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Alloimmunization of a dog erythrocyte antigen 1− dog transfused with weakly dog erythrocyte antigen 1+ blood

Abstract Background Acute hemolytic transfusion reactions because of dog erythrocyte antigen (DEA) 1 sensitization after mismatched transfusions are serious complications. Dog erythrocyte antigen 1 expression varies from negative to weakly to strongly positive. Objectives To assess alloimmunization after transfusion of weakly DEA 1+ blood to a DEA 1− dog. Animals One DEA 1− recipient and 1 weakly DEA 1+ donor, and 106 control dogs. Methods Long‐term follow‐up study. Matched for DEA 3, 4, 5, and 7, Dal, and Kai 1 and 2, weakly DEA 1+ donor packed red blood cells (RBCs) were transfused 3 times (0.45 mL/kg at Day 0, 16, and 37) to a DEA 1− recipient. Alloantibodies against RBCs from donor and 106 controls were determined in recipient's plasma samples using a commercial antiglobulin‐enhanced immunochromatographic strip and gel tube crossmatches. Alloantibody titers were determined. Results The DEA 1− recipient was sensitized after 16 days to ≥1657 days after transfusion to weakly DEA 1+ and otherwise matched RBCs. Strong to moderate crossmatch incompatibilities were observed between recipient's plasma and all 61 DEA 1+ crossmatched controls. Moderate to weak incompatibilities were also observed to DEA 1− controls. Anti‐DEA 1 and other alloantibodies were detected over the 4.5 year observation period. Conclusions and Clinical Importance Blood from a weakly DEA 1+ donor induces a strong and durable alloimmunization in a DEA 1− recipient dog. Additional alloantibodies developed against yet to be defined RBC antigens. Those results support the recommendation of typing dogs against DEA 1, considering weakly DEA 1+ as immunogenic, and crossmatching all previously transfused dogs.


| INTRODUCTION
More than a dozen blood groups have been reported in dogs and some have been classified as dog erythrocyte antigens (DEAs), but polyclonal (antisera) and/or monoclonal alloantibodies (typing reagents) are only available for some. [1][2][3][4] Based upon extensive clinical experience, we found that dogs do not have any clinically important naturally occurring alloantibodies, 5,6 albeit anti-DEA 7 has been described. 7,8 Experimental investigations and a few case studies revealed that dogs can be sensitized by mismatched blood transfusions and they develop acute hemolytic transfusion reactions, if transfused again >4 days from the same or other donors. 9,10 The DEA 1 is considered clinically the most important blood group in dogs because of its strong antigenicity and nearly equal distribution of DEA 1+ and DEA 1− dogs among many breeds worldwide. 11 Based upon our recent surveys using quantitative immunochromatographic strip methods, most dogs are either DEA 1− or moderately to strongly DEA 1+. 12,13 However, few dogs are only weakly DEA 1+, and these dogs might be mistyped as DEA 1−. Although DEA 1 mismatched transfusions have been reported to cause anti-DEA 1 alloantibodies and acute hemolytic transfusion reactions, the antigenicity of weakly DEA 1+ blood transfused to a DEA 1− dog is unknown.
In this study, we investigated the course of alloimmunization of a DEA 1− recipient dog by transfusing weakly DEA 1+ blood and an otherwise matched donor and recipient pair utilizing antiglobulinenhanced immunochromatographic strip and gel tube crossmatch kits.

| MATERIALS AND METHODS
2.1 | Animals, immunization, and blood sample collection and preparation A 6-year-old healthy DEA 1− Border Collie (Recipient, 22 kg body weight), a 2-year-old weakly DEA 1+ Appenzell Mountain blood donor dog (Donor, weighing 25 kg), and 106 healthy control blood donors (Controls, mixed breed and purebred dogs), owned by veterinary students and hospital staff, were used in this study over a 4.5-year period from 2013 to 2018. All dogs were considered healthy at time of blood collections based on annual physical examination, CBCs, and infectious disease screen. The study was approved by the Institutional Animal Care and Use and Ethics Committee of VetAgro Sup (#1267), and owner consent was obtained before Recipient and Donor enrollment into the study and blood collection.

| Alloimmunization schedule
Based upon extended blood group results, a Recipient-Donor pair was selected. During October and November 2013, 40 mL of Donor blood was collected into a 60-mL syringe containing acid citrate dextrose (ACD) solution from Blood Pack Units (Macopharma, Tourcoing, France) on 3 separate occasions. The anticoagulated blood was immediately centrifuged, the plasma was separated and frozen, and the packed red blood cells (RBCs) were washed 3 times in sterile phosphate-buffered saline (PBS). 14 In the intensive care unit under close medical supervision, 10 mL (0.45 mL/kg) of freshly washed Donor packed RBCs were transfused to Recipient via saphenous vein on Days 0, 16, and 37 ( Figure 1).
Temperature, pulse, respiration, and attitude of the Recipient were regularly assessed during and after transfusion, although signs of hemolysis or hyperbilirubinemia were not followed specifically.

| Diagnostic blood sample collections
Diagnostic blood samples were collected from the jugular or saphenous veins from all dogs into 6-mL tubes containing ACD (ACD Solution B, Becton Dickinson, Plymouth, the United Kingdom). These samples were chilled and within 1 week, they were either directly used or at that time washed 3 times in PBS for blood typing. The ACD tubes with the remaining blood were centrifuged (at 1300g for 10 minutes) on the collection day to separate plasma and RBCs for crossmatch testing and alloantibody titer determinations. The RBCs were stored at 4 C for <10 days, whereas plasma samples were frozen at −20 C until used for crossmatch tests.

| Alloantibody titration in recipient's plasma
Titer measurements for alloantibodies in Recipient's plasma from Day 106 to Day 1657 were obtained with 2-fold serial dilutions of Recipient's plasma in PBS mixed with only Donor RBCs utilizing the AGC test as described above.

| Anti-DEA 1 alloantibodies specificity in adsorption study
One milliliter of Recipient's plasma from Day 106 was separately incubated overnight at 37 C under 2 conditions with 1 mL of either strongly DEA 1+ Control or a DEA 1− Control washed RBCs. Both suspensions were then centrifuged for 10 minutes at 2000g. The resulting supernatants were again incubated as described above. 13,20 These supernatants from adsorbed plasma were crossmatched using only the AIC technique against the same DEA 1+ Control RBCs.

| Blood typing
The categorical and the semiquantitative and quantitative DEA 1 blood typing results were completely concordant between the flow cytometric and immunochromatographic strip typing techniques for Recipient, Donor, and all 106 Controls ( Figure 2 and Table 1 Table 2).

| Alloantibody titration in recipient's plasma
The Recipient's plasma alloantibody titers measured from Day 106 to Note: Results were recorded as follows: −, negative; +, positive.

| Anti-DEA 1 alloantibodies specificity in adsorption study
The presence of anti-DEA 1 alloantibodies in Recipient's plasma was assessed by crossmatching DEA 1+ Control RBCs against adsorbed plasma with the AIC technique. When previously adsorbed with DEA 1 − RBCs, the plasma from the DEA 1− Recipient showed the same variation of incompatibility against DEA 1+ Control RBCs (strip 3, Figure 6) as when not adsorbed (strip 2, Figure 6). In contrast, when previously adsorbed against DEA 1+ RBCs, Recipient's plasma became compatible with same DEA 1+ Control RBCs (strip 4, Figure 6).

| DISCUSSION
Among the known canine blood group systems, DEA 1 is considered clinically most important. However, little is known about structure or antigenicity of weakly to strongly DEA 1+ erythrocytes and onset and duration of sensitization with DEA 1+ blood. 17 In this transfusion study, we document strong and durable alloimmunization of a DEA 1 − recipient dog with blood from a weakly DEA 1+ and otherwise blood type matched donor dog after 16 days lasting for at least 4.5 years. Furthermore, alloantibodies against yet to be determined erythrocyte antigens were also induced, which appeared less strong and lasted less long. This study supports careful matching of DEA 1− recipients with DEA 1− blood and crossmatching of previously transfused dogs, whereas extended typing for other blood types for which blood typing reagents and/or tests are available will not be helpful.
Since the early experimental studies in the 1960s, it was recognized that DEA 1.1 was antigenic resulting in anti-DEA 1 alloantibody induction and acute hemolytic transfusion reaction when dogs were again transfused. 17,21 Furthermore, at least 1 clinical report documented sensitization of a DEA 1.1− dog with DEA 1.1+ blood. 11 The introduction of monoclonal anti-DEA 1 antibodies enabled the redefining of the DEA 1 blood group system with DEA 1− and weakly to strongly DEA 1 + blood types. 12 The degree of DEA 1 positivity was shown previously to remain stable overtime in a particular dog 13 and as also seen here with the weakly DEA 1+ Donor. In our previously published crossmatch survey, an accident mismatch of a DEA 1− recipient patient receiving a transfusion with strongly DEA 1+ blood became sensitized. 5 Here, we document that even weakly DEA 1+ blood transfused to a DEA 1− recipient induces anti-DEA 1 alloantibodies. Thus, weak DEA 1 typing

F I G U R E 3 Major crossmatch incompatibilities between Recipient
and Donor after transfusion of weakly DEA 1+ blood to a DEA 1− dog using an antiglobulin-enhanced immunochromatographic strip crossmatch (AIC) and an antiglobulin-enhanced gel tube crossmatch (AGC) technique. Antiglobulin-enhanced immunochromatographic crossmatch test has a band with a control antibody on the strip (labeled "C") specific to all canine red blood cells (RBCs) (thus, a red band at "C" indicates that the test is valid   Antiglobulin-enhanced immunochromatographic crossmatch tests results were recorded similarly as described in Figure 3 if the Recipient was only transfused once. We did not differentiate titers between IgG and IgM, but it would have been interesting to further characterize which class of induced Igs arose and disappeared in the Recipient's plasma. Finally, although it is anticipated that a DEA 1-related acute hemolytic transfusion reaction would occur, we do not know if the additional alloantibodies would be in vivo active and cause an acute hemolytic transfusion reaction.

| CONCLUSION
Dog erythrocyte antigen 1 typing before any transfusion and selection of DEA 1 matched or DEA 1− donors is recommended to prevent alloimmunization. 21 Weakly DEA 1+ blood is strongly immunogenic and thus weakly DEA 1+ donors should be classified as DEA 1+ dogs in any donor program. 12 The high rate of alloimmunization after transfusion outside the DEA 1 blood group system observed here and previously warrants major crossmatch testing in any dog that previously received RBC products >4 days before the next transfusion. 23,24