3.. '\ .‘g' "may! . «1.15%! , nan”:d.q: kyupd . . M53: I ‘ ‘ smut. .u Nov. 1 Sue... . _ Wan: .firafi «3 3a.. kfikmw; : . w , f. 5 emfim‘u 3, . J‘ tour ”1...: . {1 11 C?‘: 3.4... :‘P, T338” LIBRARY Michigan State 30 07 University This is to certify that the thesis entitled THE PREVALENCE AND SPECIFICITY OF ANTI—DOG ERYTHROCYTE ANTIGEN ANTIBODIES IN A POPULATION OF DOGS presented by MARGARET MEGAN LEMMON has been accepted towards fulfillment of the requirements for the MS. degree in Clinical Laboratory Science MW Professor’s Signature @11th Date MSU is an affirmative-action, equal-opportunity employer .--.-.-.-l—’—.-.-.-.—.-.-.-.—.--.-.—.—-- —-an»ca-.-.-.----a-o-—--.-u—-a—‘— ‘-l—-.--—--u—lc—.-.— -.--r-o---.--n- PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/07 p:/ClRC/DateDue.indd-p,1 THE PREVALENCE AND SPECIFICITY OF ANTI-DOG ERYTHROCYTE ANTIGEN ANTIBODIES IN A POPULATION OF DOGS BY Margaret Megan Lemmon A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Clinical Laboratory Science 2007 ABSTRACT THE PREVALENCE AND SPECIFICITY OF ANTI-DOG ERYTHROCYTE ANTIGEN ANTIBODIES IN A POPULATION OF DOGS By Margaret Megan Lemmon Antibodies to dog erythrocyte antigens (DEAs) have been reported to occur naturally in the dog population and can be induced after exposing a DEA- negative dog to the DEA. Anti-DEAs can cause transfusion reactions, including red cell loss and hemolytic transfusion reactions. To determine the current prevalence of anti-DEAs in a population of dogs, 265 serum samples from dogs of unknown medical history were screened by the tube agglutination method with a red blood cell antigen panel representing all currently definable DEAs. Reactivity was demonstrated in 184 serum samples (69.4%) that were subsequently screened with a different red blood cell antigen panel to determine the specificity of the antibody or antibodies. The prevalence of specific individual antibodies was: 1.9% anti-DEA 1.2, 5.3% anti-DEA 3, 3.0% anti-DEA 5, and 8.7% anti-DEA 7. These numbers do not reflect the antibodies associated with multiple antibody combinations. Data were adjusted to exclude spurious results, and the prevalence was: 8.4% anti-DEA 3, 5.0% anti-DEA 5, 12.4% anti-DEA 7, 19.3% had possible multiple antibody combinations, and 14.8% were unknown antibodies. This study indicated a higher overall prevalence of anti-DEAs than previous studies and a large percentage of the antibodies could not be defined. Consistent with previous studies, anti-DEA 3, 5, and 7 were identified. Copyright by MARGARET MEGAN LEMMON 2007 DEDICATION This Thesis is dedicated to my grandfather, William J. Barber, who taught me the value of continuing to learn no matter how old I am, and to my parents, D. Glenn and Margaret P. Lemmon, who have supported me in all of my endeavors. ACKNOWLEDGEMENTS I would like to thank Dr. John Gerlach for his guidance and numerous hours of help. Without his support, I would not have completed this manuscript. I would also like to extend a special thank you to Dr. Anne Hale for donating the samples utilized in this study, as well as for her countless hours of input and expert advice in dog immunohematolcgy. Finally, I would like to thank Dr. Kathleen Hoag and Dr. Michael Scott for their expertise and time given as members of my thesis guidance committee. TABLE OF CONTENTS LIST OF TABLES ................................................................................................ vii LIST OF FIGURES ............................................................................................. ix INTRODUCTION ................................................................................................. 1 BACKGROUND ................................................................................................... 3 lmmunohematology ................................................................................... 3 The History of the Dog Blood Group System ............................................ 8 The Dog Blood Group System .................................................................. 8 Description of Pre-Transfusion Testing Methodologies ........................... 14 METHODS ......................................................................................................... 20 Selection of Screening Red Blood Cells .................................................. 20 Statistics .................................................................................................. 22 Blood Collection and Storage .................................................................. 24 Serum Samples ....................................................................................... 24 Cell Washing ........................................................................................... 25 Sera Screening ....................................................................................... 25 RESULTS .......................................................................................................... 32 Detection Screen ..................................................................................... 32 Identification Screen ................................................................................ 43 Combined Results ................................................................................... 44 Controls ................................................................................................... 51 DISCUSSION ..................................................................................................... 52 Differing Definitions in Dog lmmunohematology ..................................... 62 Possible Explanations for Unknown or Inconsistent Results ................... 64 Considerations in this Study .................................................................... 69 Previous Studies ..................................................................................... 77 IdeaITransfusion Medicine Practices ...................................................... 79 CONCLUSION ............................................................................ 83 APPENDIX ......................................................................................................... 88 REFERENCES .................................................................................................. 91 vi LIST OF TABLES TABLE 1: Probability values for each DEA based on the Fisher Exact and the Harris and Hochman (H&H) Methods ...................................................... 22 TABLE 2: Detailed combined results of the Detection and Identification Screens ................................................................................................... 33 TABLE 3: Summary of the reaction conditions in the Detection Screen ........... 32 TABLE 4: Samples reacting with only one dog in the Detection Screen ........... 42 TABLE 5: Samples reacting with a two dog pair in the Detection Screen ......... 43 TABLE 6: Samples reacting with all three dogs in the Detection Screen .......... 43 TABLE 7: Summary of reaction conditions in the Identification Screen ............ 44 TABLE 8: Summary of reaction conditions for combined results of Detection and Identification Screens .............................................................................. 45 TABLE 9: Reaction patterns of screening dogs as related to individual serum antibody specificities for combined results of the Detection and Identification Screens .............................................................................. 46 TABLE 10: Reaction patterns of screening dogs as related to individual serum antibody specificities during Detection Screening ................................... 53 TABLE 11: Prevalence of anti-DEA antibodies ................................................. 55 TABLE 12: Reaction patterns of screening dogs as related to multiple serum antibody specificities for combined results of the Detection and Identification Screens .............................................................................. 59 TABLE 13: Reaction patterns of screening dogs as related to temperature of reaction in the Detection Screen, Identification Screen, and for combined results ...................................................................................................... 61 TABLE 14: Reaction patterns of screening dogs after removing the DEA Negative dog from the results ................................................................. 71 TABLE 15: p-values for each DEA after removing the DEA Negative dog ........ 73 vii TABLE 16: Reaction patterns of screening dogs with positive serum samples after removing the DEA Negative dog and samples that did not react in the Identification Screen from the results ...................................................... 74 TABLE 17: Prevalence of anti-DEA antibodies after removing the DEA Negative dog and samples that reacted in the Detection Screen but not in the Identification Screen ................................................................................ 77 TABLE 18: Comparison of anti-DEA prevalence in this study versus previous studies ..................................................................................................... 79 viii FIGURE 1: FIGURE 2: FIGURE 3: FIGURE 4: FIGURE 5: LIST OF FIGURES Gel Tube Major Crossmatch Test ................................................... 16 DEA 1.1 Card Test ......................................................................... 17 Antibody Screening Panel .............................................................. 21 Reaction Set-Up ............................................................................. 27 Reaction Grades ............................................................................ 29 INTRODUCTION Periodic surveys of American households conducted by the American Veterinary Medical Association (AVMA) have demonstrated the abundance of companion animals in America. During 1996, it was estimated that nearly 60 percent (%) of households in the United States had owned one or more pets, and out of these, nearly 32% of the households had owned dogs.1 These figures reflect a 0.8% increase in the canine pet population since the AVMA survey conducted in 1991, which amounts to 0.4 million dogs.1 With an increase in canine pet ownership, there is an increased need for veterinary care.2'3 In many cases, the companion animal is seen as a member of the family.“'5 Given the importance of the pet’s role in the family, many pet owners take steps to ensure the welfare of the animal through veterinary care. The increased demand for better pet health care has led to advancements in the quality and variety of services offered, including various surgical and medical procedures such as blood transfusions.6'7 Despite these steps forward in veterinary medicine, the field lacks the regulations governing procedures such as blood transfusions that are a prominent feature of the human health care system. The failure to regulate such procedures leads to a lack of standardization in the field of veterinary medicine. At one end of the spectrum, a veterinarian may utilize techniques and protocols similar to those in human medicine, but on the other end, a veterinarian may do only what is necessary to get by. Standardization is essential to ensure quality health care for every animal. The absence of standardization is especially significant in canine transfusion medicine. As opposed to human medicine, canine transfusion medicine is not subjected to regulations on the administration of blood products or pre-transfusion testing. Currently, a veterinarian may take a unit of blood from any dog and administer the unit to any recipient dog without pre-transfusion testing. This practice would be negligent in human medicine, but as there are currently no universal standards or regulatory guidelines in veterinary medicine, it is an accepted practice. Despite being within the limits of the law, the practice of this method of canine transfusion medicine is potentially risky. Without pre- transfusion testing, it is impossible to predict the likelihood of a transfusion reaction and may put the patient’s life in jeopardy. In most cases, canine transfusions are administered on an emergency basis, and complete pre-transfusion testing, including blood typing of the donor and recipient, serum antibody screening, infectious disease testing, and crossmatching is impossible. More in-house testing methods and a better understanding of the prevalence of antibodies against dog erythrocyte antigens (DEA) are needed to increase the safety of canine transfusion practices. BACKGROUND lmmunohematology is the field involved with the interaction between immune factors and blood components. A major focus of lmmunohematology is the preparation of blood products for transfusion. Ideally, blood is collected from a healthy donor, typed, and compatible blood products are selected for transfusion into a recipient. Before the product is transfused into the recipient, a crossmatch is performed. The major crossmatch test involves combining the recipient’s serum or plasma with the donor’s red blood cells to detect the presence of circulating antibodies in the recipient against antigens on the donor’s red blood cells. The donor’s serum or plasma can also be screened for antibodies against the recipient’s erythrocyte antigens in a minor crossmatch. Following testing, units of blood are selected for transfusion by matching the antigenic profile of the donor's red blood cells to the antigenic profile of the recipient’s red blood cells. When units of blood cannot be matched to the recipient’s antigen profile, units typed as antigen-negative may be selected for transfusion. lmmunohematology Antigens are defined as substances that can induce antibody synthesisf"9 More specifically, antigens can be composed of proteins, carbohydrates, or lipids. Canine blood group antigens have not been fully characterized for biochemical composition, although they may be comprised of glycoproteins or glycolipids.9 Antibodies are immunoglobulins (lg) that exhibit specificity for particular antigens. There are 5 classes of immunoglobulin in dogs: IgA, lgG, lgM, lgD, and IgE.8 The distinctions between the classes of immunoglobulin are the result of differences in the constant region of the heavy chain, which does not convey specificity. Rather than resulting in varied specificity, differences in immunoglobulin structure are responsible for varied function. Immunoglobulins also have varied effects depending on whether they are in vivo or in vitro. Immunoglobulins bound in vivo can result in complement activation, opsonization, neutralization, and rarely, agglutination, while immunoglobulins bound in vitro may result in complement activation and agglutination?"9 The surface of the red blood cell is covered with antigens, which give the cell its immunological identity. Plasma contains antibodies, which can be “naturally-occurring” or acquired. “Naturally-occurring”, or expected, antibodies occur without exposure to other blood types and are present in the blood from shortly after the time of birth. Acquired antibodies are produced as a result of exposure of an antigen-negative patient to that antigen.10 Exposure can occur when transfused red blood cells that are recognized as foreign induce the production of antibodies against the foreign antigens in a process called red blood cell alloimmunization.11 Exposure may also occur during pregnancy or during delivery in an antigen-negative mother carrying an antigen-positive fetus.”15 During allogeneic red blood cell transfusion, immunologic reactions due to antibodies may occur. Such reactions can be immediate or delayed, with symptoms appearing during or shortly after transfusion for immediate transfusion reactions or to approximately 21 days post-transfusion for delayed transfusion reactions, although symptoms may occur later.16 The reaction could be acute and threaten the survival of the transfusion recipient, or may lack physical manifestation. At the cellular level, immunologic reactions to transfusion are the result of complement activation or opsonization by complement and/or immunoglobulin.““‘5'17 This process of transfused red blood cell clearance is called immune-mediated red blood cell destmction.”16 Antigens on the surface of transfused red blood cells can bind to B cell receptors, which are membrane-bound immunoglobulinsf”11 Once an antigen binds to the receptor, a signal is transmitted to the B cell interior and the antigen is endocytosed by the B cell. The antigen is degraded and displayed on the surface of the B cell as peptides on major histocompatibility complex (MHC) class II molecules. When a T helper cell of the same antigen specificity recognizes the peptide-MHC class II complex, the T cell is activated, begins expressing the CD40 ligand on its surface, and secretes interleukin (IL) 4, IL-5, and lL-6, which are responsible for stimulating the B cell to proliferate and differentiate into plasma cells. The plasma cells are responsible for antibody production of the same specificity as the B cell receptor.11 Macrophages can also bind the constant region of immunoglobulins bound to surface antigens of red blood cells, and subsequently phagocytose the cell. The macrophage degrades the red blood cell into end products including peptides. The peptides can be released outside of the macrophage or they can be displayed on MHC class II molecules on the macrophage cell surface, converting the macrophage into an antigen presenting cell. The released peptides can interact with B cells, leading to B cell proliferation and differentiation. The macrophage acting as an antigen presenting cell can activate naive T helper cells with the peptide-MHC class II complex. Activated T helper cells can recognize peptide-MHC class II complexes on activated B cells, leading to antibody production.8 Immune-mediated red blood cell destruction is based on either complement (C) activation, opsonization, or a combination of both complement activation and opsonization. There are two principal ways these conditions can lead to the destruction of red blood cells: intravascular hemolysis and extravascular red blood cell desthction. lntravascular hemolysis involves complement activation leading to the activation of the membrane attack complex. The cell membrane integrity is compromised and the cell is destroyed.11 Immune-mediated red blood cell destruction by extravascular mechanisms can occur in three different ways: C3b alone, lg alone, or C3b and lg together. The mechanism of C3b alone is relatively ineffective in causing red blood cell destruction. Red blood cells with C3b present on the cell surface may adhere to phagocytic cells, although there is usually minimal phagocytosis of these cells because C3b alone does not stimulate phagocytosis.11 C3b can lead to phagocytosis when macrophage-activating immune mediators, such as C5a, are present. Often, the C3b bound to the cell surface is converted to C3d by enzymatic cleavage, and the red blood cell can usually survive normally. Red blood cells with IgG present on the cell surface usually bind to the Fc receptors of phagocytic cells and subsequently are phagocytosed. Red blood cells with both C3b and IgG present on the cell surface are more successfully destroyed than red blood cells with either C3b or IgG alone because they can more readily adhere to macrophages and induce phagocytosis.6""9"1'18 Systemic manifestations of transfusion incompatibility can be highly varied, depending on the agents involved in the event. The most severe hemolytic transfusion reactions are the result of preexisting antibodies in the recipient interacting with antigens on transfused red blood cells.19 The antigen- antibody interaction can lead to a series of events including complement activation, cytokine production, coagulation activation, and other systemic inflammatory responses. Complement activation can cause intravascular hemolysis resulting in hemoglobinemia and possibly hemoglobinuria. The anaphylatoxins produced as a result of complement activation can cause hypotension and bronchospasm, and can lead to the release or production of systemic or local mediators, such as histamine, kinins, and cytokines. The mediators may lead to physical manifestations that are similar to those of systemic allergy, such as flushing, chest pain, and vomiting.16 The full role of cytokines in the manifestations of immune-mediated hemolysis is not completely known, although certain cytokines are known to lead to fever and hypotension, and stimulate endothelial cells to increase procoagulant activity. The antigen-antibody interaction may also have a role in coagulation activation by initiating the “intrinsic” pathway through the Hageman factor. Activated Hageman factor leads to a series of processes that increase the expression of tissue factor.11 The presence of tissue factor activates the “extrinsic” coagulation pathway and is associated with disseminated intravascular coagulation.11 The outcome of these events may be uncontrolled bleeding or oozing.11 Other systemic manifestations, such as renal failure and shock, result from the mass of antigen-antibody complexes, immune mediators, and systemic effects resulting from complement activation, cytokine production, and coagulation activation."'2‘*23 The History of the Dog Blood Group System Red blood cell antigens were first discovered in humans in 1900 by Landsteiner.24 This discovery sparked the search for similar blood antigen systems in other species, and in 1910, Von Dungem and Hirszfeld made the discovery of dog blood group antigens.22 Four different antigens were described at that point. In the 19508 and 19603, the most extensive work in dog blood group research was conducted and reviewed by Swisher and Young.1‘z'13'“"25 This work described seven blood group antigens in dogs and the frequency of each antigen within a random population of dogs. The study also focused on antibodies specific for the blood group antigens.13 The Dog Blood Group System The dog blood group system is based on the antigens found on the surface of the dog red blood cell. Some of these antigens have been identified serologically, but the structures have not been well-elucidated.6'12'26 The First and Second International Workshops on Canine lmmunogenetics standardized the terminology of the blood group system.27'28 Although most dog lmmunohematology professionals adhere to the standardized terminology, some groups, including the dog immunohematology community in Japan - which was not a part of the lntemational Workshops - do not adhere to the standard terminology.”30 As decided at the First lntemational Workshop, the red blood cell antigens were called canine erythrocyte antigens, or CEAs, which was changed to dog erythrocyte antigens, or DEAs, at the Second International Workshop.”28 Each antigen is identified by the abbreviation DEA followed by a number indicating the blood group. In the case of subgroups, the antigen is identified by the term DEA followed by the blood group number, a period, and the number of the subgroup. An exception to this rule exists in the case of DEA 7; letters rather than numbers specify the subgroups. Although many blood groups have been identified, currently five blood groups are of clinical significance, which means they can be routinely identified by serologic methods and can potentially cause a stimulating event after transfusion into an antigen-negative dog. The current clinically significant groups are DEA 1, 3, 4, 5, and 7. The only clinically significant groups to have subgroups are DEA 1 and DEA 7. The DEA 1 group consists of four phenotypes: DEA 1.1, 1.2, 1.3, and null.31 Only one DEA 1 group phenotype can be expressed in any particular dog. A pattern of autosomal dominance is demonstrated in the DEA 1 group with the order of descending dominance as follows: DEA 1.1, DEA 1.2, DEA 1.3, and null.28'32 The DEA 1.1 phenotype is expressed in about 42% of the general population and DEA 1.2 is expressed in about 20% of the general population, although variation in expression occurs in different breeds.‘2'33 Greyhound and German Shepard breeds are typically DEA 1.1 and 1.2 negative, whereas Golden Retriever, Laborador, and Rottweiller breeds are usually DEA 1.1 or 1.2 positivef“3 No estimate of the frequency of DEA 1.3 has been reported. DEA 1.1 and 1.2 are consistently recognized by commercially-available antisera, but DEA 1.3 may be missed with the same antisera.3‘4 The DEA 1 group is of particular importance due to its frequency and its ability to induce antibody production in DEA 1-negative dog.13'15'2"35 Membrane proteins with molecular weights of 50 and 200 kilo Daltons (kD) have been identified for DEA 1.1 in Western blot experiments utilizing an anti-DEA 1.1 monoclonal antibody.36 An 85 kD band has been identified for DEA 1.2 in immunoprecipitation experiments utilizing polyclonal antibodies.26 The DEA 3 group consists of two phenotypes: DEA 3 and null.3'1 Autosomal dominance is exhibited for this group with DEA 3 dominant over the null phenotype.32 The DEA 3 phenotype shows breed specificity, being expressed in up to 23% of greyhounds but only 6% in the general population.12 Up to 20% of DEA 3-negative dogs may have a naturally occurring anti-DEA 3 antibody.“15 DEA 3-negative dogs that have been sensitized to the DEA 3 antigen may suffer severe transfusion reactions upon subsequent exposure to DEA 3-positive red blood cells. DEA 3-positive red blood cells repeatedly transfused to a previously sensitized DEA 3-negative dog could result in cell loss 10 within a period of five days.”30 Five bands have been identified in Western blotting experiments utilizing an anti-DEA 3 monoclonal antibody. The molecular weights of the bands were 34, 53, 59, 64, and 71 kD.37 The DEA 4 group consists of two phenotypes: DEA 4 and null.31 This group exhibits autosomal dominance, with DEA 4 dominant over the null phenotype.32 Nearly all dogs (98-99%) express DEA 4, although variance may occur among specific breeds and with variation in geographic location.” No naturally occurring antibody against DEA 4 has been reported. Red blood cells positive for DEA 4 only are the universal blood type and are usually considered safe to transfuse into dogs of other DEA types. An early study by Swisher et al. (1962) in which DEA 4-negative dogs were exposed to DEA 4-positive cells demonstrated that anti-DEA 4 antibodies were produced in response to the exposure, but subsequent exposure to DEA 4 did not result in transfusion reactions.14 A more recent study by Melzer et al. (2003), however, demonstrated a severe transfusion reaction in a DEA 4-negative dog that had been administered multiple units of DEA 4-positive blood.38 A protein of molecular weight between 32 and 40 kD has been isolated using polyclonal anti-DEA 4 antibodies in immunoprecipitation experiments.26 The DEA 5 group has two phenotypes: DEA 5 and null.31 Autosomal dominance is exhibited in the DEA 5 group, with DEA 5 dominant over the null phenotype.32 The DEA 5 phenotype shows breed specificity, being expressed in up to 23% of the general population but up to 30% in greyhounds.” Variance may also occur due to geographical location, as with the human Duffy 11 antigen”39 The majority of dogs within a geographical area, thus within the potential breeding population, usually remain within that location, and the antigen or its absence also remains as a characteristic within the location. Previous studies have reported naturally occurring anti-DEA 5 antibody in approximately 10% of randomly-selected non-transfused adult dogs in the United States.”15 Upon repeated exposure to DEA 5-positive red blood cells, DEA 5-negative dogs sensitized to DEA 5 can sequester and destroy the transfused red blood cells within a period of three days.14 The DEA 5 antigen has not been characterized. The DEA 7 group has three phenotypes: DEA Tr, O, and null.31 This is the only blood group that does not obey the rules of DEA nomenclature. Although DEA 7 has multiple phenotypes, the subgroups are not usually indicated individually in studies or in blood typing by reference laboratories. Unlike the other DEAs, DEA 7 is not a true erythrocyte antigen.40 Production of this antigen occurs in the body tissues and the soluble antigen is absorbed onto the surface of the red blood cell, similar to human blood group antigen A.31'41‘43 The DEA 7 blood group is expressed in about 45% of the general population.” Previous studies demonstrated an prevalence of anti-DEA 7 antibody in up to 50% of dogs that were negative for the DEA 7 antigen.”'44'45 DEA 7-negative dogs may be sensitized to DEA 7 through exposure to the antigen on red blood cells, and repeated exposure to the antigen in transfusion may result in sequestration of the red blood cells and cell loss within a period of 72 hours.” Three bands of the molecular weights 53, 58, and 66 kD have been isolated by immunoprecipitation experiments utilizing polyclonal anti-DEA 7 antisera.26 12 “Naturally-occurring” anti-DEA antibodies have been demonstrated against DEAs 3, 5, and 7.14'15'46 Many researchers disagree how frequently these antibodies occur, especially with anti-DEA 7.123124,“ Hale (1995) reported an prevalence of the anti-DEA 7 antibody in up to 50% of DEA 7 negative dogs, although the author’s personal observation suggested an prevalence of 20% to 50%.12 Other reports indicate an prevalence of anti-DEA 7 in 15% to 50% of DEA 7 negative dogs. Some early researchers doubted the existence of anti- DEA antibodies altogether.”48 More recently, Giger et al. (1995) suggested that dogs do not have naturally occurring antibodies of clinical significance as are present in cats and humans, which have antibodies that are expected and can cause severe hemolytic transfusion reactions.21 Today, the existence of anti- DEA antibodies is generally accepted, but their significance in dog transfusion medicine may still be debated.2"35'49-5° Currently, there are no standards or regulatory guidelines for transfusion practices in veterinary medicine. At the discretion of the veterinarian, testing can be extensive or may not be done at all.47 Although no regulatory guidelines exist, many methods are available for pro-transfusion testing, including blood typing of the donor and recipient by serologic methods, major and minor crossmatching, rapid agglutination card tests, and, more recently, the gel tube crossmatching kit. 13 Description of Pre-transfuslon Testing Methodologies The serologic method of blood typing relies on tube agglutination to determine the presence of DEAs.51 The technique involves combining polyclonal anti-DEA antisera with a suspension of washed red blood cells to be typed, followed by an incubation period at an appropriate temperature: 37 degrees Celsius (°C) for anti-DEA 1.X and anti-DEA 1.1, and 4°C for anti-DEA 3, anti- DEA 4, anti-DEA 5, and anti-DEA 7. A negative control reaction is run at both temperatures, as well, with PBS in place of the antisera. After incubation, the samples are centrifuged and the cells are gently resuspended while being viewed for agglutination. Presence of agglutinates indicates the presence of the DEA. Blood typing by serologic methods requires the skill of specially-trained technicians and uses expensive reagents. As a result, reference laboratories usually conduct this method of typing.9'”"r’1'52 Commonly, two versions of serologic typing are available: full DEA typing and abbreviated DEA typing. The full typing involves the use of all currently available anti-DEA antisera: anti-DEA 1.X, anti-DEA 1.1, anti-DEA 3, anti-DEA 4, anti-DEA 5, and anti-DEA 7, as well as anti-canine globulin (ACG).53 Full typing tests for all clinically-significant DEAs and is, therefore, the most extensive dog blood typing procedure. Some abbreviated typing panels use anti-DEA 1.X, anti-DEA 1.1, and anti-DEA 7, while others may only use anti-DEA 1.1 and anti-DEA 1.X. The corresponding antigens - DEA 1.1, 1.2, 1.3, and 7 — are of significant interest to the veterinarian because of their high prevalence in the dog population and potential for stimulating antibody production.” This typing paradigm, however, excludes 14 testing for DEA 3, 4, and 5, which all have the potential to cause transfusion reactions. ”'33 There are also known naturally-occurring serum antibodies to DEA 3 and 5, which can cause transfusion reactions, further emphasizing the importance of these DEAs in pre-transfusion testing. The crossmatch procedure is used to detect incompatibility between the blood donor and the recipient of the blood component.9'52 This technique relies on the combination of red blood cells with serum in a tube agglutination method. As with the serologic blood typing method, the samples are incubated at various temperatures, centrifuged, and gently resuspended while being viewed for agglutination. The presence of agglutinates indicates an incompatibility between the donor and recipient. Two variations of the crossmatch may be performed: major and minor. In a major crossmatch, donor red blood cells are combined with recipient semm, which is intended to simulate the reactions that may occur when the recipient receives the red blood cell component in a transfusion. In a minor crossmatch, donor serum is combined with recipient red blood cells, which is intended to simulate the reactions that may occur when the recipient receives the plasma component in a transfusion. Controls may or may not be performed depending on the standard operating procedures of the individual laboratory or clinic, although the inclusion of controls would ensure that the test is running properly and that a positive crossmatch is not due to autoagglutination. A negative control for the crossmatch procedure would consist of running the reaction as described but using PBS in place of the serum. A recipient receiving 15 whole blood would benefit from both the major and minor crossmatches, although the minor crossmatch is less frequently performed in practice. The gel tube major crossmatch kit, which is based on the principles of the tube agglutination crossmatch procedure, is a very recent addition to the canine pre-transfusion testing options. It consists of a sepharose gel matrix in a microcentrifuge tube (Figure 1). The test is performed by combining donor red blood cells with recipient serum and placing the mixture on top of the gel matrix. The tube is centrifuged and viewed for agglutinates. A positive test will show the presence of agglutinates at the top of the gel matrix. A negative test will show no agglutinates and the red blood cells will be collected at the bottom of the tube. Positive and negative control reactions are included with the kit. This test is inexpensive and very easy to perform, making it an affordable pre-transfuslon testing method for privately-owned veterinary clinics. "T than; L h ii i” I I ' IV ‘ a ‘ w). ‘ 'b Blank Positive Negative Figure 1: Gel Tube Major Crossmatch Test. A positive reaction ls demonstrated by the presence of agglutinates at the top surface of the gel column (a). A negative reaction is demonstrated by the accumulation of non-agglutinating cells at the bottom of the gel column (b). (Picture from Midwest Animal Blood Services, Inc.) 16 The rapid agglutination card test is a commercially available assay for the detection of DEA 1.1. The test consists of a murine monoclonal anti-DEA 1.1 antibody that is lyophilized onto the card.51‘54 The red blood cells to be typed are mixed with the antibody on the card and the card is viewed for agglutination. The presence of agglutinates indicates the presence of DEA 1.1 (Figure 2). Card tests may or may not include control spots. The Rapid Vet-H DEA 1.1 test includes both positive and negative control wells.54 This test is also inexpensive and easy to use, which makes it a reasonable option for veterinarians to perform in their clinics. This test lacks the ability to detect the other DEA 1 subgroups — DEA 1.2 and 1.3 — and no other DEA types can be tested by this method because there have not been any successful versions produced other than DEA 1.1. Figure 2: DEA 1.1 Card Test. A negative reaction is demonstrated by a uniform suspension of cells and the absence of agglutinates. A positive reaction is demonstrated by the presence of agglutinates. 17 Previous studies focused on determining the prevalence of anti-DEA antibodies in dog serum are informative, yet were conducted at limited reaction conditions and reflect a population of dogs that existed more than 50 years ago. In the study by Young et al. (1952), a population of only 145 dogs were screened and the testing was conducted only at room temperature.15 Additionally, the study counted as positive only these reactions that were of 1+ strength and greater, and could not be “dispersed with moderate agitation”.15 Changes in veterinary medical practices, including the increase in blood transfusions, may have an effect on the prevalence of acquired anti-DEA antibodies in the current dog population. Anti-DEA antibody prevalence and distribution may also have also been impacted by changes in breeding practices. This study was conducted with a current population of dogs, a greater number of serum samples, a more complete serum screening process, and different temperatures. The purpose of this study was to determine the prevalence and specificity of anti-DEA antibodies in a population of dog serum samples. Some previous studies indicate that naturally-occurring anti-DEAs are not clinically-significant because they do not cause hemolysis or hemolytic transfusion reactions.“‘5'51 Other studies have demonstrated the ability of naturally-occurring anti-DEAs to mediate the loss of red blood cells.” While the loss of transfused red blood cells may be of limited significance in recipients capable of regenerating their own red blood cells, it is significant in recipients with compromised capability of red blood cell regeneration. The loss of red blood cells in a recipient incapable of red blood cell regeneration will likely lead to a need for subsequent transfusions. Acquired 18 anti-DEA antibodies are a concern because, unlike naturally-occurring anti- DEAs, acquired antibodies may have hemolytic properties and may lead to more severe transfusion reactions.”'51'52 19 METHODS Selection of Screening Red Blood Cells The selection of canine blood samples was based on DEA profile. The Antibody Detection Screening panel consisted of three dogs of the following antigenic profiles: DEA 1.1,3,4,5,7 positive, DEA 1.2,4,7 positive, and DEA 1.3,4,w7 positive. These dogs were selected to represent all clinically-significant DEAs. The Antibody Identification Screening panel consisted of four dogs of the following antigenic profiles: DEA 1.1,4,5,7 positive, DEA 3,4,5 positive, DEA 1.1 positive, and DEA negative. These dogs were selected to allow for identification of the antibodies detected in the antibody detection panel by exclusion; a serum sample that did not react with a particular red cell would not be considered as containing antibodies to the DEAs represented on that red cell (Figure 3). Additionally, dogs were selected based on the probability value calculations by the Fisher Exact and the Harris and Hochman methods.55 The dog blood donors selected provide a probability (p) value of less than 0.05 for being able to identify all DEAs except DEA 1.2 and DEA 1.3. There were insufficient red blood cell donors available to give a p-value of less than 0.05 for being able to identify DEA 1.2 and 1.3. These two DEAs had a p-value of 0.147 by the Fisher method and 0.057 by the Harris and Hochman method (Table 1). 20 .o>=_moa n n. 62.303 o:_:mo.=cw u Oo< .oLBEonES Eco. u .5. Ear. SaEwEE u w. .o>=_mon x83 u +3 .2283. En; “385352 N «ow .mcozomo. Eco uflcmmofio. e new 603298 3 33 25935 2: we corner—3:02 or: L8 26=~ 2 won? =moE=o =m “38:32am: .8on cosoouco on... .moEEmm E33 2: E mguonzca no :ozmoEEcE ucn c2323 05 .2 Bo=n 8 on: (mo :0 comma “.3023 225 33an :3 too... to. 9: ._ocmq @5523. 3393.3 an 0.59". 2m oom mEEm co___D mamw cwwhum aoseoczcoo. Lmqqiw +3 :5 twee ~8on cozoouon a muo< Donn Dov km m. N new w 3m 3 35809.20 50500.2 o < 68255.30 con_m.._aE0 5:2: >2 “3530220 0. .3508. 3 < dean—53.80 359E552: EuE 3 50520220 0_ 55002 +N < 5.5anan .020 use 02053300 090. .2050 >2 50530805. m_ .8503. +n < 5:89:35» .620 new 8052mm“ cube. one 3 “5580220 0. 55000.. +v < .0255 cozouom ”m 0.59". 29 labeled with the appropriate control name. These tubes were divided into sets of two tubes, which were additionally labeled with each red blood cell donor’s name. One tube from each set of two was designated as a 37°C reaction tube. The other tube was designated as a 4°C reaction tube. The controls were run at the same reaction conditions as the serum samples, using Positive Control I (Anti- DEA 4, Lot SARA22702, diluted 1:25, Midwest Animal Blood Services, Inc.) and Negative Control Serum (Canine Negative Control, Heat lnactivated, Pooled, Michigan State University) in place of the serum samples. A negative control reaction of 0 was accepted as within specifications. A positive control reaction of 2+ or greater was accepted as within specifications. Samples run on a day in which the controls did not meet specifications were either rescreened or removed from the data set. For each control in the second panel, eight 10 x 75 mm borosilicate glass disposable culture tubes were labeled with the appropriate control name. These tubes were divided into sets of two tubes, which were additionally labeled with each red blood cell donor’s name. One tube from each set of two was designated as a 37°C reaction tube. The other tube was designated as a 4°C reaction tube. The controls were run at the same reaction conditions as the serum samples, using Positive Control ll (Goat Anti-Dog, Lot G9-3-8-05, Midwest Animal Blood Services, Inc.) and Negative Control Serum (Canine Negative Control, Heat lnactivated, Pooled, Michigan State University) in place of the serum samples. A negative control reaction of 0 was accepted as within specifications. A positive control reaction of 2+ or greater was accepted as within 30 specifications. Samples run on a day in which the controls did not meet specifications were either rescreened or removed from the data set. 31 RESULTS In this study, serum samples were screened to detect the presence of antibody against any clinically significant DEA and samples demonstrating positive reactions were rescreened to identify the specificity of the reacting antibody. Data were generated for 312 serum samples. A total of 47 serum samples were excluded due to a day of failed negative control (10 samples), failed check cells (10 samples), a clerical discrepancy (16 samples), technical error (8 samples), and non-specific complete hemolysis (3 samples). Data are presented for a total of 265 serum samples (Table 2). Detection Screen The Detection screening was conducted using dogs with the antigenic profiles of DEA 1.1,3,4,5,7 positive, DEA 1.2,4,7 positive, and DEA 1.3,4,w7 positive, as previously described. This screening resulted in 81 negative ‘ reactions (30.6%) (Table 3). Of the remaining 184 samples, a total of 125 samples (47.2%) showed positive reactions at both RT/4°C (cold) and 37°C IACG (warm) conditions, 47 samples (17.7%) were positive only at cold conditions, and 12 samples (4.5%) were positive only at warm conditions. Table 3: Summary of reaction conditions In the Detection Screen Reaction Pattern Number of Samples Percentage of Total (%) Negative 81 30.6% Positive cold 8 warm 125 47.2% Posmve cold, 47 117% Negative warm Negative cold, 12 4.5% Posmve warm Total 265 1 00% 32 o o o o o o o o o o +~ o o +m SE). +~ o o o +~ o o o o o +m o o +4 8E). o o o o o o o o o o +~ o +5 +~ 8:: o i o o +m +4 +~ +m o o o +~ o +~ 495 o o +N 0 o o +m o +4 +4. +m +~ +m +m 8:: o o o o o o o o o o o +~ o o SE: C o o o o o o o o o o o +~ +~ BE: o o o o o o 82.2 o o o o o o 85.2 +~ o o o +~ o o o o o o o o +~ RE). o o o o i o o o +5 o +m o 5 +4 55 +~ o o o +m o o o o 0 +4 o o +m 8:: o o +5 o o o +~ o +m o +m +m +m +4 8:: o o o o o o +~ o o +4 +4 +m +~ +4 2:: o o o o o o o o s o o o o o 8:: o o o o o o o o +~ o o o o o 32.2 o +5 +~ o +~ +N +~ +5 +m o +4 +4 i +4 82.2 o o o o o o +m o +~ o +m +4 ++ +4 BE: o o o o o o o o o o i o +~ +4 E82 o o o o o o 28.2 o o o o o o o o o o +m o o +4 882 o o o o o o o o o o i o o +m 88.2 o o o o o o 88.2 o o o o o o o o +4 0 +4 o o +4 88.2 o o o c o o i 0 +4 +5 +~ +5 +~ +4 8%: +5 o o o +m o o o o o +~ +~ +~ +m 35 o o +N o o o +m o +~ o +4 o o +4 spasm o o s o o o +~ o o o o o o +m 88 . o o o o o o 48m F945 2588 $4.: 2 M46 seems: $.45 E 3.3 3.4.? 3.45.... 5.4.3 3.4.3 55.45.550.238, §m<>> 900 2%; 900 cmmhowflcozmoEucmE :mmhom cozomamo meow—om 9.2505232 new 9.030300 05 “—0 3.302 32.3500 52.500 HN 2am... 33 combm 5.2505552 COOLUW COzomumD 62.5.83 4. some o o o o o o 8.2.2 o o o o o o o o o o +~ o o +~ ENE o o o o o o o o o o +N 5 +~ +m 4822 o o o o o o o o o o i o o +~ 8E2 o o o o o o o o o o +~ o o +~ RR: o o o o o o o o o o +4 o o +m 2R5. o o o o o o o o o o +~ o o +m 832 o o o o o o 8E2 o o o o i o o o o i o o +~ 22.2 o o o o +m +~ +~ o o o +5 i o BE). o o i o o o o i o +~ i +. +m 48:2 +m o o o +m o o o +~ +4 3 +~ +4 SR5. o o o o o o o o o +N 0 o +m ENE o o o o o o 8:: o o o o o o o o o .L o o +m 3:: o o o o o o 8:: +~ o o o +N o o o o o o o 5 8:: o o o o o o o o o +~ o o +m BE). 0 o o o o o o i +~ +~ +~ +m +m RE). o ++ +~ o o +~ o o +m +m +~ +m +m ES o o o o o o o o o +4 o i +m 2:: o o o o o i o o o o +m i +. 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OO crows. 543.5. m4m§2 m4mh§ hams—2 mmmtz 4Nmtz humus. swan—2 4322 +N +N +N +m mmnnfi +m +m +N amok). +_. 4. OONO +N 0000 GOOD +m COCO hams—2 mwmhz moms—2 305$. + + + + ooooNoNooooooooooNoFo +++ ++ + oooomvwooooooooowmo, + + + + oooomomooooooooooromo + + + + oooombmoooooooooomom Neon—2 +N +N +N 4. N + (‘0 4. N + (‘0 Emu—2 +m 00 O DO 4. 0') OO 0 00 4- ¢ 0 O omens. 00 O O O muons. +—. .1. +N 4. N + (V) O + N muons. +N 0 +N oioog‘oooogloo:ooooooooooog‘o O + (V) + N + (O humus. +m +N +m + N + ('0 + ('0 + N + ('0 much—2 +—. in +m + (”O + CO 4. V + ('0 + (‘0 much». OOOOO 0 00600 4. 1- OOOOO c 00000 O o o o o +N 3.022 lmim mam»: Mimi... 3 min gamma: n.m_4.w._. Him. figs EB combw cgmoEEmE 3.43 323 Emim. fir 5.4.Né 53.46... e|8 coeom :83qu 63558. ~ 22¢ s.m.4.m._.._. o. 238 37 38 o o o o o o o o o o o o o +u 8E5. o o +~ o o o o o o i o o o o $3.2 o o o o o o o o o o o o o +N 8%: o o o o o o 535. o o o o o o o o o o o o o +~ 8422 o o o o o o o o o o a o o +m 3%: o o o o o o 83.2 o o o o +~ o to, o o a a : +~ +N 5%: o o o c i o +m o o o +N 3 +m +4 843,. +4 o o o 14 o o o o o +m o o +m 2492 o o o o o o R322 o o o o o o o o o o o o o i 2422 o o o o o 0 E422 o o o o o o o o o o +~ o o +~ ate: o o o o o o o o o o .5 o o +m «222 o o o o o o {422 o o o o o o o o o o o o o a 8%: o o o o o o 83.2 o o o o +~ o o o o o o o o +m 832 o o o o o o 53.2 o o o o o o 83:2 0 o o o o o 83:2 o o o o o o 882 o o o o o o 83.2 +m o o o +m +~ o o o o i o o .5 8%: o o i o 5 o i o o o +~ o o +m 3%: o o o o o o 852 i o o o +~ o +~ o o o +m +~ +~ i. «was. +~ +N. i o +~ i! +~ o o : +~ i +N .5 55.2 hm 9.5%: 3i: 2 min 95%? mi: 2 at? $.13 $.qu him; $.43 5.9.4.3.. 9238 2m<>> So Ema; Boo coeom 8385.52 593 co=omfim €35.53 N 2.5 o o o o o 0 mmme o o o o o o o o o o o o o +N mNmmS— o o o o o o +N 0 o o +« o +P +N NNmmS. o o o o o o Ema—2 o o o o o o o o o o +N 0 o +4 owmmS. o o o o o o m rows. 0 o o o o o w 522 o o o o +N o o o o o +N 9 o +N n 592 o o o o o c o o o +—. o + _. +N +N o 522 o o +m o o o +m o +N +m +m +m +m +4 m 5&2 o o +N o o o o o +—. +N .3. o o +m 4 Ems. o o o o o o o o o o o o o b m Em.)— o o +N 0 o o +_. o +N +N +N +m +m +N N Sum—2 o o o o o o o o o o o o o +N r «mas. o o o o o o o o o o o o o +m o 522 o o o o o o o o o o o o o +_. monas— o o o o o o Roma—2 o o o o o o o o o o +N o o +N mome 0 o o o o 0 mome o o o o o o o o o o o o o +N Nome—2 o o o o o o comm.)— o o o o o o mm4w2 o o o o o o +m o o +N +w +_. +m +4 mm4w2 o o o o o o +N 0 o +N +m +F +m +4 .3492 +_. o o o +N 0 o o o o +N 0 o +N mm4m2 o o o o o o o o o o +N 0 o +m mm4w2 o o o o o o 4m4w2 o o o o o o mag—2 . 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Q oEEmw 2%; E 2%; Glob cmmbm 5:85.52 5.28 5.8200 €35.53 u can» 39 cmmzow cosmoEEmE 5mb 5:8qu 635.53 a use. +m o o o +~ o o o o o +m o o i. 33.2 o o +N 0 o o +m o i i to. +m +~ +m 882 o o o o o a 882 o o o o o o 382 o o o o o o o o o o i +~ o +~ 33.2 I +m +m +~ +m i 5 +~ o +~ +m i : +m 88.2 o o o o o o o o o o o o o i N82,. 0 o o o o o 582 +~ o o o o +~ o o o o +~ o o o 83.2 a o +~ o o o +~ o : +m i .5 +~ +m 33.2 o c o o o o o o +m o +m o o +m Rams. +m o +~ o +m o o o +N o +~ o o to, 8mm: 14 i i o +m o i o o o +~ : .. +m muons. +N 0 i o 1.. o o o i o +m .. o +~ «News. 0 o o o o o o o o o o o o i 882 o o o o o o o o o o +~ o o i is: o o o o o o o o o o i o o +~ 855. +~ o o o o o o o o o i i o +m 58.2 o o o o o o 3%: o o o o o o 1:. o o o o o o +~ $8.2 o o o o c o o o o o o o o +~ 882 o o o o i o i o o o i o o +4. 33.2 o o +~ o o o +~ o o o i o o i 332 o o +~ o o o +m o o o o +~ o o «892 o o o o o o £32 o o o o o o i o o o o i o +~ 83.2 o o i o o o o o o o +~ o o +~ 83.2 i o o o i i +~ o o o +~ o o +m $3.2. +~ on i o i i. +~ o o o a i o +~ $3.2 I35 5&2 3.4.: 2 min 3&2 Bi: 2 3.3 3.43 Sim: Nero... 3.4? 3.43.. 9233 335 300 2m<>> E 4O Cozummh 0095 II. b w__mo 4003 um. *0 £9.95: Ema—coo u I €35.83 u 2%» +N 0 +N o +m o +~ o i o i o o +m otON +m +N 3 o +m o +m o o o +N i o +m mamas. +m 3 +9 b +~ +N o o o o o o i o Ema—2 +N o o o +N +m i o o o o i o +m 3N2). +N o +~ o +m o +4 o o o i o o +~ 985. o o o o o o 8me +m o o o +m o o o o o +~ o o +m mags. +m +~ +m o +N 0 +~ o o o o o o +~ 3.52 o o o o o o o o o o i o o i 4392 +N o i o o o +~ o o o i o o +N 95.2 o o i o o o i o o o +m o o +m 95.2 o o o o o o +~ o o o o o o +N :5: +~ o o o o o +~ o o o b o o +~ 555. o o o o o o o o o o i o o o 88.2 +m +N i i +m +m o o o o o +9 o +~ mam: o o o o o o 4322 +~ o +~ o +m o +m o o o +m +~ o +m 882 +4 +m +m +m +4 +4 +m +m +N +N +m +~ +m +m mooms. +m o i o +m o +~ o o o o o o i 582 +N o o o +N 0 o o o o o o +~ o mooms. o o o o o o 88.2 +m o +m o +N o +m o +N 0 +N 3 o +m 48m: +N 0 +m o +m +~ +~ o o o +N i o +N 88.2 o o o o o o 882 +m o o o +m +~ o o o o o o o i 38.2 i o i o o o +m o o o +~ i o +m Sam—2 o oLr o o o on o o o o o +9 o o .8me Im.4.m 9:32 $4.: : m.4.m 026mm: $4.: 2 him; 3.4.3 n.m.4.m.F.F 5.4.2 3.4.2 5946.: QmEEmm 2m<>> 90 21k; nFOb cmmzom :2505500. cmmgow £23960 41 The reaction data were evaluated within these groups to detect patterns of reactivity. Within each group, the reactions were divided into categories of samples reacting with one, two, or three dogs, and were further evaluated based on the antigenic profile of the dog or dogs reacting. A total of 88 samples reacted with only one dog in the Detection Screen (Table 4). Seventy-seven samples reacted with the DEA 1.1,3,4,5,7 positive dog. Seven samples reacted with the DEA 1.2,4,7 positive dog and four samples reacted with the DEA 1.3,4,w7 positive dog. Table 4: Samples reacting with only one dog in the Detection Screen DEA Pattern Cold Warm Cold & Warm Total 1.1,3,4,5,7 29 5 43 77 1.2,4,7 4 3 1.3,4,w7 3 1 O 4 Total 36 9 43 88 Forty-eight samples reacted with a two dog pair in the Detection Screen (Table 5). Thirty-six samples reacted with the DEA 1.1,3,4,5,7 positive and DEA 1.2,4,7 positive dog pair. Ten samples reacted with the DEA 1.1,3,4,5,7 positive and DEA 1.3,4,w7 positive dog pair. Only 2 samples reacted with the DEA 1.2,4,7 positive and DEA 1.3,4,w7 positive dog pair. 42 Table 5: Samples reacting with a two dog pair in the Detection Screen DEA Pattern Cold Only Warm Only Cold 8. Warm Total 1:23:35” 8‘ 7 3 26 36 1:33:34?” 1 0 9 1° lzézfifl?‘ 0 1 2 Total 9 3 36 48 Forty-eight serum samples reacted with all three dogs in the Detection Screen (Table 6). Only two of the 48 samples reacted only at cold conditions. The other 46 samples reacted at both cold and warm conditions. Table 6: Samples reacting with all three dogs in the Detection Screen DEA Pattern Cold Only Warm Only Cold 8. Warm Total 1.1,3,4,5,7; 1.2,4,7 & 2 O 46 48 1.3,4,w7 Identification Screen A total of 184 serum samples were rescreened to identify the anti-DEA antibody detected in the Detection Screen (Table 7). Identification Screening was conducted using dogs with the antigenic profiles of DEA 1.1 positive, DEA 1.1,4,5,7 positive, DEA negative, and DEA 3,4,5 positive, as previously described. This screening resulted in 62 negative reactions (33.7%). Of the remaining 122 samples, a total of 84 samples (45.7%) showed positive reactions at both RT/4°C (cold) and 37°C/ACG (warm) conditions, 30 samples (16.3%) were positive only at cold conditions, and 8 samples (4.3%) were positive only at warm conditions. 43 Table 7: Summary of reaction conditions in the Identification Screen Reaction Pattern Number of Samples Percentage of Total (%) Negative 62 33.7% Positive cold & warm 84 45.7% Positive cold, 0 Negative warm 3O 16'3 /° Negative cold, 0 Positive warm 8 4'3 A Total 184 100% The reaction data were evaluated within these groups to detect patterns of reactivity. Within each group, the reactions were divided into categories of samples reacting with one, two, three, or four dogs, and were further evaluated based on the antigenic profile of the dog or dogs reacting. Dividing the Identification Screen data alone into categories based on these criteria did not provide useful patterns. Combined Results The results from the Detection Screen were combined with the results from the Identification Screen to give complete screening results. A total of 81 samples (30.6%) were completely negative (Table 8). Of the remaining 184 samples, 143 samples (54.0%) reacted at both warm and cold conditions. Thirty- four samples (12.8%) reacted only at cold conditions, and 7 samples (2.6%) reacted only at warm conditions. 44 Table 8: Summary of reaction conditions for combined results of Detection and Identification Screens Reaction Pattern Number of Samples Percentage of Total (%) Negative 31 30.6% Positive cold & warm 143 54.0% Positive cold, 0 Negative warm 34 12.8 A Positive warm, 0 Negative cold 7 2-6 A: Total 265 100% The combined positive reaction patterns of the Detection and Identification screens are summarized in Table 9. 45 som N N + o o o + o o Sam F F + o o o o + o m 50.4% $.34.on 4F + o o o o o + 4.8 F F o + o o + + + o o o + o o + + o o o o + o o + o + o o o + o o o + + o o o + o o + o o o o o + o o o + o o o o + o o o o + .N $924 gem ON ON 0 o + o + + + £2 F F o o + o + + o 52 N N o o + o + o + .N $9-45 58 N 28 F n o o + o o + + Em; F F o o + o + o o 50m F F o o + o o + 0 58 m .28 m .w o o + o o o + o o o o o + + + + o o o o o + + + o o o o o o + + o + o o o o o + o + + o o o o o + + o o o o o o o + o + o o o o o o + o o + :°_“_u=°o Chwuml n n h n n n I n n o u n I n n n n a £2 52. .588 9:25:34 m 4 N 9:482 N m 4 F F F F N; 4 N. F N 4 N F N m 4 N F F Nuorscw .2. measwm $3.5m a <3 <3 <3 <3 <3 <3 <3 No cozsntumfi ho 53:52 “5.631 m eon mcoeNom cosmoEEoE can cozomuoo as“ _o 3.33.. uoEnEoo .2 336503.... because EEom 3:229: B 4822 we mmou @5523 No 9:033 :ozomom ”a 2an 46 58 F F o + + o o o + u o + o o + + + + u o + o o + + + o u o + o o + + o + o o + o o + + o o o o + o o + o + o o o + o o + o o + o o o + o + + + + o o o + o + + + o o o o + o + + o + o o o + o + o + + o o o + o + + o o o o o + o + o + o o o o + o + o o + o o o o + + + + + o o o o + + + + o o o o o + + + o + o o o o + + o + + o o o o + + + o o o o o o + + o + o F.F $0;ch o o o o + + o o + 50m F F + o o o + + + o o + o o o + + 0 50m F F + o o o + o + 50m o o + o o o o + + C°_U_Ucoo Chwzfim N N m N N N . . N N . N N . N N N N . >22 32. 20:03”. mczubmcoEwo n F. n 023 02 N. m c F F F F N3 F. n F N. F. N F N m w n F F >qunzc=umoz N n F. F F F F N44F F. n F N v N F N n v n F.F @3554 Non mmEEam maEEum (mo > .NEo Dov 8ch .239an2 E09 .cam QmfimEE Fm 86mm: 8.953 u 200 . N «@04ch No 55 32 w No 8:963 9: E cozome gamma: m 9 ENE NmE N $5 No 868.98 xmoz NE... . N mow w F0 5622: 9: 9 man N «$04ch .2 8829.8 2:0qu 2238 92 EN 99:; 50m N o 50m 0 .200 F 50m m .200 N n. F 504ch o O [\NON O 0000 O 0000 O 0000 O 0000 + o+++ o +o++ o ++o+ N. F > N .200 m 50m mF .FEm>> v 6.00 NF 0 O O O O O + 50m m 200 F o 50m m ++++ ++++ o+++ +o++ ++o+ o OLDOFC‘O + +++++ + o o E0588 Nuonzi :oEucoo cozoawm N2. moiEam F0 .8339me Eczam mczahmcofiun mcEEmw No Nonfiaz min (mo w>=amoz 283 FEB moo N... No co_w:_oc_ 9: 2 26 N «£0;ch .8 852.068 9:228 2938 05 «to 99F. o\oooF mmN ._uon==< No Nanzz mczomom «moo mchoNom cozooaon macaw moEoEoonm >U0£Ea E33 .3229: o. 35.2 mm mmou 05528 No 2.333 cozowom 5F 03oF 53 As a result, anti-DEA 7 may be detected by the DEA 1.1,3,4,5,7 positive and DEA 1.2,4,7 positive pair or with all three Detection Screen dogs. Forty-eight samples (16.6%) reacted with all three Detection Screen dogs and were suspected of anti-DEA 4 or anti-DEA 7 because the common antigens among the three dogs were DEA 4 and 7. A total of 12 samples (4.4%) reacted with either the DEA 1.1,3,4,5,7 positive and DEA1.3,4,w7 positive pair or the DEA 1.2,4,7 positive and DEA 1.3,4,w7 positive pair, and were suspected of containing an unknown antibody because there were no known DEAs that were common to the dogs in each pair. An unknown antibody, a false positive, or a false negative for a different reaction pattern may be a consideration in each of the reaction patterns previously mentioned. Data were evaluated for the Identification Screen alone and no patterns of significance were detected. However, sixty-two samples (33.7%) failed to react with any of the Identification Screen dogs. The Identification Screen panel of dogs did not represent DEA 1.2 or DEA 1.3, suggesting that the non-reactive samples may contain anti-DEA 1.2 or anti-DEA 1.3. Naturally-occurring antibodies against DEA 1.2 and DEA 1.3 have not been previously reported, however, these antigens have been reported to induce antibody production in dogs negative for the corresponding antigen.33 Since the DEA exposure histories of the dogs for which serum samples were submitted are unknown, anti-DEA 1.2 and anti-DEA 1.3 may be possibilities. It must be noted, however, that these two antigens have p-values greater than 0.05, and therefore lie outside the 95% confidence interval. 54 Combined results from the Detection Screen and Identification Screen show a total of 81 samples (30.6%) as being negative for anti-DEA activity, which is based on the results from the Detection Screen. None of the samples reacted with the DEA 1.1,3,4,5,7 positive, DEA 1.1 positive, and DEA 1.1,4,5,7 positive dog group, suggesting that there were no anti-DEA 1.1 antibodies present in the samples as these dogs are the only DEA 1.1 positive dogs in the study (Table 9, Table 11). Anti-DEA 1.1 has not been reported to be a naturally-occurring Table 11: Prevalence of Anti-DEA Antibodies , 0 Previously Antibody Prevalence (/o) Described Anti-DEA 1 .1 0% no Anti-DEA 1.2 1.9% no Anti-DEA 1.3 0% no Anti-DEA 3 5.3% yes Anti-DEA 4 unlikely no Anti-DEA 5 3% yes Anti-DEA 7 8.7% yes Multiple Antibodies * * Unknown Antibodies Up to 45.3% no " An accurate prevalence of multiple antibody combinations could not be determined. Previous studies have not described the prevalence of multiple antibody combinations. antibody, but it can be induced upon exposure of a DEA 1.1-negative dog to DEA 1.1. Five samples (1 .9%) reacted with the DEA 1.2,4,7 positive dog alone and are suspected of containing anti-DEA 1.2. Anti-DEA 1.2 has not been reported to occur naturally, but agglutinating antibody can be induced in a DEA 1.2-negative dog after exposure to DEA 1.2.24'34 The serum samples are from dogs of unknown exposure history, so it is possible that an acquired anti-DEA 1.2 is present in this population. The red blood cell panel did not meet the required p- 55 value of 0.05 or less for DEA 1.2 needed for proper antibody determination, so there is a greater than 1 in 20 chance that the pattern for anti-DEA 1.2 may be another specificity.11 No samples reacted with the DEA 1.3,4,w7 positive dog alone, suggesting that there were no anti-DEA 1.3 antibodies present in the samples. Anti-DEA 1.3 has not been indicated as a naturally-occurring antibody. Antibody formation has been documented in a DEA 1.3-negative dog following exposure to DEA 1.3, although the antibody described was reactive with DEA 1.1 and DEA 1.2.33 Fourteen samples (5.3%) reacted with the DEA 1.1,3,4,5,7 positive and DEA 3,4,5 positive dog pair and are suspected of containing anti- DEA 3 or an unknown antibody. This supports previous studies in which anti- DEA 3 has been reported to occur naturally.12'15 Fourteen samples (5.3%) reacted with the DEA 1.1,3,4,5,7 positive, DEA 1.2,4,7 positive, DEA 1.3,4,w7 positive, DEA 1.1,4,5,7 positive, and DEA 3,4,5 positive dog group and are suspected of containing anti-DEA 4, an unknown antibody, or a combination of antibodies. Anti-DEA 4 has not been reported as a naturally-occurring antibody and is not likely to be seen frequently in the population since 98% of dogs express DEA 4. Eight samples (3%) reacted with the DEA 1.1,3,4,5,7 positive, DEA 1.1,4,5,7 positive, and DEA 3,4,5 positive dog group and are suspected of containing anti-DEA 5 since they are the only samples that contain DEA 5. Anti- DEA 5 has previously been reported as a naturally-occurring antibody. Two groupings of reaction patterns were suspected of containing anti-DEA 7 due to the weak expression of DEA 7 in the DEA 1.3,4,w7 positive dog — the DEA 1.1,3,4,5,7 positive, DEA 1.2,4,7 positive, and DEA 1.1.4.5,7 positive dog group 56 and the DEA 1.1,3,4,5,7 positive, DEA 1.2,4,7 positive, DEA 1.3,4,w7 positive, and DEA 1.1,4,5,7 positive dog group. The DEA 1.1,3,4,5,7 positive, DEA 1.2,4,7 positive, and DEA 1.1,4,5,7 positive dog group reacted with 3 samples (1.1%) and the DEA 1.1,3,4,5,7 positive, DEA 1.2,4,7 positive, DEA 1.3,4,w7 positive, and DEA 1.1,4,5,7 positive dog group reacted with 20 samples (7.5%), giving a total of 23 samples (8.7%) that are suspected of containing anti-DEA 7. Anti-DEA 7 has been reported to be naturally-occurring in some DEA 7-negative dogs. A total of 120 samples (45.3%) reacted but did not show activity specific to any known individual anti-DEA antibody. Any of the reaction patterns may also represent antibodies to unknown DEAs. Sixty-two samples (23%) that reacted during the Detection Screen did not demonstrate reactions in the Identification Screen. Of these 62 samples, 24 samples (38.7%) were only reactive at cold conditions, indicating a potential cold agglutinin. Six samples (9.7%) were only reactive at warm conditions, and 32 samples (51.6%) were reactive for both cold and warm conditions. Of the 62 samples, 41 samples (66.1%) were reactive with the DEA 1.1,3,4,5,7 positive dog only, which suggests the dog may express an unknown DEA. Five samples (8%) reacted with the DEA 1.2,4,7 positive dog only, suggesting another potential unknown DEA. When considering the presence of multiple anti-DEA antibodies, sixteen samples of the 120 samples with unknown specificities may be explained (Table 57 12). The multiple antibody combination of anti-DEA 1.2.3 or anti-DEA 1.3.3 explains 7 samples. The remaining 9 samples have numerous possible multiple antibody combinations: anti-DEA 3.7; anti-DEA 5,7; anti-DEA 3,5,7; anti-DEA 1.2.3.5; anti-DEA 1.2.3.7; anti-DEA 1.2.5.7; anti-DEA 1.2.3.5,7 (Table 12). Four patterns previously described for individual antibodies anti-DEA 4, anti-DEA 5, and anti-DEA 7 also correspond to multiple antibody patterns. The pattern for the individual anti-DEA 5 antibody may also represent the combination of anti- DEA 3,5. The two patterns for anti-DEA 7 may also correspond to anti-DEA 1.2.7 and anti-DEA 1.3.7. Although anti-DEA 4 is included in the possible specificities for both individual and multiple antibodies, it is not likely to be demonstrated frequently as an individual antibody as indicated in Table 11 due to the high prevalence of DEA 4 (98% positive). A more likely explanation for the pattern corresponding to the individual anti-DEA 4 specificity is the presence of multiple antibodies. There are numerous possible multiple antibody combinations corresponding to the pattern for anti-DEA 4: anti-DEA 3,7; anti- DEA 5,7; anti-DEA 3,5,7; anti-DEA 1.2.3.7; anti-DEA 1.2.5.7; anti-DEA 1.3.3.7; anti-DEA 1.3.5.7; anti-DEA 1.3.3.5,7; or any of these combinations including anti- DEA 4. When the results of the Detection Screen and Identification Screen were combined. the percentage of samples that reacted at both cold and warm conditions increased, while the percentage of samples reacting only at cold conditions or only at warm conditions decreased (Table 13). 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