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D-Dimer isolation and analysis of immunoreactivity in dogs, cats and horses

W21-048: D-Dimer isolation and analysis of immunoreactivity in dogs, cats and horses (An EveryCat-funded research grant final report)

Brown JE, Noormohammadi AH, Courtman NF. Immunoreactivity of canine, feline, and equine D-dimer with antibodies to human D-dimer. J Vet Intern Med. 2023 Nov 10. doi: 10.1111/jvim.16888. Epub ahead of print. PMID: 37950415.

https://pubmed.ncbi.nlm.nih.gov/37950415/

In human medicine, measurement of plasma D-dimer is a crucial component of workup for thrombosis, however it is much less well established in veterinary medicine. D -dimer is a product of blood clot breakdown that is elevated when blood clots are present in the body. As D-dimer can be elevated in a variety of conditions, the primary clinical use of D-dimer is to exclude thrombotic syndromes.

Modern D-dimer assays are primarily based on antibody binding. Commercially available assays use human D-dimer antibodies, and their specificity and sensitivity can vary between species. The lack of cross-reactivity of D-dimer assays with fibrinogen and fibrin is crucial for accurate results. Validating antibodies for each veterinary species can be challenging, and as such well-established assays are lacking.

The study’s objective was to investigate the immunoreactivity of D-dimer in dogs, cats, and horses using commercially available antibodies to human D-dimer.
The authors conducted a comprehensive investigation into the immunoreactivity of D-dimer in dogs, horses, and cats using a variety of commercially available antibodies designed for human D-dimer. To carry out the investigation, three groups of samples were collected: healthy canine plasma, healthy equine plasma , and pooled feline plasma from hospitalized cats.
The preparation of D-dimer involved the generation of cross-linked fibrin clots from the plasma samples, with subsequent thrombolysis. The study then estimated putative D-dimer levels using a canine-specific assay (Canine VCheck POC Assay).

The authors created in-vitro preparations of cross-linked fibrinogen degradation products to assess the immunoreactivity of D-dimer using human antibodies. Cross-linked degradation products were demonstrated through SDS-PAGE, revealing bands in the appropriate fibrinogen area, with heterogenous fibrinogen fragments in horses. The authors observed the appearance of putative D-dimer bands, suggesting in-vitro formation, although D-dimer was already present in healthy horse plasma and pooled feline plasma.

The study also conducted immunoblotting using four commercially available antibodies: Anti-Fib, DD5, DD44, and D2D. These antibodies were chosen to represent a proof-of-concept study, demonstrating the differences in their performance and highlighting the potential implications of using unproven assay kits.

The different antibodies showed varying immunoreactivity. Anti-Fib exhibited strong binding with fibrinogen in all species and additional bands in the D-dimer region, indicating potential degradation products. DD44 showed the best performance in canine samples, demonstrating specificity and sensitivity in dogs but not binding D-dimer in horses or cats. DD5 consistently reacted with fibrinogen and higher molecular weight bands. D2D did not bind higher MW peptides but showed strong binding with a protein similar to human D-dimer.

The variability in immunoreactivity across species was attributed to differences in antibody binding sites. This emphasizes the need for thorough validation studies before assuming the accuracy of human D-dimer assays in companion animals. DD44 was identified as a potential focus for developing a canine-specific assay, while challenges in clinical validation for cats were acknowledged due to notable inter-species variation in D-dimer immunoreactivity.

The study provided detailed insights into the immunoreactivity of D-dimer in dogs, horses, and cats, emphasizing the importance of selecting appropriate antibodies for accurate measurements. The research contributes valuable information to the field of veterinary diagnostics, where the specificity and sensitivity of D-dimer assays can impact the diagnosis and management of various health conditions. ~MK

See Also
Nelson OL, Andreasen C. The utility of plasma D-dimer to identify thromboembolic disease in dogs. J Vet Intern Med. 2003;17(6):830-834.

Carretón E, Morchón R, González-Miguel J, Simón F, Juste MC, Montoya-Alonso JA. Variation of d-dimer values as assessment of pulmonary thromboembolism during adulticide treatment of heartworm disease in dogs. Vet Parasitol. 2013;195(1-2):106-111.

Song KS, Kim YA, Kim HK, Park Q. Incidence and possible reasons for discordant results between positive FDP and negative D-dimer latex assays in clinical specimens. Yonsei Med J. 1999;40(2):107-111.