Quest Continues For A Blood Test To Identify Horses At Risk For Injury

A multi-part study by the University of Kentucky to help identify at-risk horses before racing will soon move into its third phase. Gluck Equine Research Center's Dr. Allen Page provided an update on the ongoing research to the Kentucky Equine Drug Research Council last week.

The EDRC was key in funding the first two phases of the study, which has yielded promising results. In the first phase, Page and fellow researcher Dr. David Horohov took blood samples from horses suffering fatal injuries in races and compared them with blood samples from competitors in the same races who did not suffer injuries. The researchers looked for messenger RNA (mRNA) markers that were present in the injured horses but not the uninjured ones. Messenger RNA is responsible for carrying instructions from genes leading to the production of proteins. The number of these instructions, or gene copies, change if the body is increasing or decreasing inflammation somewhere.

Initially, the research identified 21 markers and determined that three of them were present in 88 percent of horses suffering injuries. The tests developed by the research had a 76 percent sensitivity rate, meaning they could practically identify an at-risk horse 76 percent of the time. Page said he wants to get that sensitivity rate higher, as it's important to minimize false positives that would keep healthy horses out of races.

The study's second phase is in progress now and will sequence RNA from the phase one samples, which will help researchers look at a total of 22,000 markers. So far, about 150 markers are promising enough to merit additional research to see if they could be combined with the results from the first phase findings for more accurate and sensitive testing.

Page is now preparing to launch the third phase of the study. In this phase, researchers want to take pre-race blood samples (rather than post-race samples) to validate that the markers could be used effectively as predictors in a practical application. The team will collect blood samples taken at the time of furosemide administration or pre-race TCO2 testing and bank those samples. If a horse is fatally injured in a race, the team will go back and analyze that pre-race sample to see whether it showed the same indicators they've seen in post-race analysis.

In this phase, the relative infrequency of fatal racing breakdowns means the team will have to cast their net wide.

“Obviously, the rate of catastrophic injury is quite low,” said Page. “So if we want to look at pre-race samples, it requires a large number of samples be collected.”

Page projects the team will need to collect 10,000 blood samples, expecting that will result in about 12 or 13 samples related to fatal orthopedic breakdowns (sudden deaths or accidental deaths will not be included). Those samples will then be stored for future research, so the process will not have to be repeated or funded again for subsequent projects.

“We're really happy and encouraged by the results we've got so far and fully anticipate that by using these pre-race samples, we'll be able to validate what we've done and potentially come up with a commercially viable and useful test that we can use in the racing industry to help further decrease the catastrophic injury rates,” said Page.

The sample collection for the project's third phase will take about a year to complete.

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Study Finds Three Biomarkers That Could Help Predict Fatal Racing Injuries

As the quest continues for a better way to identify racehorses at risk for fatal injury, a study from the University of Kentucky's Gluck Equine Research Center may provide scientists and veterinarians a roadmap of where to look. At a regularly scheduled meeting Oct. 22, the Kentucky Horse Racing Commission received initial results from a study by Gluck researchers Dr. Allen Page and David Horohov looking at inflammatory biomarkers. Biomarkers are proteins, which can be produced at different levels in the body depending on changes and normal processes like inflammation.

Researchers have looked at biomarkers before to try finding some that would signal inflammation that may still be subclinical, or not producing recognizable symptoms yet in the horse. Previous efforts have yielded mixed results, in part because the body undergoes some amount of normal inflammation in response to exercise even when the horse isn't battling an underlying injury. Other biomarkers don't show up until the injury occurs, which makes them useless from a predictive standpoint.

Thanks to funding provided by the Kentucky Equine Drug Research Council (KEDRC), Page and Horohov are in the midst of a two-phase research project — the first looking at a new set of biomarkers and the second looking at messenger RNA, which signals the production of proteins like biomarkers. The second phase is still in progress, and Tuesday's meeting focused on the results from the first phase.

Page and Horohov gathered data from racetracks in four racing jurisdictions, testing blood taken pre-race for TCO2 testing and comparing results between horses who suffered fatal musculoskeletal injuries and competitors from the same races who did not. They examined 21 markers and found three — IGF-1, MMP-2, and IL1RN — which were present in different levels in injured horses versus non-injured horses.

These results made sense to the researchers. IGF-1 is known to play a role in bone development and repair, and it was increased in injured horses, suggesting chronic inflammation was present. Matrix metalloproteinase-2, or MMP-2, is thought to assist with tissue repair and fracture remodeling and was also elevated in injured horses. Interestingly, IL1RN is more commonly known as IRAP–a anti-inflammatory material derived from a horse's own blood and given therapeutically by veterinarians to reduce inflammation and aid in healing an injured horse. IRAP was decreased in fatally injured horses, suggesting the body's natural anti-inflammatory process had been disrupted for some reason. Horses with higher levels of IRAP were actually seven times less likely to suffer fatal injuries.

While those results are encouraging, Page cautioned that it will still be challenging to practically apply the new information. The three biomarkers weren't perfect predictors of impending injury; 24 percent of the time, a horse would not appear to be at risk based on its biomarker levels when in fact it did suffer a fatal injury and 12 percent of the time, tests suggested the horse was at risk of a fatal injury but the horse finished the race without a catastrophic breakdown.

Overall, the three biomarkers provided about 88 percent accuracy at identifying horses at risk.

The test is also expensive and in a research setting the tests took around 48 hours to complete. Page pointed out that those estimates are based on his team's study, where samples had to be tested for 21 different biomarkers. Reducing the number of markers tested would shorten that time, but it would still likely take 24 hours to get results.

Then there's the question of what to do with horses whose blood indicates they may be at risk. It remains unclear whether the commission legally can or should mandate imaging, particularly if the horse doesn't appear lame and no one is sure where the problem might be. Then there's the question of false positive tests — if a horse's entry was contingent upon a biomarker test, horses could be pulled from races who weren't actually at elevated risk, and it would be hard to know which were which.

Commission members acknowledged it will be a challenge to determine how biomarker data could be practically applied to a time sensitive screening process.

Page said he's hopeful the second phase of the study, which looks “upstream” at mRNA responsible for the production of proteins like biomarkers, may provide more specific guidance.

See Page's presentation here:

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