Blood Collection Tube Size and CO2 Concentration (AAEP 2012)

Veterinarians screen racehorses regularly for evidence of performance enhancers, aiming to use sample collection methods that yield accurate and consistent results. Blood-draw supplies vary among veterinarians, however, and could conceivably impact test results, so a Purdue University research team recently evaluated how such variables impact a common measurement used in drug testing--total plasma carbon dioxide (CO2) concentrations.

Stacy Tinkler, DVM, Dipl. ACVIM, clinical assistant professor of equine community practice at the Purdue University College of Veterinary Medicine, and colleagues compared blood tube size and material, and she presented their results at the 2012 American Association of Equine Practitioners convention, held Dec. 1-5 in Anaheim, Calif.

Regulators test total CO2 concentrations in racehorses' blood to determine if trainers or caretakers administered alkalinizing substances, also known as "milkshakes." These mixtures of bicarbonate and/or other alkaline substances, considered moderate performance enhancers in human athletic arenas, are administered to horses shortly before they compete. Some believe the mixtures delay lactic acid buildup in muscles, allowing horses to run farther before tiring, although "the jury is still out on whether or not … administration actually has a beneficial effect on performance in the horse," Tinkler said. Regulators test for excess blood carbon dioxide before or after a race, depending on the particular track’s testing protocol.

Veterinarians know that under-filling tubes when collecting blood for total CO2 concentration measurement can yield lower values. And Tinkler noted that even when tubes are filled completely and according to manufacturer’s instructions, they can yield different air-to-blood ratios, depending on their size and expected draw volume.

They recognized that such variability could possibly even occur among tube material types, so Tinkler and her colleagues took a closer look. . "The object of this study was to determine the effect of blood collection tube size and material on the measured value for total CO2 concentrations in equine plasma," she said.

She and colleagues measured plasma total CO2 concentrations in 20 horses’ blood collected in eight different types of tubes:

  • 2-milliliter (mL) glass tubes;
  • 2-mL plastic tubes;
  • 3-mL plastic tubes;
  • 3-mL plastic plasma separator tubes;
  • 4-mL plastic tubes;
  • 6-mL plastic tubes;
  • 10-mL glass tubes; and
  • 10-mL plastic tubes.

The team found that the air-to-blood ratio was significantly higher (meaning there was more air and less blood in the tubes) and the total CO2 concentration was significantly lower in the 2- and 3-mL tubes as compared to other tube sizes. They recommended using vacutainer (a sterile glass or plastic tube with a closure that creates a vacuum inside the tube, facilitating the blood draw) tubes that are 4 mL or larger when testing total carbon dioxide concentration. They detected no significant difference in total CO2 measurements between plastic or glass tubes, she said.

"In other words, larger tubes (4-mL or larger) had better vacuums and, therefore, pulled more blood and had lower air-to-blood ratios (meaning less air and more blood in the tubes)," Tinkler explained. "It turns out that the total CO2 concentration is higher (i.e., more accurate) in these larger tubes with more blood and less air in them. Smaller tubes with more air space (i.e., higher air-to-blood ratio) had more space for carbon dioxide to move out of the blood and into the air space so when you measure the total CO2 in the blood sample, it is falsely lower.”

Tinkler concluded, "Blood should be harvested into collection tubes with a small air-to-volume ratio whenever an accurate measurement is required."

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