Sample Handling's Effects on Plasma CO2 Levels (AAEP 2012)

By its very nature the practice of “milkshaking,” or administering bicarbonate or other alkalinizing substances to racehorses as performance enhancers, can be tough to pinpoint—horses metabolize the substances quickly, and testing laboratories must look for elevated blood total carbon dioxide (CO2) concentrations as evidence. So to preserve an accurate snapshot of the horse’s blood CO2 levels at collection time, and ensure consistent test results, proper sample storage and handling are crucial.

Purdue University researchers recently evaluated sample handling and storage time's effects on total CO2 concentrations in plasma. Stacy Tinkler, DVM, Dipl. ACVIM, clinical assistant professor of equine community practice in the university’s College of Veterinary Medicine, reported their results at the 2012 American Association of Equine Practitioners convention, held Dec. 1-5 in Anaheim, Calif.

Veterinarians and trainers believe mixtures of bicarbonate and/or other alkaline substances, administered to a horse shortly before he competes delays lactic acid buildup in a horse's muscles, allowing them to run farther before tiring. Physicians consider it a moderate performance enhancer in human athletes performing short, intense exercise.

Tinkler explained that veterinarians can test for excess carbon dioxide in the blood before or after a race, depending on the particular track’s testing protocol; however, inconsistent sampling, processing, and storage methods commonly cause laboratories to underestimate total CO2. "This lab error could work in favor of those who may be administering alkalinizing agents to horses illegally because it might falsely lowers the total CO2 value below the detection limit, and some horses that have been given a 'milkshake' will compete, potentially lending them an unfair advantage over other horses," she said.

Storage temperature fluctuations and sample pickup and delivery delays—common scenarios with off-site blood testing—could conceivably impact blood CO2 concentration stability. Tinkler and colleagues simulated these conditions by using different handling protocol and storage times before testing. The team collected blood from six adult horses and randomly assigned the samples to be centrifuged immediately or just prior to analysis after a predetermined period of time (24, 48, 72, and 96 hours) in storage at 4°C, about 39°F, Tinkler said.

The team found that plasma total CO2 concentrations decreased over time in storage, Tinkler reported. She also noted that the values decreased faster when samples were centrifuged later, as opposed to processing them immediately after collection.

In order to achieve the most accurate results, Tinkler concluded, "blood samples … should be centrifuged immediately after collection, stored at 4°C, and analyzed as soon as possible to ensure accurate values for plasma total CO2 concentrations."

Disclaimer: Seek the advice of a qualified veterinarian before proceeding with any diagnosis, treatment, or therapy.