Transfusion Medicine: Antigens, Antibodies, and Serologic Testing in Dogs and Cats

Topics in Companion An Med 34 (2019) 3646
Review Article
Transfusion Medicine: An Update on Antigens, Antibodies and Serologic
Testing in Dogs and Cats
Rebecca Zaremba, DVMa,b,*, Aimee Brooks, DVM, MS, DACVECCa,b,
Elizabeth Thomovsky, DVM, MS, DACVECCa,b
Keywords:
Blood typing
crossmatch
antigen
antibody
standard laboratory
immunochromatographic strip
gel-tube assay
transfusion reaction
a
Department of Veterinary Clinical Sciences,
Purdue University College of Veterinary
Medicine, West Lafayette, IN, USA
A B S T R A C T
Red blood cell transfusions have become an integral component in the treatment of anemic patients and
the number of transfusions continues to increase from year to year in both veterinary and human medicine.
Although crucial, red blood cell transfusions are not benign and can lead to serious adverse reactions. Therefore, determining the most appropriate donor unit to use for transfusion can be challenging. As transfusion
medicine evolves, greater numbers of blood surface antigens are being documented and pretransfusion testing protocols are becoming more complex. In order to better understand this evolving field, this review
presents the basic immunology of pretransfusion testing, including methodologies of blood typing and crossmatch testing, and their application in canine and feline medicine.
© 2019 Elsevier Inc. All rights reserved.
b
Purdue University College of Veterinary
Medicine, West Lafayette, IN, USA
Introduction
Background and Terminology
The first documented successful red blood cell (RBC) transfusion in
any species was an experimental dog to dog transfusion performed in
1665 by Dr Richard Lower. Experimental transfusions in cats as part
of blood typing research were performed in the early 1900s by Dr
Reuben Ottenburg.1 Today, RBC transfusions are a critical and potentially life-saving therapy for both human and veterinary patients.
However, RBC transfusions are not without potential risk.2,3 The most
significant complication related to a RBC transfusion is an acute
hemolytic transfusion reaction (AHTR). During an AHTR, the immune
system of the recipient acutely destroys transfused red blood cells
from the donor. Published rates of AHTRs during RBC transfusions in
dogs are reported to be between 0.2% and 1.0%, but these numbers
may be an underestimation.3,4,5 Since AHTRs are caused by immunologic incompatibilities between donor and recipient, they are best
avoided by pretransfusion screening. In order to select the most
appropriate pretransfusion screening test for a patient, it is imperative to understand the options, underlying immunology, and limitations of each test.
Serological testing for transfusion compatibility is based on the
use of antibodies (historically derived from serum) to detect the presence of an antigen.
* Corresponding author.
E-mail address: [email protected] (R. Zaremba).
Abbreviations: NeuAc, Acetylneuraminic acid; AHTR, Acute hemolytic transfusion
reaction; ABRI, Animal Blood Resources International; CMAH, Cytidine
monophosphate-N-acetylneuraminic acid hydroxylase; DEA, Dog Erythrocyte Antigen;
NeuGc, Glycolylneuraminic acid; HBOC, Hemoglobin-based acellular oxygen carriers;
IMHA, Immune Mediated Hemolytic Anemia; ICS, Immunochromatographic Strip; IgG,
Immunoglobulin G; IgM, Immunoglobulin M; MAC, Membrane Attack Complex; PCV,
Packed Cell Volume; PBS, Phosphate buffered saline; PCR, Polymerase chain reaction;
RBC, Red Blood Cell; SL, Standard Laboratory Tube Agglutination Assay
http://dx.doi.org/10.1053/j.tcam.2018.12.005
1938-9736/© 2019 Elsevier Inc. All rights reserved.
Antigens and Alloantigens
An antigen denotes any structure that induces an immune response in
an individual, usually through the production of antibodies directed
against the antigen. Antigens are often proteins, carbohydrates, or lipids
on the surfaces of cells to which antibodies can bind. The specific portion
of the antigen that is bound by the antibody is called an epitope. Different
locations (epitopes) in a single protein on the surface of a cell can be recognized by several different antibodies. A mixture of antibodies that all
recognize the same antigen at different epitopes is called a polyclonal mixture (or a polyclonal antibody) and is usually made by exposing an animal
to the antigen and harvesting its serum. In contrast, a sample containing
multiple copies (clones) of a single type of antibody that is all identical
and all bind to the exact same epitope on that antigen is a monoclonal
mixture (or monoclonal antibody). Monoclonal antibodies are usually
made in the laboratory by replication of the lymphocytes producing the
specific antibody in a host animal.6,7 While serologic testing for specific
antigens on a RBC can use either polyclonal or monoclonal antibodies,
monoclonal antibodies are more specific for a particular antigen.
As opposed to an antigen on a pathogen (ex. bacterium), which is
expected to consistently induce an immune response in all individuals,
the antigens on the surface of RBCs are often called alloantigens. Alloantigens are “self” markers to which the individual usually does not react.
However, these alloantigens may produce an antigenic response in the
immune system of a different (non-self) individual. The alloantigens of
interest for the purposes of this article are those found on the donor or
recipient RBCs. Common RBC alloantigens that can induce a significant
immune reaction in a transfusion recipient are the basis of the blood typing systems used in humans and animals.
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
The antigenicity of a particular substance refers to how strongly it
is able to stimulate the adaptive immune response through the production of antibodies or interactions with T-cells. The more antigenic
the substance, the greater the response.8
37
alloantibodies react with blood from other individuals of the same species even during the first transfusion, making pretransfusion testing in
these species critical prior to transfusion.
Agglutination
Antibodies and Alloantibodies
An antibody is a protein that is either bounded on the surface of B
lymphocytes, produced by plasma cells and secreted free into plasma,
or mounted on mucosal surfaces.9 Each antibody is made up of 2
heavy chains and 2 light chains (Fig 1).
The class and properties of the antibody determines its effect on
the immune response. For example, Immunoglobulin M is a strong
mediator of agglutination between RBCs, due to its pentamer confirmation (Fig 1). The antibodies discussed in transfusion medicine can
be multiple different classes, but are often referred to as “alloantibodies.” Much like alloantigens, alloantibodies can be present in healthy
individuals and cause no problems with that individual’s blood, but
may react with foreign blood types. This is in contrast to “autoantibodies” which will attack antigens on an individual’s own cells.
The presence or lack of alloantibodies, as well as what degree of
immune response they induce, dictates whether or not blood can be
transfused without pretransfusion testing. In some cases, alloantibodies may be present, but if they produce a weak or nonexistent immune
response when exposed to the antigen they are considered to be of
“low clinical significance.” For example, although research10-12 indicates that pre-existing alloantibodies against certain blood types (e.g.,
dog erythrocyte antigen [DEA] 7) may exist in transfusion-na€ıve dogs,
they are widely considered to lack clinically significant naturally occurring alloantibodies. This has led to the practice of referring to the first
canine transfusion as “safe,” meaning the risk of significant acute
hemolytic reaction is low in a dog given a transfusion for the first time
from any donor. However, even in dogs, exposure to transfused antigens recognized as foreign by the immune system will inevitably lead
to alloantibody formation within 3-7 days of exposure.2,13 This makes
subsequent transfusions in any species increasingly likely to cause a
reaction.2,3,13 In contrast, humans and cats have naturally-occurring
alloantibodies to nonself-blood antigens from infancy. These
Screening for agglutination as evidence of RBC cross-linking by
antibodies is the basis of many forms of pretransfusion testing. It
is also evidence of the presence of autoantibodies in disease states,
such as immune-mediated hemolytic anemia (IMHA).
Agglutination, considered a hallmark sign that antigen and/or
antibody reactions are occurring, takes place when antibodies bind to
antigens on more than one RBC cross-linking them and resulting in
clustering of those RBCs together. While weak interactions may only
be visible microscopically (microagglutination), large amounts of
antibody can produce grossly visible macroagglutination (Fig 2).
Agglutination creates grape-like clusters of cells that will not disperse
when diluted with saline. This must be distinguished from the electrostatic interaction of RBCs causing rouleaux formation; such interactions appear as stacking of RBCs that disperse with dilution.14
Feline blood in particular is prone to rouleaux formation. Differentiating rouleaux from agglutination is important to avoid false positives
when assessing blood for incompatibility.15
Temperature will change the binding affinity of antibody and/
or antigen interactions and affect whether or not agglutination
occurs. The most important “agglutinins” (antibodies that cause
agglutination) are believed to be those that occur at body temperature (37°C), (“warm agglutinins”). However, some agglutination
reactions are only seen at room or cooler temperatures (“cold
agglutinins”). The clinical importance of cold agglutinins in veterinary medicine is under further research. In human medicine,
cold agglutinins are considered less important than warm agglutinins since clinically significant reactions take place at normal
body temperatures.1,16 Therefore, the temperature at which pretransfusion testing is performed is important when assessing the
strength of antibody and/or antigen interactions and may not
accurately reflect what is happening in the body if performed at
cooler temperatures.
Fig. 1. Antibody structure and agglutination
Antibodies are composed of 2 heavy and 2 light chains. The heavy chain type determines the immunoglobulin class (IgM, IgG, IgA, IgD, or IgE) and also come together at the
“tail” of the antibody to create the class-specific Fc region. The light and heavy chain regions that make up the “arms” of the antibody contain the variable regions that determine
the antigen binding specificity (Fab’ region). Binding of antibodies to antigens on multiple RBCs physically holds the RBCs together by crosslinking and creates the microscopic and
macroscopic sign of agglutination. As illustrated above, IgM is particularly good at inducing agglutination because its pentamer structure exposes more Fab’ sites for crosslinking.
38
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
Fig. 2. Agglutination
Agglutination is the result of an antigen/antibody reaction which can be identified grossly (macroagglutination, A) or visible microscopically (microagglutination denoted by
black arrows, B).
Immunology of RBC Destruction
Acute transfusion reactions are the complication of greatest concern when transfusing RBCs. They occur when a clinically significant
preformed alloantibody recognizes and binds an alloantigen on a
RBC. This antibody binding “tags” the cell for destruction (Fig 3). The
immune system may destroy tagged cells by phagocytosis (the RBC is
engulfed and digested by an immune cell), or by hemolysis, where
the cell membrane is disrupted and cellular contents leak out. Phagocytosis usually occurs in the spleen and liver and is mediated by neutrophils and macrophages. The phagocytosed cell contents are
partially digested by the immune system, resulting in the classic signs
of extravascular hemolysis (icterus, bilirubinuria). Hemolysis of the
antibody-tagged RBCs is triggered by release of cytotoxic factors from
other immune cells, such as natural killer cells, or by activation of
complement.
Complement is a major mechanism of intravascular hemolysis
seen in acute transfusion reactions. Intravascular hemolysis causes
RBC contents to spill into the bloodstream, resulting in
hemoglobinemia and hemoglobinuria. Since the ability to trigger
complement and cause hemolysis indicates that antibody-antigen
interactions are occurring, hemolysis is also considered a “positive”
result for incompatibility along with agglutination in many transfusion screening tests. The complement cascade is a chain reaction of
zymogens, similar to the coagulation or digestive enzyme cascades.
Complement can aid in the destruction of RBCs in several ways. The
first is by “tagging” cells (similarly to the way antibodies “tag” or
opsonize cells) for phagocytosis by macrophages and other immune
cells. Complement also works to attract more inflammatory cells to
the site of activation, facilitating destruction. Finally, complement can
directly lyse cells by assembling the membrane attack complex; the
membrane attack complex will create pores in a cell and disrupt its
membrane (Fig 3).
If hemolysis occurs secondary to a transfusion, this is considered
an AHTR, which is a type II hypersensitivity reaction. These reactions
are clinically significant and occur within minutes to hours of a transfusion, resulting in destruction of transfused cells. Typically, with an
AHTR, preformed antibody against the transfused antigen is present
Fig. 3. Opsonization and RBC destruction
Antibodies or complement components bind to RBC antigens (opsonization) and this interaction will lead to 1 of 3 pathways: (A) Antibody Fc region or complement component
is recognized by immune cells which mediate phagocytosis. (B) Antibodies or complement components bound to RBC antigens can activate the complement cascade. The end result
is formation of the membrane attack complex (MAC), creating pores in the RBC membrane and causing lysis. (C) The Fc region is recognized by natural killer cells (NK cells), as well
as macrophages, neutrophils and eosinophils. This interaction leads to either the release of granzymes (degrades RBCs) or perforins (creates pores in RBCs) from immune cells, leading to apoptosis (programmed cell death) and/or hemolysis.
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
in large enough quantities to trigger the immune response. While
either (or both) intra- or extravascular hemolysis can occur, intravascular hemolysis is particularly dangerous as it also triggers a systemic
inflammatory response which can result in multiple organ dysfunction and hemodynamic instability. The kidneys are particularly vulnerable to damage from the pigmenturia created by heme released
during intravascular hemolysis.17
Not every patient with alloantibodies has an AHTR. Animals with
very low levels of alloantibodies may not mount an immediate
response to a transfusion, but will upregulate alloantibody production after the transfusion (i.e., a delayed transfusion reaction). Similarly, any patient exposed to a RBC alloantigen recognized as foreign
will create antibodies against the antigen, usually within 3-5 days of
initial exposure. In both cases, this production of alloantibodies in
response to the transfusion can lead to a gradual destruction of
the transfused RBCs within the course of a few days.12 Therefore,
while there may be no clinical signs of a reaction during the transfusion, the transfused RBCs may have a shortened lifespan leading to a
rapid decline in the post-transfusion packed cell volume (PCV), and
possibly signs consistent with delayed hemolysis (icterus, pigmenturia). Delayed transfusion reactions are generally not life threatening,
but the intensity of the response is dependent on the amount and
strength of antibody and/or complement reaction.10,11 Readers are
referred elsewhere for more in-depth reviews of the diagnosis and
treatment of transfusion reactions.3,18,19,20
Blood Types
Blood types are markers on the RBC surface that researchers have
identified as inducing an immune response in other individuals, that
is they are alloantigens. The human ABO blood types are comprised
of carbohydrate antigens attached to lipids or proteins on the cell surface. While the ABO system is the most well-known in humans as it
causes the most clinically significant reactions, over 30 RBC blood
type systems containing >300 antigen variants have been discovered
in humans, and more continue to be found.21,22 Other human red cell
antigens are proteins, cellular signaling molecules, or ion channels.
Therefore, “blood types” may have many other functions for the cell
in addition to being recognizable as antigens to the immune system.
What makes a particular RBC surface marker a “blood type” antigen is
simply the fact that an antibody has been discovered that reacts to it
in a certain proportion of a population. In many cases, blood type
antigens are identified by antibody binding years before more information is known about the alternative function and/or identity of the
antigen; in other cases, the antigen is already known to play another
role in the cell at the time it is identified as a blood type.
In a perfect world, a patient would be transfused with donor RBCs
that have the exact same blood type markers as the recipient to
reduce the risk of the recipient recognizing the cells as foreign. However, given the number and complexity of possible types as noted
above, having the ability to test for, much less finding, identical blood
in an available donor pool is unlikely in a clinical setting. Therefore,
blood typing focuses on finding compatible blood by screening for
the most antigenic blood types to minimize the risk of transfusing an
antigen likely to cause a reaction. For example, in the human ABO system, “O” blood is compatible with type A and B recipients as they lack
antibodies to the “O” (unmodified, universal) glycoprotein. Therefore,
a type A person would be compatible with type A or O blood, even
though the antigens are not identical. This is why type O individuals
are considered “universal donors.”
Canine
The original research on canine blood types was performed by
intentionally giving incompatible transfusions to research dogs and
then collecting their blood. The separated serum or “antisera” from
39
Table 1
Canine RBC Antigens and Clinical Significance
Canine RBC Antigens
Clinical Significance
DEA 1
DEA 3
DEA 4
DEA 5
DEA 7
Dal
Kai 1
Kai 2
High antigenicity, high frequency, AHTRs reported
Delayed reactions, nonhemolytic
High antigenicity, low frequency, 1 AHTR reported30
Delayed reactions, nonhemolytic
Delayed reactions, nonhemolytic
Unknown
Unknown
Unknown
Typing for DEA 3, 6, and 8 is currently unavailable due to a lack of available antisera.
those dogs contained polyclonal antibodies that were then used in
vitro to identify multiple RBC antigens that were subsequently classified under the DEA system.2,23 Originally, the DEA system described
blood types 1.1, 1.2, 1.3, 3, 4, 5, 6, 7, and 8. Currently, blood typing is
commercially available for DEA 1, 4, 5, and 7 only 2,13,23,24 (Table 1),
due to limited supplies of antisera for testing. While this limits our
ability to screen for all known blood types in dogs, this may be of limited clinical significance as DEA 1 is currently the only commonly
screened blood type likely to cause an AHTR.
Depending on the population studied, 33%-60% of canines are DEA 1
positive.13,25 While previous work had subdivided DEA 1 into separate
antigens—DEA 1.1, 1.2, and 1.3—recent studies using a newer monoclonal DEA 1 antibody describes the DEA 1 antigen as an autosomal dominant inheritance pattern with 4-5 possible alleles.6,26 This means that,
rather than being positive or negative for DEA 1.1, 1.2, or 1.3, dogs are
either positive or negative for DEA 1, but the strength of that “positivity”
depends on which form of the gene they have inherited. By this system,
dogs are either negative for DEA 1 or positive ranging from 1+ to 4+.6 It is
currently unknown whether a DEA 1(1+) donor would cause as strong a
reaction as a DEA 1(4+) donor in a DEA sensitized dog. Therefore, when
screening, it is easiest to consider all DEA 1 dogs as either negative or
positive, regardless of the strength of positivity.
Other DEA antigens of lesser clinical significance in dogs include 3,
4, 5, and 7. Alloantibodies existing against DEA 3, 5, and 73,23,27-29
have been documented, but unlike DEA 1 alloantibodies, they have
not been found to cause immediate transfusion reactions. Delayed
transfusion reactions such as shortened RBC lifespans have been
described following subsequent transfusions of DEA 3, 5, or 7 positive
blood to sensitized recipients.3,23,28,29
Unlike DEA 3, 5, and 7, 1 case report has demonstrated that DEA 4
sensitization can cause AHTRs in subsequent transfusions.30 However, DEA 4 is a high-prevalence antigen in canines (98% of dogs are
positive for this antigen).30 Therefore, in order for a dog to have a
DEA 4 mediated AHTR, the recipient would have to be DEA 4 negative
(2% of the population) AND receive a second transfusion from a DEA 4
+ donor. Thus, it is extremely unlikely to clinically see an AHTR
related to DEA 4 sensitization. Therefore, due to the unlikelihood of a
reaction related to DEA 4+ and the infrequency of DEA 4¡ dogs in the
population, canines who are negative for DEA 1, 3, 5, and 7 and
positive for 4 are often described as “universal donors.”2,3,30
While the DEA system was the nomenclature agreed upon for
canine blood types by the international workshop for canine immunogenetics in 1972, additional antigens continue to be discovered
which do not conform to the DEA system.31 The Dal antigen was discovered in 2007 after a Dalmatian receiving repeated transfusions for
chronic kidney failure became sensitized to blood to which he should
have been DEA compatible. This incompatibility was seen during
crossmatching with 80 potential DEA compatible donors. The cause
of the reactions seen during crossmatching was a new RBC antigen
later named Dal.12 Further researches has shown that Dal has an autosomal dominant mode of inheritance and that breeds commonly used
for blood donation such as Greyhounds, Labradors, and Golden
40
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
Retrievers were 100% positive for Dal. In contrast, 12% of Dalmatians,
42% of Dobermans, and 57% of Shih Tzus in a population of 1130 dogs
were Dal negative, putting Dal¡ dogs at greater risk for sensitization
to Dal following a first transfusion.32 As bedside blood typing for Dal
is not currently available, it is unknown if and what kind of transfusion reaction a Dal-sensitized dog would have if given Dal+ blood during a second transfusion.
Two additional blood antigens, Kai 1 and Kai 2, were identified in
2017 and have been evaluated in the United States and South
Korea.25,33 Dogs are most commonly Kai 1+/Kai 2¡ (94%), with only
1% of dogs (mostly Lhasa Apsos) being Kai 1¡/Kai 2+. Dogs cannot be
positive for both Kai 1 and 2, but can be negative for both.25 While no
naturally occurring alloantibodies to the Kai system have been discovered, alloantibodies against Kai were detected in previously transfused dogs.25 The clinical significance of the Kai alloantibodies in
sensitized recipients has not yet been determined.
Despite a growing understanding of canine blood antigens, there
are likely many antigens still unidentified. This makes it impossible to
“perfectly match” a canine transfusion to prevent reactions. In one
study, even after extended typing and matching for DEA 1, 3, 4, 7
and Dal, 4 dogs still became crossmatch incompatible to their original
donors at the time of a subsequent transfusion, indicating that these
dogs were exposed to additional un-screened antigens.13 In another
study, up to 46% of transfused dogs were incompatible by crossmatch
to their original DEA 1 matched donors after their first transfusion.34
Furthermore, dogs who have never received a transfusion can still be
incompatible to donor blood via crossmatch. In one population, 17%
(25 of 149) of transfusion na€ıve dogs were incompatible by crossmatch
with at least 1 of 3 potential DEA 1 and 7 negative donors.16 These
findings emphasize that blood type matching alone in dogs does not
guarantee transfusion safety due to the presence of untested and
unclassified blood antigens.
Feline
The most described feline blood antigen system is similar to the
ABO system used in humans, characterizing cats as A, B or (rarely) AB.
Also similar to humans, cats have preformed alloantibodies against
other blood types prior to any transfusion exposure. However, unlike
humans, there is no “O” or universal donor type in cats. This means
that cats MUST be transfused with blood of a compatible type, or significant life-threatening transfusion reactions are likely to occur.
While the weak anti-B antibodies of type A cats may cause delayed
RBC survival, the strong anti-A antibodies of type B cats can cause
fatal reactions if they receive even 1mL of type A blood.35
Unlike dogs, the genes encoding the feline AB blood system have been
characterized. Type A cats have functional cytidine monophosphateN-acetylneuraminic acid hydroxylase (CMAH) enzymes that convert acetylneuraminic acid (NeuAc) to glycolylneuraminic acid. Type B cats have
mutations in the CMAH gene that reduce the enzyme’s function, leading
to a lack of conversion of the NeuAc on the red cell membrane. Type AB
cats have partial function of the CMAH enzyme leading to approximately
equal amounts of NeuAc and glycolylneuraminic acid on the RBC membrane.36 Genetic testing for mutations of the CMAH gene exists, but it is
currently impossible to distinguish between the A and AB phenotypes.37
Additionally, 3 of 112 cats who were genetically tested had blood phenotypes that differed from the blood type predicted by their genotype. This
suggests that there are undiscovered mutations affecting CMAH function.37 While genotyping cats may help breeders reduce unwanted blood
types in their populations, due to discordant results and lack of availability, it cannot currently replace serologic blood typing in felines.
While the AB system is the predominant blood typing system for
cats, additional antigens outside of the AB system that cause hemolytic reactions have been described.38 The Mik antigen is the first
non-AB system blood type described in cats. It was discovered in
2007 after a Mik¡ renal transplant recipient was administered Mik+
blood leading to a hemolytic transfusion reaction. When 65 type A
cats from a donor population were screened, 3 additional cats were
Mik¡ and had incompatible crossmatches with Mik+ type A cats in
the population.38 As these donor cats were all transfusion na€ıve, this
implies that alloantibodies against Mik are naturally occurring in cats
and could cause AHTRs during the first transfusion. Unfortunately,
testing for Mik is not available as a bedside test at this time.
Although Mik is the only non-AB feline blood type so far described,
crossmatch studies show mixed evidence as to the likelihood of other
unknown antigens causing transfusion reactions in cats. One study
crossmatched 112 cats and found no conclusive evidence for non-AB
blood type incompatibility.37 However, 2 other retrospective studies
found that 25% of cats develop incompatible crossmatch results after
a first transfusion despite the transfusion being type AB compatible
blood, some as soon as 2 days post-transfusion. These findings may
be secondary to transfusion of Mik+ blood products or antigens that
have yet to be discovered.39,40 There are also mixed results as to
whether using type and crossmatch compatible blood for feline transfusion is better than using just type compatible blood.39,41 In recent
studies, whether or not a transfusion was crossmatch compatible did
not consistently predict a transfusion reaction in the recipient. Therefore, although there is clear evidence that cats have natural and
induced alloantibodies against non-AB blood type antigens, the risk
of having clinically significant transfusion reactions caused by those
antibodies is still unclear and seems to be rare.
Due to limited availability of feline type-specific blood, as well as
the time delay of blood typing in critical cases, xenotransfusion of
cats with dog blood has been successfully described.42-44 A retrospective review of xenotransfusion in cats did not find evidence of major
incompatibility when pretransfusion testing was performed between
cats and canine donors, and no severe acute reactions have been
reported for cats receiving a first-time transfusion of canine
blood.42,44 However, all cats destroyed the transfused blood within
4-7 days post-transfusion and any cat receiving a second xenotransfusion >6 days after the initial transfusion had significant, often fatal,
anaphylactic reactions. More recent publications crossmatched feline
and canine blood samples against each other and found that 50%-75%
of cats were incompatible via major crossmatch, and almost 100% of
all minor crossmatches were incompatible.43,45 The transfusion and
health history of these animals were not described. These authors
concluded that compatibility of canine blood, therefore, cannot be
guaranteed in all first-time feline recipients. As transfusion of compatible feline blood is obviously preferred, xenotransfusion with
canine blood should be reserved as an emergency technique for critical, time-sensitive cases where species-specific type compatible
blood cannot be obtained. Practitioners should never give a second
transfusion of dog blood after 4 days from initial xenotransfusion to
the feline recipient as it can be fatal.44
Methods of Blood Typing
Blood typing can either be performed in-house or at a commercial
laboratory. Commercial laboratory testing using the gold-standard
tube method provides more comprehensive blood typing information
than is available in-house and is often used in blood donors. Currently
available in-house commercial blood typing kits include card agglutination tests for canine DEA 1 and feline A, B, and AB and immunochromatographic strip (ICS) assays for the same blood types. A gel-tube test
for felines is also available.
Card agglutination kits consist of marked areas impregnated with
monoclonal antibodies that bind to the antigen of interest; antibodies
for DEA 1 for dogs, and for A and B for cats. The recipient’s blood is
first mixed with phosphate buffered saline to maintain pH and osmolality of the cells, then the mixture is dropped onto the card where it
mixes with the antibody. If the antigen is present (i.e., the patient is
positive for that blood type), visible agglutination of the red cells
should occur on the card as they are crosslinked by antibodies. The
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
41
Fig. 4. Card blood typing
(A) The positive control is exhibiting agglutination but the patient test is not exhibiting agglutination. This would be interpreted as a DEA 1 negative test. (B) The canine patient
test is exhibiting agglutination similar to that in the positive control. This patient would be reported as DEA 1 positive. (C) This feline patient test is exhibiting agglutination in the
type A box indicating that the patient is type A. None of the patients are auto-agglutinating, as shown by the lack of agglutination in the “autoagglutination saline screen” box.
cards are easy to use and yield an obvious visual positive (agglutination) or negative (no agglutination) result (Fig 4). However, if autoagglutination is present in the animal, such as in IMHA, interpretation
of results may be confounded. Once the sample begins to dry, agglutination can no longer be distinguished from sample dehydration
and clumping, thus the test must be interpreted immediately.
Although card typing is reasonably accurate, it did show the lowest
accuracy (91.4%) in comparison to other methods of canine and feline
blood typing (ICS, gel tube assay) in recent studies.46,47 Therefore,
when typing for nonurgent purposes such as donor screening or
breeding animals, more accurate commercial laboratory methods are
preferred.46,47
A second option for in-house blood typing is the ICS assay which
impregnates a paper strip with a line of monoclonal antibody for DEA
1 in dogs or 2 lines with antibodies for Type A and B in cats (Fig 5).
Recipient blood is mixed with buffer in a test well and is then allowed
to wickup the strip. If RBCs bind to the antibody within the line,
they are trapped within the line on the strip. The hemoglobin in the
red cells thus creates a visibly darker red stripe, indicating a positive
result (Fig 5). The presence or absence of the red strip may be less
subjective in interpretation than agglutination. However, because the
color change depends on the presence of hemoglobin to create a visible result, the color change may be faint in severely anemic animals,
creating difficulty in interpretation. This method has been shown to
be 100% specific for detection of DEA 1 and feline A blood types, and
97.1% specific for feline type B.46,47 Therefore, when the indicator
strip is positive, the test can be considered correct (a true positive).
However, the ICS assay was shown to be only 88%, 97.7%, and 95.7%
sensitive for those antigens, respectively, so rare false negatives are
possible. This method is also purported to be less affected by autoagglutination when compared to other methods in recent studies.46,48
If extended typing (beyond DEA 1 or feline A, B, and AB) is
required, the tube method is typically employed as this is considered
the gold standard.24,46,47 In this test, the RBCs are incubated with
antibodies against a specific blood type in a tube, and then centrifuged and resuspended to assess for hemolysis or agglutination, similar to a crossmatch tube assay (see below).
An in-house version of the tube method has been modified by
adding a dextran-acrylamide gel matrix impregnated with an antibody against the blood type of interest. Recipient blood is placed on
top of the matrix and the tube is then centrifuged according to manufacturer guidelines. A gel-based tube method was shown to be the
most accurate bedside test (99.4% accuracy in cats, 100% in dogs) for
DEA 1 and feline AB blood typing in recent studies; unfortunately,
this test is no longer available from the manufacturer. A similar test
from another manufacturer has not been directly compared to other
methodologies, but is currently available for cats.46,47 In this test, if
the cells are able to move freely through the gel to pellet at the bottom, then they are a type B cat. However, if there are RBCs and agglutinates dispersed throughout the gel then they are considered a type
AB cat.
Currently, Animal Blood Resources International is the only commercial lab in the United States to perform extended blood typing for
canines, with typing for DEA 1, 4, 5, and 7 available at the time of
writing. Typing for DEA 3, 6, and 8 is currently unavailable due to a
lack of antisera.2,23 Typing for cats is currently widely available only
for blood types A, B, and AB. Typing for the newly discovered antigens
Dal, Kai, and Mik are currently available only through select research
facilities if antibodies are available.24
Crossmatch Testing
Crossmatching is a serologic method used to detect incompatibilities
between donor and recipient blood. It screens for a general reaction
between a specific donor and recipient and can therefore detect antibody
reactions to known blood-type antigens, as well as those not tested for
42
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
Fig. 5. ICS assay blood typing
(A) Example of a DEA 1 positive blood type as indicated by the presence of a red line in the “DEA 100 space (arrow). (B) Example of a type A feline as indicated by the presence of a
red line in the “A” space (arrowhead) and lack of line in the “B” space (open arrowhead). For all ICS tests, a solid red line in the control space (denoted by the letter C) must be present for the test to be valid.
by blood typing.2,3 Blood typing can determine the presence of a known
alloantigen (i.e., DEA 1 is present). In contrast, crossmatching can determine if a general antibody and/or antigen reaction against RBCs is occurring in the mixed sample, but does NOT identify which antibody is
reacting with which antigen. The basis of all current crossmatch testing
involves mixing recipient and donor samples and screening for antibodies binding to RBC surface antigens. The crossmatch is generally done in
2 parts: a major and minor crossmatch.
In the major crossmatch, donor RBCs are mixed with recipient
plasma and evaluated for evidence of antibody in the plasma binding
to donor RBCs, represented by hemolysis or agglutination.3,49 The
minor crossmatch is similar, but mixes donor plasma with recipient
RBCs to detect antibodies in donor plasma that recognize recipient
RBCs.3,49 The end goal of crossmatch testing is to avoid immunemediated transfusion reactions that result in immediate hemolysis.3,50,51 However, even with a compatible crossmatch test,
appropriate RBC survival and/or complete elimination of transfusion
reactions are not guaranteed as low titers of anti-RBC antibodies may
not cause enough agglutination for detection, but may still result in a
transfusion reaction.2,3,49 Additionally, crossmatching evaluates the
current immunological state of the samples; it cannot be used to
determine compatibility for future transfusions. Even if the same
donor that was “compatible” initially is used, if the patient has
subsequently become sensitized to that blood, a second crossmatch
would be needed to detect this.2,3 Interpretation of some assays may
also be hindered by disease processes that cause autoagglutination
such as IMHA.2,3 Also, as discussed with Mik testing, while crossmatching can detect the presence of pre-existing alloantibodies that
are not screened for by typing, it cannot determine whether or not
those antibodies will actually cause a clinically significant transfusion
reaction. Finally, it is important to remember that other types of
transfusion reactions, such as volume overload or blood product contamination, cannot be prevented by these screening methods.
Crossmatch testing is generally recommended in all cats prior to
RBC transfusion, and in any dogs with a history of previous transfusion or unknown transfusion history.2,3,37 Because canines are generally said to lack naturally occurring alloantibodies to other blood
types, it is not uncommon for veterinarians to forego typing and
crossmatching of dogs prior to the first transfusion. A survey of veterinary referral and teaching hospitals indicated that only 15% of practitioners routinely crossmatched dogs prior to transfusion.52 However,
while the risk of AHTRs was not increased by bypassing crossmatching, using crossmatch-compatible blood may improve the duration of
RBC survival in the recipient. One retrospective study in dogs and 2
studies in cats have shown that transfusion of crossmatch-compatible
blood had increased post-transfusion PCV as well as improved
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
Table 2
Standard Laboratory Tube Agglutination Assay Technique for Crossmatching
1. Obtain blood in both ethylenediamine tetra-acetic acid (EDTA) tube and in a
nonadditive tube from both donor and recipient. If using a stored RBC unit,
blood for crossmatch testing can be taken from the pigtail.
2. Centrifuge and separate plasma and serum from the RBCs. Transfer the
serum into a separate tube without additive (red top). Discard the plasma from
the EDTA tube.
3. Collect 0.25-0.5mL of RBCs from the EDTA tube and add 1.5mL of phosphate
buffered saline (PBS)
a. Centrifuge for 45 seconds, decant the saline, and repeat 3 times
4. Resuspend the cells in PBS to make a 3%-5% solution to make “RBC suspension”
(Approximately 4mL total volume)
5. Label tubes to make the following mixtures:
a. Major crossmatch: 2 drops patient serum with 2 drops donor RBC suspension
b. Minor crossmatch: 2 drops patient RBC suspension with 2 drops donor serum
c. Control: 1 drop patient RBC suspension with 2 drops patient serum
6. Centrifuge all tubes for 20 seconds followed by gentle mixing to resuspend cell
button
7. Incubate all tubes for 30 minutes at 35-39°C.
8. Centrifuge tubes for 20 seconds followed by observing the tube for hemolysis
and tapping tube to resuspend cell button
9. Add a drop of suspension to a slide and examine for macroscopic agglutination
or hemolysis
10. Evaluate same drop of suspension microscopically on low power
11. Interpretation of results:
a. If either hemolysis or agglutination is seen macroscopically, or if agglutination
is seen microscopically, the donor is considered incompatible
b. If the control tube is positive for hemolysis or agglutination the test should be
repeated and if still positive the test is considered incompatible
duration of PCV increase in the recipient17,39,40 compared to type
matched, but not crossmatched transfusions. Decisions about the
need for crossmatching in a clinical case need to be balanced with
the methodology of crossmatching used and urgency of the patient’s
clinical state since crossmatching all recipients and donors may not
be practically or financially possible.
Crossmatch Test Methods
Standard Laboratory Tube Agglutination Assay
Currently, the gold standard method of crossmatching in the United
States is the standard laboratory tube agglutination assay (SL) which is
typically performed at a clinical pathology facility. For a description of
this technique as performed at our institution, see Table 2. In short, the
SL technique involves separating RBCs and plasma, washing RBCs (to
remove any autoantibodies) and mixing recipient and donor RBCs and
plasma in 3 separate tubes—the first for the major crossmatch, the second for the minor crossmatch, and the third tube as a control (recipient
blood and recipient plasma) to screen for autoagglutination. After the
samples are combined, the contents of each tube go through multiple
steps of washing, centrifugation, and incubation at 35-39°C. The goal of
these steps is to remove any antibody not bound to a red cell antigen
and ensure temperatures are appropriate for “warm agglutinin” binding.
The tubes are then visually scored for macroagglutination, microagglutination and hemolysis on a subjective scale of 0 to 4+.2,3,49,50 Hemolysis
and/or agglutination caused by antigen-antibody interaction indicates
incompatibility between donor and recipient. If hemolysis or agglutination is noted in the patient control tube even after the wash steps, then
the patient is autoagglutinating (such as in IMHA) and the test cannot
accurately determine compatibility between patient and donor.50
Gel-Tube Assay
The gel-tube assay methodology is a point-of-care crossmatching test designed to be used in the clinic. Test tubes used to assess
43
the major, minor, and patient control samples come prefilled
with dextran-acrylamide gel. Similar to the tube test described
previously, serum and RBCs are separated and RBCs are washed
to remove any autoantibodies binding to the RBCs before combining the RBCs with serum in the tube. The gel allows compatible
RBCs to filter to the bottom of the tube after room temperature
incubation and centrifugation, but traps agglutinated RBCs above
or in the gel.41,44 An incompatible test is indicated by the majority
of RBCs clumped within the suspended matrix or on top of the
gel after centrifugation (No cells reaching the bottom of the well)
(Fig 6). The number of cells on top of or within the gel is rated on a
scale of 0 to 4+ (With 0 being a compatible resultno cells on top
of the gel).50
Overall, moderate agreement between the gel tube and the
gold standard SL method has been noted, with one study finding a
relative specificity of 96% (sensitivity could not be determined due
to the low number of incompatible tests). 53 However, another
study reported all dogs tested (35 of 35) were found to be compatible by the gel tube assay, but 45.7% (16 of 35) were found to be
incompatible via the gold standard SL test.50 The authors concluded this may be due to the gel matrix allowing small (1+)
amounts of agglutination to sift through the gel, while the SL test
was also able to detect these aggregates by visual observation at
the microscopic level.12,53 Higher levels of agreement were noted
between the SL and gel-based method when agglutination was
more significant (3+).12,53
ICS
Similar to the ICS test methodology used for blood typing, a
point-of-care (ICS) crossmatch assay is commercially available for
dogs and horses.34,48 After washing RBCs and mixing RBCs and
plasma with buffer in a well, the mixture is allowed to wickup a
paper strip impregnated with anticanine antiglobulins in a detector
line area. The anticanine antiglobulins can detect immunoglobulin
G, immunoglobulin M, and/or C3 complement component bound to
the RBCs and trap these RBCs within the detector line, turning it red
(incompatible). In other words, a red line indicates that the donor
and/or recipient sample interaction has resulted in coating of the
red cells with immunoglobulins or C3 and is therefore incompatible
(Fig 7).34,48
A reported advantage of this test per the manufacturer is that
large clumps of autoagglutinated RBCs should not be able to wickup
the paper strip, reducing the chances of autoagglutination (as in cases
of IMHA) causing false positive results.34,48,54 One study using the ICS
method to assess transfusion compatibility in dogs found that all
autoagglutinating dogs (n = 8) were able to be screened as compatible
to a donor by ICS crossmatch methodology.34 ICS testing is currently
only available for dogs, but a feline test is under development by the
manufacturer.55
Comparison of the pros and cons of each type of crossmatch testing described are outlined in Table 3.
Summary and Future Directions
While tube testing is still commonly employed for blood typing and crossmatching in human medicine, advances in blood
banking safety and technology are expanding. Automated techniques such as the use of microplate technology to screen multiple
samples simultaneously in a short time frame is improving the
efficiency of donor screening and may reduce costs associated
with blood typing and crossmatching when performed on a large
scale.56
44
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
Fig. 6. Canine gel-tube assay
(A) The majority of the red blood cells (RBCs) have traveled through the matrix and to bottom of well. This is indicative of a negative (compatible) crossmatch. (B) The RBCs have
agglutinated and cannot migrate through the gel, indicating a positive (incompatible) crossmatch.
In addition, molecular blood typing using polymerase chain reaction to screen for human blood type variations in donors and recipients
is allowing for standardized, accurate, and extended blood typing in
humans.21,57 Combined with database management systems, polymerase chain reaction testing greatly expands the ability to store and
quickly search this data for compatible blood products, even in human
patients with rare blood types.57 Also, research into synthetic receptors
for cell surface antigens to replace monoclonal antibodies may improve
consistency and accuracy in screening for RBC antigens.57
While veterinary medicine lags behind human medicine in blood
banking scale and technology, future research into the structure and
function of RBC surface antigens in veterinary species will improve our
ability to characterize blood groups and hopefully lead to development
of molecular techniques for blood typing and compatibility testing in
our species of interest. More widely available testing methodologies in
more species will improve access to blood typing and crossmatching.
Finally, in a synergy between human and veterinary medicine, continued work to improve the safety of hemoglobin-based acellular oxygen
carriers in human medicine may lead to development of products that
can replace RBC containing transfusions in the future, especially in
patients shown to be incompatible with available blood products.57
In the interim, the recent increase in commercially available tests
for blood typing and crossmatching will hopefully allow veterinarians
to minimize transfusion reactions and make blood transfusions safer
for our patients. The importance of access to accurate typing and
crossmatching methodologies is underlined by the discovery of novel
RBC antigens in both dogs and cats. Understanding the available testing as well as the limitations of these tests to predict and prevent clinically relevant transfusion reactions is important for anyone
administering RBC products to canine and feline patients.
Acknowledgments
-Photos of agglutination courtesy of Dr. Craig Thompson, DVM,
DACVP, Purdue University, West Lafayette, IN.
-Photos of card blood typing and gel-tube assay crossmatch courtesy of DMS Laboratories, Inc., Flemington, NJ, www.rapidvet.com
-Photo of immunochromatographic strip dog blood type courtesy
of IndyVet Emergency & Specialty Hospital Blood Donation Center,
Indianapolis, IN.
R. Zaremba et al. / Topics in Companion An Med 34 (2019) 3646
45
Fig. 7. ICS assay crossmatch- Canine
(A) Example of a compatible major crossmatch. This is attributed to the lack of color change/red line in the “XM” space (arrow). (B) Example of an incompatible minor crossmatch. This is attributed to the (faint) color change/red line in the “XM” space (arrowhead). A solid red line in the “C” (control) space must be present for the crossmatch to be considered valid.
Table 3
Comparison of Crossmatch Techniques
Crossmatch Technique
Advantages
Disadvantages
Standard laboratory (SL) tube
agglutination assay
Can be completed in any practice
Can be used in any species Current gold standard
in veterinary medicine
Gel-tube assay
Easy to perform without need for additional training
Time to complete: 20-30 min
Requires less blood than SL method
Stable reaction
Easy to perform without need for additional training
Time to complete: 20-30min Requires less blood
than SL method
Possible reduced likelihood of false positive due to
auto-agglutination
No universal protocol for completion and/or interpretation
Time consuming (at least 1 h to complete) Requires advanced training
Subjective results Not a stable reaction
Affected by autoagglutination
Requires 2 kits to perform both major and minor crossmatch
Only available in canine and feline species Affected by autoagglutination
Moderate agreement with SL method
No publications available assessing use in cats
Requires 2 kits to perform both major and minor crossmatch
Interpretation is color-based and may be affected by severe anemia,
icteric and/or hemolyzed plasma
Currently only available in canines
Not yet compared to the SL method
Immunochromatographic
strip (ICS)
Supplementary materials
Supplementary material associated with this article can be found
in the online version at doi:10.1053/j.tcam.2018.12.005.
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