Antiphospholipid syndrome (APS) is a well-characterized syndrome in people, consisting of clinical signs of thrombosis, pregnancy-related complications, and thrombocytopenia. The international classification criteria for definite APS involve clinical findings along with the laboratory evidence of presence of antiphospholipid antibodies, which among others include the detection of lupus anticoagulants and anticardiolipin (aCL) antibodies. Lupus anticoagulants are circulating blood coagulation inhibitors or antibodies directed mainly but not exclusively against phospholipid-binding plasma proteins. The anticoagulant effect of lupus anticoagulants is strictly an in vitro phenomenon identified in a functional coagulation assay. The detection of aCL antibodies is a sensitive but not specific test for APS, and is quantified by ELISA. Increased concentrations of aCL antibodies can also be detected in people with infectious disease, immune-mediated conditions, neoplastic disorders, and in healthy individuals.[4, 5] People with APS are not necessarily positive for both lupus anticoagulant and aCL antibodies, and studies have shown that patients positive for lupus anticoagulants are more likely to experience thrombotic incidents than people only positive for aCL antibodies. However, removal of aCL antibodies from the diagnostic criteria has resulted in failure to diagnose APS in a subset of patients, which lead to the inclusion of both parameters in the diagnostic criteria for APS. Precise laboratory diagnostic guidelines from the Scientific subcommittee of the International Society of Thrombosis and Hemostasis (ISTH/SCC) have been proposed to diagnose the presence of lupus anticoagulants.[8, 9]
Although the presence of aCL antibodies has been reported in both healthy and diseased dogs in a single study, it remains unknown if these dogs had normal or abnormal hemostasis in the form of clinically detectable bleeding, hemorrhagic diathesis, or laboratory evidence of prolonged coagulation tests. Only a single case report has suggested the presence of the APS in the dog. Consequently, the importance of the APS, lupus anticoagulants, and aCL antibodies in dogs remains unknown.
Previously, we have found breed specific differences in hematologic serum and biochemical and hemostatic analytes in adult healthy Bernese Mountain Dogs. The thromboelastographic (TEG) parameter MA had a moderately higher de novo generated reference interval, which in most other dogs would indicate platelet or fibrinogen associated hypercoagulability, but most strikingly, we found that activated partial thromboplastin time (aPTT) was markedly prolonged with a new interval of 6–100 seconds, whereas the remaining parameters of hemostasis (fibrinogen, d-dimers, prothrombin time, and the TEG evaluation [R, K, angle]) were within standard reference limits. In this study, none of the dogs had clinical evidence of coagulopathy. Single coagulation factor analyses performed for FVIII, FIX, FXI, FXII were within reference range for all samples, and therefore no apparent cause of the prolonged aPTT in these dogs could be detected. In people, an isolated prolonged aPTT without evidence of bleeding should be fully investigated, as it could be because of a thrombophilic condition caused by antiphospholipid antibodies rather than an indication of bleeding. The objective of this study was to investigate if these healthy Bernese Mountain Dogs with prolonged aPTT met the laboratory classification criteria for APS by (1) examining the dogs for lupus anticoagulants by following the human ISTH/SCC guidelines, and (2) identification of aCL antibodies in the dogs.
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- Material and Methods
In this study, the aPTT synthASil tests were within reference range at both visits for all Bernese Mountain Dogs, indicating that contact factor deficiency was not the cause of the prolonged aPTT-SP. Little attention has been made to antiphospholipid antibodies in dogs, and the diversity of these antibodies and their individual significance therefore remain unknown.
Healthy people can have aCL antibodies or lupus anticoagulant and remain asymptomatic. Lupus anticoagulant is more prevalent in healthy young females. The majority of dogs in this study were females. Although thrombotic events are rare in dogs, they have been documented and include stroke, pulmonary thromboembolisms, and deep vein thrombosis.[20-22] In future studies, it may therefore be of use to test dogs with these types of diseases for the presence of antiphospholipid antibodies. Although all dogs in the study had prolonged aPTT-SP, only 68% of the dogs were identified as having lupus anticoagulants in the Bernese Mountain Dog and healthy donor mixing study. According to the human studies, a more pronounced relative correction than 70% suggests a factor deficiency, and a less pronounced relative correction suggests the presence of an inhibitor. Single factor analysis and TEG were previously performed in the dogs in this study not identifying factor dysfunction, and the aPTT synthAFax was within the reference interval for all dogs. In humans, it has previously been suggested that low concentration of lupus anticoagulants can remain undetected with a 1 : 1 portion patient and healthy donor mixing study, or slow binding of the proteins is not detected in an immediately run sample. Some studies therefore suggest a 4 : 1 patient healthy donor mixing study or a time-delayed aPTT measurement. Currently, the true lupus anticoagulant concentration of the remaining 7 dogs is therefore not clear, in particular, because these dogs had abnormal aCL IgG antibody concentration. In people, apart from using the aPTT test, the presence of lupus anticoagulants can be evaluated by performing the screening and confirmatory diluted Russell's Viper Venom tests with various concentrations of phospholipid. Presently, however, these tests have not been validated in the dog.
High levels of aCL antibodies are involved with thrombosis and recurrent pregnancy loss, and can also be present in human cancer patients.[24-28] The dogs neither had clinical signs of thrombosis, nor were they pregnant at the time of blood sampling or there were indication of increased pregnancy morbidity in this small population of dogs. Although the extensive examination protocol of the dogs did not detect tumors, occult neoplasia could not fully be excluded. Bernese Mountain Dogs are prone to disseminated histiocytic sarcoma, which is a neoplasia associated with the immune system,[29-31] and it is possible that levels of aCL antibodies exceeding the range of the control dogs without reason represent a dysfunctional immune system, which previously has been suggested in this breed. Presently, however, aCL antibodies should be evaluated in a greater number of Bernese Moutain Dogs to determine if this is a generalized breed trait or localized to a particular geographic area. The aCL antibody test was semi-quantitative. Although a quantitative test for dogs using human aCL antibodies has been described, we were not successful at validating this procedure in laboratory. As a means of standardization in this study, 3 internal controls (high levels, medium levels, and low levels of aCL antibody) were used for inter- and intraplate control procedures run using each ELISA plate, a method that has been previously described. Furthermore, total IgG was assessed to judge whether aCL test signal could be because of unspecific binding, and showed the lack of correlation between total IgG and the aCL test. There was no association between the dogs having the presence of lupus anticoagulants and high concentration of aCL antibodies. In humans, the presence of both lupus anticoagulants and aCL antibodies in a patient is not necessarily more strongly associated with the risk of thrombosis; however, there are suggestions that co-factor (β2 glycoprotein I) dependent antiphospholipid antibodies are more significant for morbidity in APS than co-factor-independent antiphospholipid antibodies. Specifically because of these conditions, when testing aCL antibodies in humans, the sample diluent in the ELISA test is considered greatly as, for example, adult bovine serum contain β2 glycoprotein I as opposed to BSA. Furthermore, within the last couple of years, it has appeared that high concentrations of antiβ2 glycoprotein I antibodies can be more seriously associated with clinical signs than aCL antibodies in patients with thrombosis.[3, 34, 35] Co-factors have only been examined once in dogs previously, and the importance of these factors was doubtful. The significance of co-factors in dogs therefore remain unknown, and the presence of antiβ2 glycoprotein I antibodies in dogs needs to be evaluated.
In conclusion, we suggest that lupus anticoagulants and aCL antibodies contribute to the prolongation of aPTT in a subset of apparently healthy adult Bernese Mountain Dogs. The significance of the findings in the dogs remains unknown at present, and needs further confirmation in a larger set of dogs. Further studies including canine specific quantitation of aCL antibodies, further antiphospholipid antibody identification, and association with disease processes are in progress.