2) Separation (Vet Clinic Guide)

PRP GUIDE FOR VETS | 1 – Blood Extraction | 2 – Separation

It is clear now that the foundation of PRP therapy is the concentration of platelets from the patient’s blood. Inside a single drop of blood, there are approximately 4 platelets; after separation, a drop of plasma should contain 10 to 15 platelets. This separation process is rather simple, since it mainly involves the use of the centrifuge and manual separation of the PRP, or the correct management of the commercial PRP Kits. So, ironically, the easiest step (operatively speaking), is the one that owns the magic: to transform simple blood into a pure and natural regenerative medication.

Types of centrifuge

  • Swinging Centrifuge. This type of centrifuge is ideal for PRP preparation. The main characteristic of a tilting centrifuge is that the bucket container takes a 90° position in relation to the centrifuge shaft, instead of maintaining a fixed position. This centrifuge should be used at 700 RPM for 10 minutes. The con of this equipment is that it is a little expensive (3500 USD approximately).
  • Fixed-Angle Centrifuge. As its name indicates, this centrifuge rotates on a fixed angle of approximately 45°. This is more commonly used in PRP preparation because most of the veterinary practices already have one of these, and when the intention is to buy a new one, the cost is significantly lower than that of the swinging centrifuge. The main con of the angled centrifuge is that the platelets might crash against the tube walls, possibly causing their activation. However, if this happens, the growth factors are still preserved for another 15 minutes, so if this is the equipment used, the Veterinarian should perform the application within the mentioned timeframe.

RPM Protocols

There is a large variety of protocols for the centrifugation timing and RPM (revolutions per minute) settings, which happens because of the different ratios of the centrifuges. It is impossible to assign a single RPM protocol for all centrifuges, so in order to standardize, the Relative Centrifugal Field (RCF) is the unit every operator should base on, and then convert that value to RPM according to their centrifuges’ characteristics. The established protocol indicates 250 RCF for 10 minutes for PRP preparation.

The conversion from RCF to RPM is very simple, and depends solely on the centrifuge ratio (R); this can be obtained by opening the centrifuge lid, placing a ruler or measuring tape at the shaft and measuring from there to the distal part of the tube. To convert manually, the following formula may be used:

RCF = 1.118 x 10-5 x R (RPM)2

Since the formula may be unpractical or complex, there are standardized graphics called nomograms, which help convert these values quickly. The nomograms should be used from left to right, locating first the centrifuge ratio (R), then the RCF (250 for PRP), and then drawing a line between the two points. This line should be prolonged onto the RPM column, which will indicate the amount of RPM needed in 10 minutes for that specific centrifuge.

Extraction of PRP

Once centrifuged, the tube will show three different layers (bottom to top):

  • Red Cells. Erythrocytes and hemoglobin.
  • Buffy-Coat. Leukocytes.
  • Plasma.
  • F1: Platelet-Rich Plasma
  • F2: Platelet-Normal Plasma
  • F3: Platelet-Poor Plasma

When using a commercial PRP Kit, the tubes are different from the conventional ones, so the extraction should be done according to the manufacturer instructions. If the extraction of PRP will be done manually, authors agree on two different options, depending on the type of centrifuge being utilized:

  • If a swinging centrifuge is being used, a needle should be used to discard the F3 layer of the plasma, and then a new needle to get F2 and F1, as well as the buffy-coat from each tube. This F2-F1-BC mix can be placed inside a new dry tube (red tube or Falcon), resuspended with with 0.75 – 1 cc of Platelet-Poor Plasma, and then returned to the centrifuge for another 10 minutes. Once the second centrifugation is finished, the concentrated platelets will be located at the bottom of the tube, so Platelet-Poor Plasma can be removed. Resuspention of the platelet concentrate with remaining plasma will allow the PRP to be drawn with a syringe.
  • When using a fixed-angle centrifuge, the double centrifugation is not recommended because it is unknown if the platelets have already crashed and become active, so there is a timeframe to observe. Thus, the PRP extraction should be performed using a syringe to draw the F1 layer of the plasma, being very careful to place the bezel to the inside part of the tube and trying not to touch the buffy-coat or red cell layers. From this point on, the growth factor availability becomes shorter, so the next steps must be followed as soon as possible.

Read about the next step “Activation” here.