and vasoconstriction (129, 142).
Multiple studies examined whether free hemoglobin induces inflammation, particularly in in-
nate immunity by functioning as a Toll-like receptor (TLR) ligand. The consensus suggests that
hemoglobin is not a TLR ligand and does not directly induce inflammation (143), but rather
binds multiple damage-associated molecular patterns (DAMPs) and pathogen-associated molecu-
lar patterns (PAMPs), enhancing their TLR-mediated proinflammatory function (143, 144). For
example, hemoglobin binds LPS and enhances LPS-induced inflammation in vitro (e.g., TNF-α
secretion) (145) and LPS- and sepsis-induced mortality in vivo (145, 146). Unexpectedly, globin
(i.e., lacking heme and iron) has the opposite effect, binding LPS and ameliorating its conse-
quences in vitro and in vivo (145). Although hemoglobin also interacts with HMGB1 (a DAMP)
and various PAMPs to enhance inflammation, these effects are probably due to heme, because
they are suppressed by hemopexin (147); the possible therapeutic benefits of hemopexin infusions
are discussed below. Finally, although NO scavenging by free hemoglobin primarily modulates
vascular tone (148), NO also affects inflammation and immunity (149).
If free hemoglobin continues to circulate, it degrades intravascularly. Fe2+ rapidly oxidizes

to Fe3+ , producing methemoglobin, and heme is readily released by methemoglobin (150–

152). Hemoglobin tetramers also rapidly dissociate into dimers (135). Interestingly, CO-bound
hemoglobin does not release heme; thus, CO production by HO-1 metabolism of heme might
be beneficial (153), suggesting that, given heme’s toxicity (see below), inhaling (low amounts of !)
CO might provide therapeutic benefit during HTRs. However, few data currently support this
approach (154).
Hemopexin avidly binds highly toxic free heme with 1:1 stoichiometry (141). These bind hep-
atocyte CD91 and are ingested by receptor-mediated endocytosis (141). Circulating hemopexin
levels are limiting (155) and decrease during intravascular hemolysis. Nonetheless, hemopexin
is an acute-phase reactant (156), and its hepatic synthesis can be markedly induced (141, 152).
If hemopexin is depleted, heme binds other proteins, but with lower affinity, including albumin
(155), low-density lipoprotein, high-density lipoprotein, and α1-microglobulin (141).
If not bound, free heme produces multiple effects, particularly on endothelial cells and circulat-
ing cells. Thus, free heme induces bone marrow release of neutrophils (157) and directly activates
them, inducing the oxidative burst, IL-8 expression, and chemotaxis (158), the latter perhaps
through G protein–coupled receptor signaling and increased reactive oxygen species (ROS) levels
(159). Free heme also activates endothelial cells, upregulating intercellular adhesion molecule 1
(ICAM-1) and P-selectin expression, inducing adhesion of (activated) neutrophils (160).
Free heme induces inflammation in vitro, signaling through nuclear factor κB (NF-κB) to
upregulate cytokine production [e.g., IL-1β, monocyte chemoattractant protein 1 (MCP-1),
macrophage inflammatory protein (MIP)-2, MIP-1α, and TNF-α] (143, 161). Heme also syner-
gizes with multiple TLR ligands, including LPS, enhancing inflammation in vitro and in vivo (143,
162); in addition to NF-κB, these effects involve ROS production, Syk, and the mitogen-activated
protein kinase pathway. These findings suggest that heme may be a TLR ligand. Although this
process is still actively being investigated, data suggest that heme directly binds TLR4 and in-
duces cytokine production in a LPS-independent manner and independent of HO-1-mediated
metabolism (163–165).
Whether cellular delivery of heme derives from free heme, the heme-hemopexin-CD91
pathway, the hemoglobin-haptoglobin-CD163 pathway, or extravascular hemolysis (see below),
it is catabolized by HO-1 to yield biliverdin and CO (148). A mismatch between heme delivery
and its catabolism yields dramatic consequences, as seen in HO-1-knockout mice, including renal
dysfunction, cytokine synthesis, hepatocyte iron overload, and enhanced sepsis mortality (152,
166, 167).
Given the importance of heme and/or hemoglobin clearance during intravascular hemoly-
sis, purified and/or recombinant haptoglobin and/or hemopexin infusions be might expected to
be therapeutically beneficial; this may be true (131, 132, 147, 166). Finally, during intravascular

hemolysis, RBC cytosolic contents (168, 169) are released into the circulation, along with the mem-
branes (170) and cytoskeleton (i.e., stroma) of RBCs. Other than measurement of clinical param-
eters (e.g., lactate dehydrogenase and potassium levels) and studies with stroma-free hemoglobin
(112), little is known about the importance of these components in HTR pathophysiology.
Responses to Extravascular Hemolysis

IgG-mediated DHTRs primarily induce extravascular hemolysis. IgG- and C -coated RBCs are
cleared by FcγR- and CR-mediated pathways, respectively. In intact hosts, splenic macrophages
predominantly clear opsonized RBCs, but in splenectomized recipients, Kupffer cells mediate
hepatic clearance. In addition, other cells, such as endothelial cells and neutrophils, can in-
gest opsonized RBCs (171, 172). FcγR- and CR-mediated signal transduction systems induc-
ing phagocytosis are well understood; inhibiting these pathways (173) or depleting macrophages
(e.g., by liposomal clodronate) prevents opsonized RBC clearance (174). Following ingestion, the
endosome-lysosomal system degrades RBCs, releasing hemoglobin, heme, and iron, followed by
cytosolic heme metabolism by HO-1 and intracellular iron storage and/or export mediated by
ferritin and ferroportin, respectively.
The FcγR pathway is proinflammatory, inducing cytokine secretion, even following phagocy-
tosis of inert particles. However, increased free iron concentrations in the labile intracellular pool
also enhance cytosolic ROS production, which may signal through NF-κB and/or inflammasomes
to induce proinflammatory cytokine secretion.
Macrophage death is a dramatic consequence of defective heme metabolism in vitro and in vivo,
or of overexuberant ingestion of heme, hemoglobin, or opsonized RBCs (152, 175, 176). It occurs
by direct toxicity and/or by autocrine-induced programmed necrosis (164). Although a markedly
diminished macrophage population should inhibit further clearance of heme, hemoglobin, and
damaged or opsonized RBCs (or pathogens), this phenomenon has been little appreciated, partic-
ularly in HTRs.
Cytokine Storm as a Unifying Concept in Hemolytic Transfusion

Reaction Pathophysiology
Similarities between the clinical presentations of HTRs, sepsis, and the systemic inflammatory
response syndrome suggested that the mechanisms underlying HTRs could substantially be due
to cytokine storm (177, 178). The work described above concerning hemolysis suggests that it
induces and enhances inflammation. This section focuses on evidence that HTRs induce cytokine
storm. Although no further discussion ensues regarding other major consequences of HTRs (i.e.,
DIC, renal dysfunction, shock, and death), there is some consensus that these are also explained
by cytokine storm.
Multiple studies using in vitro HTR models identified monocytes or macrophages (179)
as the probable source of proinflammatory (e.g., TNF-α, IL-8, MCP-1) (180–184) and anti-
inflammatory cytokines (e.g., IL-1ra) (181, 185). These concepts were confirmed in human pa-
tients with AHTRs or DHTRs (186–188); these studies are difficult to perform because clinical
HTRs occur sporadically and unexpectedly, and controlled trials are unethical. However, settings
exist in which inducing an HTR-like event is therapeutically indicated (189). The best exam-
ple involves intravenous infusions of purified anti-D Rh immunoglobulin into RhD+ recipients
to treat immune thrombocytopenic purpura. These infusions can induce significant, potentially
life-threatening, drops in hemoglobin (190, 191), thus mimicking HTRs. In addition, increased
levels of multiple cytokines were induced in vivo (192, 193). Given the controlled nature of this
intervention, future mechanistically based studies are possible. Even more control is possible using
mouse models of IgG- and IgM-mediated HTRs (54); passive immunization with IgG anti-RBC
alloantibodies, followed by incompatible transfusions, led to accelerated clearance in vivo by ex-
travascular hemolysis (54), accompanied by cytokine storm (194), which required FcγR function
but not C (34).
Sickle Cell Disease as a Special Case

Sickle cell disease patients receive multiple RBC transfusions throughout their life, have high
levels of RBC alloimmunization, and are at significant risk for experiencing HTRs. Interestingly,
HTRs in these patients can present clinically as stereotypical vaso-occlusive crises (195). Given
that patients with sickle cell disease have an underlying proinflammatory state (196), their chronic
hemolysis may enhance HTR-induced inflammatory responses. However, chronic hemolysis also
upregulates HO-1 function and ferritin, which may diminish inflammation. Murine sickle cell dis-
ease models have enabled mechanistic studies of these issues, particularly suggesting that cytokines,
such as TNF-α, induce vaso-occlusive crises in vivo (197). Interestingly, although IgG-mediated
HTRs induce vaso-occlusive crises in sickle cell disease mice, TNF-α levels are not increased (198),
in contrast to the situation in wild-type mice (194). Nonetheless, HTRs in sickle cell disease mice
increase IL-8 levels, as in wild-type mice (194) and humans (183, 188). In addition, blocking IL-8
prevented HTR-induced vaso-occlusive crises in sickle cell disease mice (198). Interestingly, el-
evated IL-8 levels are a biomarker for vaso-occlusive crises in human sickle cell disease patients
(199). Multiple genetic cytokine polymorphisms have been described, particularly in individu-
als of African descent, that modify inflammatory responses (200, 201); this variation in cytokine
responsiveness may affect the clinical outcome of incompatible transfusions in these patients.
Another dramatic, and potentially fatal, complication of HTRs in sickle cell patients is hyper-
hemolysis (202). In this syndrome, not only are transfused incompatible RBCs rapidly cleared, but
there is also brisk hemolysis of the patient’s own “innocent bystander” RBCs. Although the under-
lying pathophysiology is not understood, various mechanisms, including cytokines and eryptosis,
have been proposed (203, 204).
Finally, the case of sickle cell disease described at the beginning of this article illustrates how
a patient’s underlying disease, along with potential genetic polymorphisms in C (C proteins,
CRs, and C -regulatory proteins), FcγRs, haptoglobin, hemopexin, metabolic enzymes (e.g.,
HO-1), and cytokines, can modify the clinical expression and downstream consequences of
HTRs. Understanding these mechanisms and modifying factors aids in identifying the correct
diagnosis and provides clues regarding appropriate therapy (see below).
TREATMENT OPTIONS FOR HEMOLYTIC

TRANSFUSION REACTIONS
The best available treatment for HTRs is prevention, and most efforts in transfusion therapy
for ∼100 years involve practicing personalized medicine: providing the right blood to the right
recipient at the right time. Thus, extensive efforts are made to store blood appropriately, to type
donors and recipients accurately, to screen recipients for alloantibodies, to perform serological
cross-matches, and to monitor recipients closely posttransfusion. However, HTRs still occur, and
unfortunately, posttransfusion treatment options have not changed for ∼30 years despite little
evidence regarding efficacy. Current treatments include corticosteroids, which are effective in
AIHA but may or may not be effective for HTRs (205, 206), fluids and diuretics (perhaps along
with urine alkalinization), standard treatment of DIC, if present, and general supportive care.

Based on the concepts presented here, the current treatments appear logical, but they have un-
clear utility. Nonetheless, in the context of the pathophysiological mechanisms described, other
treatments are possible, some of which are being tested in settings of (antibody-mediated) hemoly-
sis. These include modifying the effects of intravascular hemolysis using NO inhalation (207, 208),
oral arginine (209), haptoglobin and hemopexin infusions (131, 132, 147, 166), upregulation of
HO-1 function (210), and acetaminophen to reduce heme-induced oxidation (211). In addition, C
and CR inhibitors (212–216) can potentially diminish intravascular and extravascular hemolysis.
Macrophage depletion or functional inhibition could also prevent extravascular hemolysis and its
consequences; this was accomplished in models using clodronate (174, 217), ethanol (218, 219),
glycine supplementation (220, 221), and glutamine depletion (222, 223). FcγR function can also
be blocked directly (224, 225) or by inhibiting subsequent signal transduction (173, 226). Finally,
modulating the inflammatory response and cytokine storm is attractive and may be one mecha-
nism of corticosteroid action; novel approaches include intravenous gammaglobulin (227), ethyl
pyruvate (228, 229), and IL-10 or IL-1ra infusions (230). Although difficult to study during spo-
radic human HTRs, these approaches can be evaluated in animal models and in more controlled
human settings (e.g., Rh immune globulin infusion for immune thrombocytopenic purpura).
FINAL THOUGHTS REGARDING THE CLINICAL CASE

The biological landscape of the mechanisms underlying immune-mediated destruction of incom-
patible transfused RBCs is wide, including C protein action, FcγR ligation, the effects of RBC
fragments and contents, and the metabolism of multiple hemoglobin breakdown products, includ-
ing signaling pathways involving iron and oxidative stress. These almost certainly do not work in
isolation, and evidence of cross talk continues to grow. Downstream effects involve direct effector
molecules and signal transduction pathways that induce both damaging and protective responses,
thereby modifying vascular biology, cytokine storm, coagulation, and end organ function. As
knowledge continues to grow, and the armamentarium of interventions increases, the HTR treat-
ments recommended by expert panels may evolve. To the extent possible, these decisions should be
guided by evidence. Although the patient described at the beginning of this article fortunately had
a favorable outcome, it remains unclear whether his recovery was due to the multiple therapeutic
interventions provided, whether he recovered in spite of these, or whether they had no effect.
We hope that future therapies will be more mechanistically based, more specific, and more goal
directed (e.g., using IL-8 inhibitors or targeted C inhibitors), rather than providing everything
that one uses for any transfusion reaction (e.g., diphenhydramine is more appropriate for allergic
transfusion reactions). Finally, given the sporadic nature of HTRs, it will be challenging to assess
whether new interventions are superior to existing recommendations, especially given the lack of
evidence regarding the efficacy of current recommendations.
DISCLOSURE STATEMENT
J.C.Z. has a sponsored research project with Immucor, Inc., for the development of RBC typing
reagents, which is unrelated to the general topic of this review. The authors are not aware of any
other affiliations, memberships, funding, or financial holdings that might be perceived as affecting
the objectivity of this review.
ACKNOWLEDGMENTS
We wish to dedicate this paper to the memory of Dr. George Garratty for his many con-
tributions to our understanding of immune-mediated hemolysis, his enthusiasm for all things

immunohematological, and his passionate and devoted support to young investigators in transfu-
sion medicine.
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Arthritis Rheumatol. 36:1305–14

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The Roles of Cellular and Organismal Aging in the Development of

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Driver and Passenger Mutations in Cancer
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Engineered In Vitro Disease Models
Kambez H. Benam, Stephanie Dauth, Bryan Hassell, Anna Herland, Abhishek Jain,
Kyung-Jin Jang, Katia Karalis, Hyun Jung Kim, Luke MacQueen,
Roza Mahmoodian, Samira Musah, Yu-suke Torisawa,
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AA Amyloidosis: Pathogenesis and Targeted Therapy
Gunilla T. Westermark, Marcus Fändrich, and Per Westermark p p p p p p p p p p p p p p p p p p p p p p p 321
Hepatitis C Virus–Associated Cancer
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Diseases of Pulmonary Surfactant Homeostasis
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The Inflammasomes and Autoinflammatory Syndromes

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DNA Replication Stress as a Hallmark of Cancer
Morgane Macheret and Thanos D. Halazonetis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 425
Birth and Pathogenesis of Rogue Respiratory Viruses
David Safronetz, Heinz Feldmann, and Emmie de Wit p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 449
Protein Glycosylation in Cancer
Sean R. Stowell, Tongzhong Ju, and Richard D. Cummings p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 473
Pathobiology of Severe Asthma
Humberto E. Trejo Bittar, Samuel A. Yousem, and Sally E. Wenzel p p p p p p p p p p p p p p p p p p p p 511
Genetics and Epigenetics of Human Retinoblastoma
Claudia A. Benavente and Michael A. Dyer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 547
Indexes
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Cumulative Index of Article Titles, Volumes 1–10 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 567
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The Annual Review of Virology captures and communicates exciting advances in our understanding of viruses
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TABLE OF CONTENTS:
• Forty Years with Emerging Viruses, C.J. Peters • Polydnaviruses: Nature’s Genetic Engineers, Michael R. Strand,
• Inventing Viruses, William C. Summers Gaelen R. Burke
• PHIRE and TWiV: Experiences in Bringing Virology to New Audiences, • Human Cytomegalovirus: Coordinating Cellular Stress, Signaling,
Graham F. Hatfull, Vincent Racaniello and Metabolic Pathways, Thomas Shenk, James C. Alwine
• Viruses and the Microbiota, Christopher M. Robinson, Julie K. Pfeiffer • Vaccine Development as a Means to Control Dengue Virus
Pathogenesis: Do We Know Enough? Theodore C. Pierson,
• Role of the Vector in Arbovirus Transmission, Michael J. Conway, Michael S. Diamond
Tonya M. Colpitts, Erol Fikrig
• Archaeal Viruses: Diversity, Replication, and Structure, Nikki Dellas,
• Balance and Stealth: The Role of Noncoding RNAs in the Regulation Jamie C. Snyder, Benjamin Bolduc, Mark J. Young
of Virus Gene Expression, Jennifer E. Cox, Christopher S. Sullivan
• AAV-Mediated Gene Therapy for Research and Therapeutic Purposes,
• Thinking Outside the Triangle: Replication Fidelity of the Largest RNA R. Jude Samulski, Nicholas Muzyczka
Viruses, Everett Clinton Smith, Nicole R. Sexton, Mark R. Denison
• Three-Dimensional Imaging of Viral Infections, Cristina Risco,
• The Placenta as a Barrier to Viral Infections, Isabel Fernández de Castro, Laura Sanz-Sánchez, Kedar Narayan,
Elizabeth Delorme-Axford, Yoel Sadovsky, Carolyn B. Coyne Giovanna Grandinetti, Sriram Subramaniam
• Cytoplasmic RNA Granules and Viral Infection, Wei-Chih Tsai, • New Methods in Tissue Engineering: Improved Models for Viral
Richard E. Lloyd Infection, Vyas Ramanan, Margaret A. Scull, Timothy P. Sheahan,
• Mechanisms of Virus Membrane Fusion Proteins, Margaret Kielian Charles M. Rice, Sangeeta N. Bhatia
• Oncolytic Poxviruses, Winnie M. Chan, Grant McFadden • Live Cell Imaging of Retroviral Entry, Amy E. Hulme, Thomas J. Hope
• Herpesvirus Genome Integration into Telomeric Repeats of Host • Parvoviruses: Small Does Not Mean Simple, Susan F. Cotmore,
Cell Chromosomes, Nikolaus Osterrieder, Nina Wallaschek, Peter Tattersall
Benedikt B. Kaufer • Naked Viruses That Aren’t Always Naked: Quasi-Enveloped Agents
• Viral Manipulation of Plant Host Membranes, Jean-François Laliberté, of Acute Hepatitis, Zongdi Feng, Asuka Hirai-Yuki, Kevin L. McKnight,
Huanquan Zheng Stanley M. Lemon
• IFITM-Family Proteins: The Cell’s First Line of Antiviral Defense, • In Vitro Assembly of Retroviruses, Di L. Bush, Volker M. Vogt
Charles C. Bailey, Guocai Zhong, I-Chueh Huang, Michael Farzan • The Impact of Mass Spectrometry–Based Proteomics on Fundamental
• Glycan Engagement by Viruses: Receptor Switches and Specificity, Discoveries in Virology, Todd M. Greco, Benjamin A. Diner,
Luisa J. Ströh, Thilo Stehle Ileana M. Cristea
• Remarkable Mechanisms in Microbes to Resist Phage Infections, • Viruses and the DNA Damage Response: Activation and Antagonism,
Ron L. Dy, Corinna Richter, George P.C. Salmond, Peter C. Fineran Micah A. Luftig
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PM10CH04-Zimring ARI 2 December 2014 8:47

• Other articles in this volume

• Top downloaded articles

Annu. Rev. Pathol. Mech. Dis. 2015. 10:83–110 Keywords

This article’s doi: Abstract

downstream consequences [e.g., renal dysfunction, disseminated intravascular coagulation (DIC),

www.annualreviews.org • Pathobiology of Transfusion Reactions 85

THE ROLE OF COMPLEMENT IN HEMOLYTIC

C3-deﬁcient animals; however, it is unclear whether C3-independent pathways are important in

FC RECEPTORS AND ANTIBODY EFFECTOR FUNCTIONS

γ-chain (FcγRI and FcγRIIIA). In contrast, FcγRIIB (CD32B) contains an immunoreceptor

NEGATIVE FEEDBACK LOOPS IN ANTIBODY BINDING

www.annualreviews.org • Pathobiology of Transfusion Reactions 87

secondary antibodies (18).

INTEGRATED HEMOLYTIC TRANSFUSION REACTION MODEL

MAC formation, resulting in extravascular hemolysis. Nonetheless, in patients with incompatible

Effector Mechanisms in Hemolytic Transfusion Reactions

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Interestingly, monoclonal anti-M antibodies directly damage human RBCs in vitro by a C -

throphagocytosis correlated with an agglutinating autoantibody (61, 62). Although sequestration

Additional Interesting Animal Models

Table 1 Models for the study of HTRs

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Effects of Recipient Genetic Variability

DOWNSTREAM CONSEQUENCES OF RED BLOOD CELL HEMOLYSIS

Responses to Intravascular Hemolysis

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If free hemoglobin continues to circulate, it degrades intravascularly. Fe2+ rapidly oxidizes

to Fe3+ , producing methemoglobin, and heme is readily released by methemoglobin (150–

www.annualreviews.org • Pathobiology of Transfusion Reactions 97

Responses to Extravascular Hemolysis

Cytokine Storm as a Unifying Concept in Hemolytic Transfusion

Sickle Cell Disease as a Special Case

TREATMENT OPTIONS FOR HEMOLYTIC

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FINAL THOUGHTS REGARDING THE CLINICAL CASE

100 Zimring · Spitalnik

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102 Zimring · Spitalnik

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104 Zimring · Spitalnik

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106 Zimring · Spitalnik

J. Infect. Dis. 202:624–32

system. DNA Cell Biol. 32:36–40

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renal damage after acute hemolysis in hemopexin-deﬁcient mice. Blood 94:3906–14

108 Zimring · Spitalnik

immune globulin intravenous administration in immune thrombocytopenic purpura patients. Blood

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110 Zimring · Spitalnik

Contents Volume 10, 2015

The Roles of Cellular and Organismal Aging in the Development of

Emerging Concepts in Alzheimer’s Disease

The Inﬂammasomes and Autoinﬂammatory Syndromes

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An online log of corrections to Annual Review of Pathology: Mechanisms of Disease articles

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