How an orthobiologic is prepared can influence the final product. | Haylie Pfeffer/The HorseHow an equine orthobiologic is prepared and the patient its collected from can influence the final product used to inject the horse, including key signaling proteins known as growth factors and cytokines, said Brenna Pugliese, DVM, MS, Dipl. ACVS, a PhD candidate in the Schnabel Laboratory at North Carolina State Universitys College of Veterinary Medicine, in Raleigh. Pugliese explained how equine practitioners should prepare orthobiologic products to maximize their quality and efficacy in her presentation at the 2025 American Association of Equine Practitioners Convention, held Dec. 6-10, in Denver, Colorado.Although output can vary widely among products and individual patients, preparation method plays a central role in determining the biologic composition of orthobiologics used for joint injections. Growth factor and cytokine concentrations are influenced not only by processing techniques but also by medications, exercise, metabolic status, and blood collection practices, said Pugliese.The Effect of Medications on Equine Orthobiologic ProductsPugliese said that non-steroidal anti-inflammatory drug (NSAID) administration is one patient factor that can affect platelet and leukocyte (white blood cell) function. While researchers have not demonstrated changes in the cytokines examined to date in horses receiving NSAIDs, she recommended discontinuing these medications 24 hours prior to blood collection for orthobiologic preparation to help maximize product efficacy.As far as other drugs, acepromazine (a tranquilizer) has not been found to affect platelet biology in dogs, said Pugliese, and researchers have suggested cytokine concentrations are not substantially altered at five minutes following sedation. Even so, Pugliese recommends veterinarians draw blood for orthobiologics prior to sedation when safe to do so.For horses receiving long-term medications, such as sedatives during long-term stall rest, timing becomes more important. Reserpine has been shown to prime platelets for activation, while trazodone can affect platelet aggregation (accumulation). Pugliese recommended a two-week washout period for both drugs before blood collection for orthobiologic preparation. Horses that have undergone standing or recumbent surgery should also have a 24-hour washout period before collection, while a one-week washout is more appropriate following vaccines.Health Factors That Affect OrthobiologicsAs noted earlier, exercise and metabolic status can also influence orthobiologic output, as can hydration. Intense exercise has been shown to increase tumor necrosis factor (TNF) levels while decreasing interleukin-1 receptor antagonist (IL-1Ra, inhibits inflammation) and can cause plasma alpha-2 macroglobulin (A2M) concentrations to fluctuate, which might affect the final orthobiologic product. Dehydration can also concentrate certain blood components. Research evaluating orthobiologic preparation in horses with pituitary pars intermedia dysfunction (PPID, once called equine Cushings disease) is limited, said Pugliese, and data are currently lacking for horses with equine metabolic syndrome (EMS). Therefore, experts currently recommend PPID and EMS horses be metabolically regulated prior to veterinarians drawing blood for orthobiologics.Proper aseptic technique during blood collection for orthobiologics remains critical. Clipping the collection site is not required unless the horse has a long hair coat, in order to ensure sterile preparation, Pugliese said. To minimize vessel trauma, she recommended using an 18- or 19-gauge butterfly needle, entering the vessel once, and advancing the needle to the hub. Using an extension set can help reduce trauma and eliminate the need for reinsertion. With appropriate preparation, the process can be performed by a single practitioner or one with an assistant.Take-Home MessageOrthobiologic quality depends on both preparation technique and patient factors. Managing medications, timing blood collection appropriately, and using careful, low-trauma collection methods can help optimize growth factor and cytokine content, supporting more consistent and effective joint therapies.