Chapter 9
Canine Recipient Screening
Lynel J. Tocci
Department of Emergency and Critical Care, Lauderdale Veterinary Specialists, Fort Lauderdale, Florida, USA
Introduction
Canine blood transfusions are lifesaving interventions that have taken on an essential role in veterinary patient management. Pre-transfusion testing of the recipient, including blood typing and crossmatching, should be performed with the ultimate objective of preventing an immune-mediated transfusion reaction. This facet of human medicine is highly regulated by the US Food and Drug Administration (USFDA) and the AABB (formerly the American Association of Blood Banks). Additionally, accrediting agencies like the College of American Pathologists (CAP), as well as the Joint Commission on Accreditation of Healthcare Organization (JCAHO), provide standards and clinical practice guidelines to help reduce the risk of error and ensure quality and safety of transfusion. Similar stringency does not exist in veterinary medicine to date, so it is up to veterinarians to optimize transfusion safety from start to finish. Blood and blood components are biological products and their intended use in patient treatment requires professional judgment based on clinical patient evaluation. Pre-transfusion testing cannot guarantee normal red blood cell (RBC) survival and transfusion reactions can be life threatening. Because of the risks associated with transfusions, it is the responsibility of the veterinary team to remain current with transfusion practice in order to prevent transfusion-related complications. This chapter will review the principles and procedures for canine recipient screening prior to RBC transfusion.
Blood group genetics
Blood groups are defined by inherited antigens on the surface of the RBC. These antigens are species specific, made up of sugars or proteins attached to the RBC membrane. RBC antigens contribute to the recognition of self and can elicit the production of an alloantibody when introduced into the circulation of a dog whose RBCs lack the antigen (Andrews and Penedo 2010). Inherited antigens on the surface of the canine RBC membrane are referred to as dog erythrocyte antigens (DEAs). Their detection and description are based on immunohematologic serology tests using polyclonal and monoclonal antibodies. Depending on the antigen, immunogenicity and clinical significance can vary.
Canine blood groups
Canine blood groups were first recognized by Von Dungern and Hirszfeld in 1910, but it was not until 40 years later that Swisher and Young further defined canine blood groups (Swisher and Young 1961). They described seven antigens: A, B, C, D, E, F, and G. In 1972 and 1974, there were two international workshops on canine immunogenetics and a canine blood group system nomenclature was established (Vriesendorp et al. 1973, 1976). By convention, each DEA is followed by a number beginning with 1 for each locus, then a period followed by a number for the allele at the locus. There is no DEA 2 and although DEA 6 and 8 were once described, there are no longer antisera for these available. In the United States, the currently recognized canine blood groups are DEA 1, DEA 3, DEA 4, DEA 5, and DEA 7, but based on serologic testing more than 12 canine blood groups have been described. Additionally, in 2007 a high-frequency antigen named Dal was discovered, which has no correlation with the recognized DEA antigens and has yet to receive a DEA designation (Blais et al. 2007).
All blood groups are a two-allele system with a positive and negative type, except the DEA 1 blood group system that, until recently, was thought to include at least two alleles, DEA 1.1 and DEA 1.2, and possibly a third allele DEA 1.3. Based on polyclonal studies, the DEA 1.1 antigen was considered dominant over DEA 1.2 and only DEA 1.1 negative dogs could be DEA 1.2 positive. Flow-cytometric studies using a monoclonal antibody demonstrated that the DEA 1.1 blood group has varying strength, from weak to moderately to strongly positive, and that the DEA 1.2 and 1.3 blood groups originally identified are actually a continuum of the DEA 1.1 blood type (Acierno et al. 2014). Based on this current research, what historically was classified as DEA 1.1 has become DEA 1. The same researchers have also discovered an autosomal dominant mode of inheritance of at least four alleles of DEA 1, wherein strongly to weakly DEA 1 positive alleles are dominant over the DEA 1 negative allele (Polak et al. 2015).
The incidence of the DEA 1 antigen varies in dogs by geographic region and breed. In Turkey, the frequency of DEA 1 is 50–71.2% in four native breeds, whereas in Spain the prevalence of DEA 1 in greyhounds is 51.7% (Ergul et al. 2011; Mesa-Sanchez et al. 2014). Interestingly, in Portugal, the overall percentage of DEA 1 positive dogs is 56.9%, but all Boxers, German Shepherds, and Dobermans are DEA 1 negative and all Saint Bernards are DEA 1 positive (Ferreira et al. 2011). Overall, it is estimated that approximately 50% of dogs are DEA 1 positive (Kessler et al. 2010; Giger 2014). Dogs previously considered DEA 1.1 negative and 1.2 positive are more likely weakly DEA 1 positive and are considered rare (Acierno et al. 2014).
The DEA 1 blood group is extremely antigenic and considered the most clinically significant blood group in the dog, similar to the ABO blood group system in humans. Unlike the ABO system in which naturally occurring isohemagglutinins exist, a naturally occurring anti-DEA 1 isohemagglutinin does not exist in DEA 1 negative dogs. However, an acute hemolytic transfusion reaction has been reported in a DEA 1 negative dog that developed anti-DEA 1 antibodies after a previous transfusion (Giger et al. 1995). DEA 3 has low (5–10%) prevalence in dogs in the United States, although the incidence might be higher in greyhounds (Giger et al. 1995; Iazbik et al. 2010). Naturally occurring anti-DEA 3 antibodies exist in 20% of DEA 3 negative dogs (Hale 1995). DEA 4 is a high-frequency antigen found in 98% of the US canine population; naturally occurring anti-DEA 4 antibodies do not exist in DEA 4 negative dogs. However, a hemolytic transfusion reaction was reported in a DEA 4 negative dog with anti-DEA 4 antibodies after a previous sensitization (Melzer et al. 2003). DEA 5 is a low-frequency antigen with a 12–22% incidence, but up to 30% of greyhounds might be positive. Like DEA 3, naturally occurring anti-DEA 5 antibodies have been reported. Neither anti-DEA 3 nor anti-DEA-5 antibodies have been reported to cause acute hemolytic transfusion reactions, although delayed hemolysis and decreased survival of transfused RBCs is possible. DEA 7 is present in up to 45% of dogs. Naturally occurring anti-DEA 7 antibodies are present in 20–50% of DEA 7 negative dogs and will cause decreased RBC survival when DEA 7 positive RBCs are transfused to DEA 7 negative dogs (Hale 1995; Hohenhaus 2004).
A corresponding antibody to the Dal antigen has also been reported and was induced post transfusion in a Dal negative Dalmatian. This is an antibody of extremely low frequency as the only Dal-negative dogs identified have been Dalmatians, Doberman Pinschers, and Shih Tzus (Blais et al. 2007; Goulet et al. 2014). Further studies are needed to determine the frequency of this antigen’s occurrence universally and in other breeds.
Pre-transfusion testing
Pre-transfusion testing is a process involving both serologic and non-serologic tests to help ensure the safety and adequate survival of blood components in a recipient. It is performed to minimize the risk of transfusion of incompatible donor RBCs that might result in an immune-mediated hemolytic transfusion reaction. In human patients, the USFDA regulates pre-transfusion testing by the authority of the Clinical Laboratory Improvement Amendment of 1988 (CLIA ’88). The steps include a clinician-ordered transfusion request, patient identification, sample collection, patient blood typing, antibody screening, and crossmatching (Roback et al. 2011). To date, no such requirements exist in veterinary medicine. As in human medicine, for canine recipients the process should include the steps listed above, as well as a review of the medical record and clinical history of the patient. The goal is to determine if the dog has ever been blood typed and/or transfused, as well as to document current medications and disease diagnosis. A recommended protocol to follow before RBC administration is presented in Box 9.1.
Sample collection
Pre-transfusion testing begins with proper sample collection. Each blood sample should be labeled with a unique patient identification (name, medical record number) and sample collection date. Unlabeled or mislabeled specimens should not be used for testing. Care should be taken when phlebotomy is performed to avoid hemolysis. Performing testing with a hemolyzed sample can mask antibody-induced hemolysis. In patients experiencing in vivo hemolysis from diseases such as immune-mediated hemolytic anemia (IMHA), hemolysis cannot be avoided but should be noted. To avoid contamination with interfering fluid additives or medications, blood samples should not be collected from intravenous lines or above the catheter infusion site.
For blood typing, whole blood is collected into an EDTA anticoagulant tube. Whole blood is also collected into a serum separator or clot tube for crossmatching. RBCs from the donor are required for the major crossmatch. Most stored RBC units contain integrally attached segments or “pig tails” to provide the donor RBCs needed for crossmatching. If segments are unavailable, an EDTA sample from the donor can be used.
Sample age
With regards to crossmatching, the pre-transfusion sample must reflect the patient’s current immunologic status. Patients that have never been transfused can have samples drawn well in advance of possible transfusion needs (i.e., as part of pre-surgical blood work). Based on recommendations in human medicine, when performing pre-transfusion testing for a patient that has been transfused during the previous 3 months or when the transfusion history is uncertain, the sample should be no more than 72 hours old (Carson 2011). Additionally, research performed to investigate the effect of duration of storage on crossmatch results using equine blood demonstrated that using stored samples (1–4 weeks old) increased the likelihood of false incompatible crossmatch results (Harris et al. 2012). While this study suggests that using older stored samples for crossmatch testing will not increase the risk of giving incompatible blood, it highlights the need for fresh samples to ensure accurate compatibility testing results.
Other considerations
Standards in human blood banking require the confirmation of a patient’s blood type when each new blood sample is received for testing, as well as verification of the donor unit blood type (Carson 2011). This is not a requirement in veterinary medicine and can be cost prohibitive, therefore in most cases blood typing can be performed hours to days before the need for transfusions. Ideally, all dogs should be blood typed at least once prior to receiving a blood transfusion and the blood type conspicuously noted in the electronic and/or paper medical record.
Unlike in humans, pregnancy does not sensitize dogs to RBC antigens, as demonstrated by an investigation of 35 previously pregnant dogs that determined the incidence of pregnancy-induced alloantibodies. All dogs were pregnant 4 weeks to 24 months prior to sampling and no evidence supported pregnancy-stimulated alloantibody production when compared to control dogs (Blais et al. 2009). Given these results, previous pregnancies are not considered to affect pre-transfusion testing in dogs.
Pre-transfusion tests
Pre-transfusion serologic tests include blood typing and crossmatching. The principle of these tests is to demonstrate in vitro antigen–antibody reactions with the endpoint being a visible reaction, most often agglutination. This allows a blood type to be identified or compatibility between a donor and a recipient to be determined. Agglutination takes place in two stages: the first is sensitization and occurs when an antibody reacts with an antigen on the RBC; the second is bridge formation when antibodies attached to RBCs crosslink and cause visible aggregates.
Allogeneic blood transfusions have the potential to introduce foreign antigens into the recipient. Depending on the antigenicity of the foreign antigen introduced, sensitization and antibody production can occur. In dogs, DEA 1 is an extremely antigenic blood group and transfusion of DEA 1 positive RBCs to a DEA 1 negative dog will result in the formation of alloantibodies in as little as 3–4 days from exposure (Abrams-Ogg 2001). For this reason, patient and donor DEA 1 typing should be determined prior to transfusion in order to transfuse DEA 1 compatible RBC components. However, in the event of a life-threatening emergency, especially when DEA 1 negative donor blood is transfused, DEA blood typing of the recipient can be initially omitted and performed at a later time.
The pre-transfusion crossmatch is designed to help ensure the RBC transfusion will be effective, while minimizing the risk of adverse reactions (immediate or delayed RBC destruction). This technique will be described in more detail below (see the section on compatibility testing).
Blood-typing methods
Immunohematology is the study of antigens and antibodies associated with blood transfusions, as well as complications of pregnancy such as neonatal isoerythrolysis. Reactions between RBC antigens and known antibodies can be detected using testing methods. The principle of blood typing is therefore relatively simple and most often based on the hemagglutination of patient RBC antigens with known antibody or antisera. In human immunohematology, slide, tube, solid phase microwell, and gel column testing methods are used. Similar serologic methods are available for blood typing in dogs. The International Society of Animal Genetics is responsible for the standardization of blood-typing reagents. Monoclonal antibodies against DEA 1 have been developed at Kansas State University and the University of Lyon. Unfortunately, the availability of blood typing antisera beyond DEA 1 is limited, thus reserving extended typing of canine RBC antigens to specialty laboratories. The commercial reference laboratory in the United States (Animal Blood Resources International, Dixon, CA, and Stockbridge, MI) offers extended typing for DEA 4, DEA 5, and DEA 7. Specific extended antigen typing is performed using polyclonal antibodies and the tube agglutination method.
Tube test method
The tube test method is the reference standard for determining blood type in human immunohematology. It requires the use of antigen-specific antisera and is performed by trained medical technologists in clinical laboratories. In veterinary medicine, the tube test method has been difficult to standardize and is primarily performed in reference laboratories using polyclonal antisera and an antiglobulin phase. It is considered the reference method, but a standardized gel test similar to the human version was introduced to veterinary medicine in 2003 (Figure 9.1). This test was designed for trained technologists in clinical laboratories and results were 100% correlated with the tube test (Giger et al. 2005). Unfortunately, this test is no longer available.
Figure 9.1 Canine gel blood-typing test (no longer available). Microtubes 1–3 are DEA 1 negative because all patient RBCs pass through the gel and form a cell button. Microtube 4 is DEA 1 positive because agglutinated cells form a cell layer at the top of the gel.
Blood-typing cards
One of the standardized point-of-care canine blood-typing tests that is commercially available for DEA 1 typing outside of the commercial laboratory is the blood typing card, which first became available in the 1990s from DMS Laboratories New Jersey (DMS RapidVet-H, DMS Laboratories Inc., Flemington, NJ). These cards are stored at room temperature and have lyophilized murine monoclonal DEA 1.1 reagent on the patient test area that is reconstituted with phosphate-buffered saline diluent prior to performing the test (Figure 9.2). There is a positive control well and an autoagglutination well; patient whole blood is collected in an EDTA tube and is added to the testing wells. Dogs with DEA 1 positive RBCs will agglutinate in the patient test well, whereas dogs with DEA 1 negative RBCs will not (Figure 9.3). If there is agglutination in the autoagglutation well, the test is invalid and accurate DEA 1.1 typing cannot be interpreted with this test (Figure 9.4).
Figure 9.2 Canine DEA 1 blood-typing card (DMS RapidVet-H, DMS Laboratories Inc., Flemington, NJ).
Figure 9.3 Example of DEA 1 positive and DEA 1 negative blood typing. a. Erythrocytes with the DEA 1 antigen form visible line agglutination in the patient test well. b. Erythrocytes without the DEA 1 antigen do not agglutinate in the patient test well.
Figure 9.4 Interpretation of the canine blood-typing card, including autoagglutination, obtained from the manufacturer’s package insert (DMS RapidVet-H, DMS Laboratories Inc., Flemington, NJ).
Immunochromatographic cartridges
More recently, an immunochromatographic cartridge test became available from Alvedia (Quick Test DEA 1, Alvedia, Lyon, France). This method relies on the migration of RBCs on a monoclonal antibody-containing membrane on an absorbent paper strip (Figure 9.5). The absorbent paper strip is dipped into anticoagulated (EDTA) recipient whole blood and swirled in diluent in a plastic well. The immunochromatographis strip is then placed into the RBC suspension and the RBCs diffuse through the strip (Figure 9.6). Erythrocytes with the corresponding antigen form a visible line on the membrane to indicate DEA 1 positive (red line at DEA 1) or negative (no red line at DEA 1) (Figure 9.7). Only free RBCs can move up the test strip, so in theory, autoagglutination should not affect this test.
Figure 9.5 Immunochromatographic cartridge test kit (Quick Test DEA 1, Alvedia, Lyon, France).
Figure 9.6 Diffusion step: the immunochromatographic strip is placed into the patient RBC suspension.
Figure 9.7 a. DEA 1 positive cartridge test: erythrocytes with the DEA 1 antigen form a visible line on the strip (red line at DEA 1). b. DEA 1 negative cartridge test: erythrocytes without the DEA 1 antigen do not form a visible line on the DEA 1 antigen area.
Comparison of blood-typing tests
A study compared the gel-based method to both the card and cartridge methods for DEA 1 typing and found the results from both tests are reliable (Seth et al. 2012). The cartridge method was also compared to the gel-based method in another study and 98.8% agreement was found between these methods. The only discrepant result was observed in a dog with IMHA: the gel method typed the dog as DEA 1 negative and the cartridge method typed the dog as DEA 1 positive (Blois et al. 2013). The cartridge method was also compared to flow cytometry for testing of DEA 1 expression in dogs and it was found that both the cartridge and flow cytometric methods had close correlation (Acierno et al. 2014).
Automated blood typing
In 2011, a new automated canine blood-typing method was developed (QuickVet/RapidVet Analyzer, Scandinavian Micro Biodevices ApS, Farum, Denmark). This is the first automated blood-typing test in veterinary medicine and, in contrast to other point-of-care tests, no user interpretation is necessary (Figure 9.8). The system consists of a one-time-use cartridge (QuickVet/RapidVet DEA 1.1 blood typing cartridge, Scandinavian Micro Biodevices ApS, Farum, Denmark) with three capillary channels, which is used with an analyzer (Figure 9.9). The cartridge is preheated to 37°C and then a diluted recipient blood sample is added. The blood sample moves through the cartridge channels by capillary action and the analyzer continuously measures the light transmitted. The analyzer software determines whether agglutination has occurred by comparing the curve of transmitted light for the reaction channel and the reference channel. The test takes approximately 5–10 minutes and the result is positive, negative, or inconclusive; all results are archived in the analyzer.
Figure 9.8 Quick Vet/Rapid Vet analyzer (Scandinavian Micro Biodevices ApS, Farum, Denmark).
Figure 9.9 Quick Vet/Rapid Vet canine DEA 1.1 blood-typing cartridge (Scandinavian Micro Biodevices ApS, Farum, Denmark).
Strict pretesting guidelines require sample dilution and hemolyzed or auto-agglutinating samples cannot be used. A clinical evaluation of this new method compared it to the gel column test that is no longer available. Of the 93 samples compared, 90.3% (84/93) were in agreement, with a sensitivity of 96.2% (50/52) and a specificity of 82.9% (34/41). The test was considered suitable for in-house DEA 1 typing, but the importance of proper sample dilution and the need for prior hemolysis and agglutination testing of the sample, which will interfere with accurate test results, was emphasized (Kohn et al. 2012). To date, no studies have compared the automated method to the card or immunochromatographic cartridge methods.
Commercial laboratory blood-type testing
In situations when performing in-hospital blood typing is not feasible, patient and donor samples can be sent to commercial veterinary diagnostic laboratories (e.g., Antech Diagnostics, IDEXX Laboratories, Animal Blood Resources International) to perform canine DEA 1 typing. Turnaround times might vary, so planning ahead is imperative when using a commercial laboratory.
Performing blood typing in practice
Despite the identification of many DEAs in dogs, the DEA 1 blood group is the most clinically important, similar to the ABO system in humans, due to its high degree of antigenicity. In dogs that are DEA 1 negative, naturally occurring isohemaglutinins (antibodies) do not exist. However, transfusion of DEA 1 positive RBCs to a DEA 1 negative dog will result in rapid sensitization and anti-DEA 1 antibody production. Thereafter, a dog with anti-DEA 1 antibodies that subsequently receives a DEA 1 positive RBC transfusion can have a fatal hemolytic transfusion reaction. It is for this reason that all dogs requiring a transfusion should be blood typed prior to the transfusion of any blood components containing RBCs. DEA 1 positive dogs are “universal recipients” and can receive DEA 1 positive or DEA 1 negative RBCs. Since blood is a limited resource and DEA 1 negative canine donors are not always readily available, it is ideal not to transfuse DEA 1 negative RBCs to DEA 1 positive dogs, but rather DEA 1 negative RBC transfusions should be conserved for DEA 1 negative recipients.
Canine blood donors must be typed for DEA 1 at minimum because the practice of transfusing untyped canine RBCs is not considered acceptable or safe. Extended DEA typing of canine donors is controversial and must be performed through a commercial laboratory (Giger 2014). Although universal canine donors have been described in different ways, most commonly they are negative for DEA 1, 3, 5, and 7, and positive for DEA 4. The practice of selecting and screening canine blood donors is discussed in more detail elsewhere in this textbook (see Chapter 13).
Crossmatching methods
In dogs, the lack of clinically significant naturally occurring alloantibodies makes the risk of an overt hemolytic reaction low during the first transfusion. However, crossmatching is important in previously transfused patients because sensitization might have occurred after the prior transfusion. A previous study demonstrated that seven of nine dogs transfused with DEA 1 type specific RBCs became sensitized to other RBC antigens (Kessler et al. 2010), therefore a crossmatch must be performed before a RBC transfusion in all canine patients previously transfused, unless there is an urgent life-threatening need for blood. Blood that is crossmatch compatible should be selected for previously transfused canine patients or those in which the previous transfusion history is unknown or unavailable. When crossmatch compatible units cannot be found, notifying “on-call” blood donors or considering alternatives to allogenic RBC transfusion can be pursued.
The major crossmatch is the serologic method designed to determine compatibility between donor RBCs and the recipient (patient) plasma. The intent is to prevent incompatible transfusions that could result in an immune-mediated hemolytic transfusion reaction. Donor RBCs are incubated with recipient serum and observed for agglutination or hemolysis. If agglutination or hemolysis is observed, an incompatibility exists and the donor blood should not be used for transfusion. Causes of incompatibility occur if the recipient has a naturally occurring or induced alloantibody directed against an antigen present on the donor RBCs. If no agglutination or hemolysis is noted, the crossmatch is considered compatible and the donor blood is acceptable for transfusion. It is important to note that a compatible serologic crossmatch does not guarantee normal RBC survival or completely eliminate the risk of the transfusion. Delayed transfusion reactions, as well as reactions to donor plasma proteins or white blood cells, are not detected by crossmatch testing.
The minor crossmatch is the serological test designed to determine compatibility between donor plasma and recipient (patient) RBCs. The transfusion of plasma containing blood products (e.g., fresh frozen plasma, whole blood) has the potential to cause destruction of recipient RBCs if the donor has an alloantibody. In human medicine, donors are screened for antibodies after blood collection and plasma or plasma components are not used if an antibody is present. Compatibility testing (minor crossmatch) is therefore not required for transfusion of plasma products to people. Most commercial veterinary blood suppliers screen canine donors as well, thereby eliminating the need for minor crossmatching. Likewise, given that canine blood donors are selected on the basis of not having received a previous transfusion, the likelihood of canine donors having clinically relevant alloantibodies that might affect a minor crossmatch is very low.
In human medicine, the crossmatch was first described in 1907 and has been modified multiple times. Major milestones in its evolution include the use of multiple techniques, such as tube and gel, as well as several types of enhancement media such as albumin, enzymes, polyethylene glycol, and antiglobulin. If a slide or tube method is used, the experience of the person performing the test is of high importance; the test is time-consuming, can be cumbersome, and is falling out of favor in human clinical laboratories. The use of gel technology is increasing due to its ease of use and standardization.
A variety of crossmatch methods are available in veterinary medicine. The standard tube test is time-consuming and cumbersome. Because the experience of the person performing the test is of the utmost importance, it is the author’s opinion that the tube test should be reserved for licensed technologists or other similarly trained personnel. The tube test can be performed in-house and the tube crossmatch procedure is described in Box 9.2. Crossmatching can also be performed with point-of-care methods using gel agglutination and immunochromatographic tests. Both of these methods offer advantages over the standard tube crossmatch and are considered user friendly for any veterinary practice.
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