PRP GUIDE FOR VETS | 1 – Blood Extraction
The first step to prepare PRP is the blood extraction from the patient. One might think this is the easier part, since blood withdrawal is a daily procedure for Veterinarians, but this is actually the phase where the most mistakes are made, so there are a few things worth pointing out before the extraction:
Even when the patient has already been diagnosed, anamnesis and clinical history should always be updated. Note that there is no age restriction for this procedure, nor does it affect the blood volume of the individual.
The patient must be free from NSAIDs therapy 7 days prior to the procedure, since most of them alter the platelet functions.
Blood count is a must on every patient, so that a proper assessment of the quality of the blood can be made. PRP should not be prepared from patients with platelet altered pathologies or low hematocrit. It is very important to take note of the basal platelet status.
When all previous points have been completed, the patient must be scheduled for the blood extraction, indicating the owners that a 4 hour-fast period will be necessary to ensure clean, transparent plasma, which will facilitate the separation of the platelets later on.
Before starting with the procedure, the veterinarian must evaluate the patient to verify if it is a cooperating animal or if sedation drugs should be administered. Also, at this point the operating personnel have to decide (according to the patient’s health status, weight and size proportions) if the blood extraction can be made from the cephalic veins or if a direct jugular withdrawal would work best. Usually, jugular puncture is indicated in small dogs and cats, while cephalic withdrawal may be an option for a healthy, normotensive, large breed dog.
Once the extraction procedure is about to begin, the operating personnel should consider that timing is essential from this point on.
The ligation or venous pressure cannot be maintained longer than 1 minute. If this time is exceeded, tissue factors and thromboplastin will be released, activating the platelets, making them release their growth factors, and leading us to obtain blood with empty, useless platelets. Following the same logic of these arguments, it is very important to highlight that the withdrawal should be by vacuum extraction, so that there is no contact between the platelets and environmental oxygen.
The use of EDTA (purple) tubes is not recommended for PRP preparation, since this anticoagulant is used for blood counts only, and has no preserving elements. In fact, EDTA alters the platelet membranes and releases growth factor prematurely.
Red or “dry” tubes are also NOT recommended for PRP. The reason behind this is that, even though most people believe red tubes are completely dry or empty, it is not entirely true. They DO contain additives that work as platelet activators. Reading carefully the label on these tubes, the words “clot activator” will be found. Also, the plastic of the tubes contains micronized silicone, which can also generate platelet activation. All of this results on a platelet-free serum after the centrifugation process, which cannot be used for PRP.
Yellow tubes also contain clot activator, and green heparin tubes are not ideal for platelets either.
One of the best choices is the blue tube, which contains sodium citrate 3.2%. It offers higher stability, and the concentration ensures a proportion of 1:9 (1 part of anticoagulant and 9 parts of venous blood). Sodium citrate at this concentration works searching for calcium ions and form chelates, so the inactivated calcium keeps the blood from clotting. Also, the materials on the plastic of this tube (polypropylene, polyethylene terephthalate) help maintain the growth factors inside the platelets. It is important to mention that other sodium citrate tubes with different concentrations, such as the black tube, should not be used for PRP because the full separation of platelet plasma from serum would not be achieved. When working with sodium citrate tubes, the inversion of the tube for blood dilution should be only 3 to 4 times.
The very best anticoagulant for PRP is ACD (Acid Citrate Dextrose-A), which is commonly used in blood banks. This is the only one that works better than sodium citrate, but it is only available in USA and Europe, so other countries should stick to the sodium citrate anticoagulant, or buy commercial PRP Kits that do contain ACD.
How much blood is needed?
In Veterinary Medicine, we have a huge advantage in matters of calculating the blood volume needed for PRP preparation, since the dosages of PRP needed are basically the same (proportionally speaking) as in Human Medicine. Plus, the volume of blood needed to produce enough PRP for a human or a giant sized dog is so little, that obtaining that same amount of blood from a small breed dog or a cat is just as low-risk.
The majority of the available commercial PRP Kits require 20 cc of blood to produce an approximate of 2 cc of PRP. If prepared manually, filling 6 to 8 blue tubes sums about 24 cc of blood. There are no specific formulas (yet) to calculate the blood extraction volume per species in Veterinary Medicine, but papers and experienced Veterinarians suggest that the 20 cc extraction should be limited to animals weighing more than 10 kg, while smaller animals should be drawn from 5 to 10 cc in the case of dogs and cats. Literature suggests extraction of 2.5 cc in rodents and similar sized species, and even lower volumes in the case of small reptiles and birds.
Once the blood extraction is completed, it is very important to remember that the PRP should be prepared within the first 24 hours since its withdrawal, considering that the sooner it is done, the higher concentration of inactivated platelets will be obtained during the next steps.