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The dog erythrocyte antigen 1 blood group in nondomesticated canids and compatibility testing between domestic dog and nondomesticated canid blood

Abstract Background The dog erythrocyte antigen (DEA) 1 blood group is considered as the most immunogenic and clinically important in dogs. Little is known in nondomesticated canids. Objectives To type DEA 1 in nondomesticated captive canids and to evaluate potential interspecific blood transfusions between domestic and nondomestic canids. Animals One hundred forty captive nondomesticated canids belonging to 13 species from 19 French zoos, and 63 domestic dogs. Methods Prospective study. Blood samples were typed for DEA 1 using immunochromatographic and flow cytometric techniques. A neutral gel column test was used for crossmatching. Results Of 140 nondomesticated canids, 72.9% were DEA 1+ and 27.1% were DEA 1− using immunochromatographic technique and 74.3% were DEA 1+ and 25.7% were DEA 1− by flow cytometric technique. Crossmatch (XM) between 140 nondomesticated canid red blood cells (RBCs) and plasma from a previously DEA 1+ sensitized DEA 1− dog revealed 112 incompatibilities (80%). Crossmatches between 130 nondomesticated canid serum and 1 or up to 8 donor dogs' RBCs revealed 99 of 130 (76%) compatibilities. Crossmatches between 115 nondomesticated canid RBCs and donor dogs' serum revealed 59 of 115 (51%) compatibilities. Conclusions and Clinical Importance Dog erythrocyte antigen 1 blood type is present in nondomesticated canids with variable prevalence depending on species. The majority of tested nondomesticated canids appear to have no naturally occurring alloantibodies against domestic dogs' RBCs. Therefore xenotransfusion of blood from domestic dogs can be considered when species specific blood is not available. Cross matching is essential before xenotransfusion.

The crossmatch (XM) technique is used to determine donorrecipient compatibility before blood transfusion. Based upon extensive clinical experience, dogs have no 6,7 or no clinically important naturally occurring alloantibodies, as anti-DEA 7 antibodies that have been recently described. 8,9 Crossmatching before a first transfusion is not systematically required in dogs and is recommended when transfusion history is unknown, when a transfusion has been performed more than 4 days before or in a case of hemolytic reaction consecutive to a previous transfusion sensitization. Even when DEA 1-matched blood is used for transfusion, some XM incompatibilities have been found 26 days after transfusion. 6 Based on this observation, alloantibodies against blood groups other than DEA 1 are produced; therefore, XM must be performed after a first blood transfusion even when DEA 1 blood-typing was performed.
Xenotransfusion, that is, transfusion of blood from 1 species to another species, might be used only when an intraspecies donor is unavailable. 10,11 Although ethically questionable, this practice is occasionally used in cats with dog blood. Xenotransfusion from domestic dog to nondomesticated canids have only been reported twice. 12,13 Canidae is a family of carnivore mammals composed of 35 species; including wolf (Canis lupus) from whom domestic dog (Canis lupus familiaris) is a descendant. Based on phylogenetic studies, 4 clades have been discovered: "related to wolf," "related to fox," "South-American canids," and "2 species of Urocyon gender." 14 At this time, little is known about blood groups in nondomesticated canids and their incidence in incompatible transfusion.
The purpose of this prospective study was to assess the blood group DEA 1 for the first time in nondomesticated canids by using 2 technics of blood typing which are well known in domestic dogs 15 : immunochromatographic strip and flow cytometry. Thereafter, we also aimed to determine DEA 1+ and DEA 1− distribution depending on the species. The second objective was to investigate blood compatibilities between domestic dogs and nondomesticated canids and to determine if domestic canine blood could be used for xenotransfusion in nondomesticated canids when homologous blood is not available.
For each specimen, approximately 3 mL of blood was collected into both ethylenediaminetetraacetic acid (EDTA) and dry tube. Dry tube was centrifuged to separate serum from red blood cells (RBC) and both tubes were sent to Dianov laboratories. Samples were preserved at 4 C and analyzed less than 1 week after sampling. 16 Crossmatches between 2 animals were realized when the samples from these animals were treated in the same week.
Dog blood samples used for XM tests were collected during blood collection by the SIAMU (Intensive Care Unit, VetAgro Sup Veterinary campus) blood bank donors. Sixty-three dogs were collected. These dogs were healthy and had not received any previous blood transfusion. This prospective study was approved by the Ethical Committee of VetAgro Sup (#1908).

| Laboratory methods
For each canid, aliquots of EDTA-anticoagulated whole blood samples were used for DEA 1 blood typing by immunochromatographic strip kit (Canine QuickTest DEA 1, Alvedia, Limonest, France) and the remaining blood was centrifuged at 1000g for 10 minutes to collect packed RBCs (pRBCs) for flow cytometric DEA 1 typing and stored at 4 C for XM testing within a week. Serum was separated on dry tubes and kept frozen at −20 C in a microtube between 1 week to a month for afterward XM testing.

| Gel column crossmatch without antiglobulin
Crossmatch tests were performed and interpreted according to the manufacturer's instructions (ID-Cards NaCl, Enzyme Test and Cold Agglutinins, Bio-Rad, DiaMed GmbH, Cressier, Switzerland) and as previously described. 6 In a 3 mL polystyrene test tube, 50 μL of 1% donor pRBCs in low ionic strength solution (Bio-Rad, DiaMed GmbH) were added at the top of the gel card column with 25 μL of recipient serum, briefly mixed, and incubated at 22 C for 10 minutes. After incubation, the gel column (GC) cards were centrifuged in a special GC centrifuge (ID Centrifuge 6S, Bio-Rad, DiaMed GmbH) at 85g for 10 minutes, and the location of the migrated RBCs was recorded. In the absence of agglutination, the RBC passed through the gel to the bottom which was scored as "compatible," whereas agglutination on the top of or within the gel was considered "incompatible." Autocontrols (using RBCs and plasma from same canid) were included for all XM tests performed.
Four varieties of XM assays have been performed in order to confirm that anti-DEA 1 alloantibodies were the same in wild canids and in an immunized dog. For 1 sort, the plasma of a DEA 1− dog previously transfused with DEA 1+ blood, consequently possessing anti-DEA 1 alloantibodies (sensitized dog plasma = SD plasma, thereafter) 17 was tested with nondomesticated canids' pRBCs. One sort of XM assays was between nondomesticated canids' serum and dogs' pRBCs (major XM). The third one was between nondomesticated canids' pRBCs and dogs' plasma (minor XM). And the last one was between nondomesticated canids' pRBCs and other nondomesticated canids' serum.

| Statistical analysis
Descriptive data are presented as average, range, and percentage. The blood typing test results were compared using McNemar test. The statistical analyses were performed using a commercially available statistical program (R, Saint-Louis, Missouri), and a P ≤ .05 was considered significant.

| DEA 1 typing results
For the immunochromatographic method, control bands were always present. The control band intensity was weak for some individuals:

| Autocontrol test results
There was no autoagglutination observed in any auto-control tests when crossmatching serum and pRBCs from the same canid.

| Crossmatch between nondomesticated canids' pRBCs and nondomesticated canids' serum
between flow cytometry and immunochromatographic strip techniques. 6,15 12,13 An island fox (Urocyon littoralis clementae) was bitten by a rattlesnake, causing anemia and a severe thrombocytopenia. A xenotransfusion using domestic dog whole blood was performed. No transfusion reactions were observed and the fox fully recovered. 12 An arctic fox (V lagopus) presented an immune-mediated hemolytic anemia. Xenotransfusion with pRBCs from a domestic dog was successfully used twice 24 hours apart. Pretransfusion major XM was compatible but 6 days posttransfusion, major XM was incompatible. 13 Moreover, our results added to precedent reports 10,13 argue to the use of future xenotransfusion is risky because of alloantibodies induction leading to severe hemolytic reactions thus, when xenotransfusion is considered, XM is essential.