Front Matter

406 Canine Sports Medicine and Rehabilitation

not provide more platelets than a larger volume
of PRP with a lower platelet concentration. The
ideal final volume of PRP is influenced by the
volume that can be delivered to a site of injury
and whether any aliquots of PRP are to be fro‑
zen for future use. In the latter case, production
of a larger volume of PRP might be preferable.

Preparation

Platelet‐rich plasma can be prepared from canine
blood with basic laboratory supplies and spe‑
cific centrifugation protocols (Perazzi et  al.,
2013). Blood is collected and usually, but not
always, anticoagulated. Acid citrate dextrose‐A
is the most commonly used anticoagulant
although sodium citrate and citrate phosphate
dextrose‐A can also be used (Franklin et  al.,
2015). These are the only two anticoagulants
that have been shown to support the metabolic
needs of platelets. Use of heparin is less common,
and ethylene‐diamine tetracetic acid (EDTA)
should not be used (Marx, 2001). Centrifugation
with a soft spin (190–1000 g, depending on the
protocol and the centrifuge; Perazzi et al., 2013;
Silva et  al., 2013; Franklin et  al., 2015), is per‑
formed to separate the contents with red blood
cells at the bottom, plasma at the top, and the
buffy coat at the interface. Depending upon the
centrifugation protocol, the canine platelets are
in the deeper portion of the plasma and the
buffy coat. Accordingly, the deeper portion of
the plasma, and potentially some portion of the
buffy coat, are collected to obtain a PRP. If a
double centrifugation is performed, all of the
plasma and some portion of the buffy coat is

transferred to another tube and a faster spin is

performed to pellet  all cellular components,

including the platelets, within platelet‐poor

plasma (PPP) (Figures 16.1 and 16.2). The final

volume of the desired PRP is determined, any

excess volume of PPP is removed and dis‑

carded, and the pellet is suspended in the

remaining volume of PPP to produce the final

PRP (Figures 16.1 and 16.2).

The location of the platelets relative to the

plasma layer and buffy coat is the most impor‑

tant concept to understand. If one retrieves

only the plasma layer and either very little or

none of the buffy coat, the platelet concentra‑

tion will be relatively low and the leukocyte

concentration will also be low. With increasing

retrieval of the buffy coat, the platelet concen‑

tration will increase but so will the leukocyte

concentration. Retrieval of much of the buffy

coat results in extremely high platelet and leu‑

kocyte concentrations. Importantly, unpub‑

lished data from one author’s (SF) lab confirm

that the platelets are more superficially located

within the preparation than the leukocytes,

which would be expected based upon their

smaller size in comparison to the leukocytes.

This means that it is feasible to obtain relatively

high platelet concentrations with low or negli‑

gible leukocyte concentrations if one can select

this fraction of the plasma and buffy coat—

something that is simple in principle but can be

challenging to execute.

Although preparation of PRP can be done

using basic supplies, many practitioners in

human and veterinary medicine elect to use

commercially available centrifugation or filtra‑

tion‐based PRP concentrating systems because

Transfer

blood to tube

and centrifuge.

2

1

3456

Collect blood^7

from patient.

Plasma

Platelets

Leukocytes

Red blood cells Inject PRP into

joint.

Collect

plasma and

platelets.

Centrifuge

plasma and

platelets again.

Remove

excess

platelet-poor

plasma (PPP)

Resuspend platelets in remaning

PPP to make platelet-rich

plasma (PRP).

Figure 16.1 Double‐spin platelet‐rich plasma (PRP) preparation protocol.