Study validates canine urine 5-HIAA ELISA against LC-MS/MS: full analysis

A new BSAVA PetSavers-funded paper in the Journal of Small Animal Practice takes a practical step toward making urinary 5-HIAA testing more usable in canine medicine. The study validated a commercially available ELISA assay for the serotonin metabolite 5-hydroxyindole acetic acid in canine urine against LC-MS/MS, the gold-standard analytical method, and found acceptable precision with good overall agreement, though the authors flagged the need for further validation at higher analyte concentrations before full clinical adoption. (synapsesocial.com)

The backdrop is a familiar one in veterinary diagnostics: biologically interesting markers often exist before there is a practical way to measure them outside specialist settings. BSAVA notes that serotonin has been implicated in several canine diseases, including myxomatous mitral valve disease, pulmonary hypertension, and dilated cardiomyopathy. But serum serotonin is difficult to work with because of its short half-life, making urinary 5-HIAA a more appealing downstream marker. Until now, however, measurement has relied on more complex and costly analytical platforms that are not widely available in routine practice. (bsava.com)

According to the study summary, Castillo, Adam Swallow, Tom Williams, and Penny Watson evaluated urine from 26 dogs undergoing routine diagnostic investigations at a referral center and compared ELISA results with LC-MS/MS. The ELISA achieved acceptable precision, defined in the summary as a coefficient of variation under 20%, and showed good agreement with the reference method, with a reported bias of 0.92 µmol/L. The key limitation was at the upper end of the range: the assay appeared reliable at lower concentrations, but higher concentrations need further validation before the test can be treated as fully ready for broad clinical application. (synapsesocial.com)

That finding is consistent with what other veterinary assay-validation studies have shown. In canine urine, ELISA methods can be attractive because they are simpler and more affordable than chromatographic methods, but matrix effects, dilution issues, and recovery performance can all affect reliability. A 2022 study on urinary metanephrines in dogs, for example, found acceptable performance for one analyte but unsatisfactory dilution recovery for others, underscoring why species- and matrix-specific verification matters before clinicians lean on biomarker results. A more recent Frontiers study validating a urinary 5-HIAA ELISA in healthy dogs similarly reported that canine urine created a marked matrix effect that required dilution to mitigate overestimation. (journals.sagepub.com)

Direct outside commentary on the new JSAP paper appears limited so far, but BSAVA framed the work as clinically relevant because it points to a “more accessible, less invasive, and relatively low-cost” testing option for primary clinicians. That’s a measured claim, and probably the right one. The paper does not establish that urinary 5-HIAA should now be used routinely in screening or diagnosis for any specific canine disease. Instead, it strengthens the analytical case that the assay may be usable, within limits, as the field works toward clearer clinical applications. (bsava.com)

Why it matters: For veterinary professionals, the main significance is infrastructure, not just biomarker biology. If urinary 5-HIAA can be measured accurately enough with a commercial ELISA, more practices and diagnostic labs may be able to access serotonin-pathway testing without relying exclusively on LC-MS/MS. That could support research studies, referral workups, and eventually more standardized monitoring in diseases where serotonin metabolism is suspected to play a role. But the limitations are important: analytical agreement with a reference method is only one step. Clinicians still need validated reference intervals, disease-specific decision thresholds, and a better understanding of preanalytical variables before they can interpret results confidently at the individual-patient level. (synapsesocial.com)

There’s also a broader lesson here for practice teams and laboratory directors. Veterinary medicine continues to adapt human-origin assays for canine use, and this can work well, but only after careful validation. Studies in canine urine biomarkers repeatedly show that sample handling, matrix interference, and assay design can materially change performance. In that sense, this paper is a reminder that “commercially available” does not automatically mean “clinically interchangeable” with a reference method across all concentration ranges. (journals.sagepub.com)

What to watch: The next milestones will be studies that connect urinary 5-HIAA results to specific canine disease states, define clinically useful cutoffs and reference intervals, and test how the assay performs in dogs with genuinely high concentrations rather than mostly lower-range samples. If those data hold up, urinary 5-HIAA could move from an interesting research analyte toward a more practical tool in canine internal medicine and cardiopulmonary workups. (synapsesocial.com)