Autologous Blood Plasma Therapy (Chapter 2)

That is probably why platelet count of at least 1,000,000 platelets/μl in 5 mL of blood plasma proposed by Robert Marx as a working definition of PRP became a kind of standard. It is widely agreed that the therapeutic properties of plasma are mainly based on the synthesis and secretion of growth factors and cytokines contained in platelet granules and released from the latter upon platelet activation, modifying the pericellular microenvironment. The most important of them are vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factors 1 and 2 (IGF-1, IGF-2), matrix metalloproteinases-2 and -9, as well as interleukin 8. We will consider in more detail their characteristics and the mechanism of their action on tissues in the next chapter.

However, especially in the context of wound healing, it is necessary to bear in mind that a favorable microenvironment in the wound and mechanical stimuli induce the cell differentiation and reparative processes in tissues with or without the presence of stimulating growth factors.

Some authors, seeing parallelism between PRP therapy and prolotherapy, mention that the mechanism of autologous plasma action is based not only on the ability of platelets to deliver growth factors directly to sites of injury, but also on the principle of initiating tissue healing and repair through the local administration of irritating substance and, therefore, inciting inflammation.

Since 2008, publications in which it was emphasized that not only platelets, but also leukocytes play a significant role in the therapeutic potential of autologous plasma, began to appear. When centrifuging whole blood, white blood cells are concentrated in a layer called "buffy coat." Platelets, as a rule, are found directly on its surface. It was revealed that leukocytes promote collagen type III expression while reducing the expression of collagen type I, which can promote fibrosis. At the same time, it was proven that white blood cells provide protection against infectious agents and help to reduce the growth of microorganisms. Thus, just like with platelets, the question of the optimal concentration of leukocytes in plasma arose; this question remains open as of this writing. Some studies suggest that white blood cells contribute to the overall growth factor content of PRP and change the relative proportions of the contained growth factors. Therefore, it was proposed to consider PRP containing a significant number of leukocytes as a separate therapeutic preparation.

Since, as discussed earlier, there was no consensus on either the preparation of PRP (speed and time of centrifugation, the use of an anticoagulant) and its content (platelets, leukocytes, growth factors), or applications, several authors have tried to characterize and classify the numerous techniques available on the market.

In 2009, Ehrenfest et al. proposed a classification of PRP preparations dividing it into four categories, depending on their leukocyte content and fibrin architecture: pure platelet-rich plasma (P-PRP), leukocyte- and platelet-rich plasma (L-PRP), pure platelet-rich fibrin (P-PRF), and leukocyte- and platelet-rich fibrin (L-PRF). It is understood that these products have different effects, biology, and potential uses; therefore, when choosing a product, the purpose of its use must be taken into account.

In 2012, Mishra et al. proposed another classification based on two features: 1) the presence or absence of white blood cells, and 2) whether or not the PRP is activated. This classification included four types of PRP: L-PRP solution (liquid form), L-PRP gel, P-PRP solution, and P-PRP gel. The only new parameter of this classification is the evaluation of the platelet concentration (type A: platelets > 5 × baseline; type B: platelets < 5 × baseline). This last parameter seems disputable, as many researchers had abandoned the concept of platelet concentration in the previous years for a logical reason: platelet concentration depends only on the volume of liquid serum used to keep the platelets in suspension. The quantity of serum varies significantly depending on the protocol and the intended application and has no impact on the expected effect. The concept of the absolute quantity of platelets seems more logical, even if most publications failed to detect a clear and reproducible effect of this parameter on the clinical outcomes.

The platelet quantity (absolute number) is also mentioned in another classification system called PAW (Platelets, Activation, White cells), which was proposed to organize and more accurately compare protocols and results described in the literature. It is based on three parameters: the absolute number of platelets, the manner in which platelet activation occurs, and the presence or absence of white cells. However, this system, like the previous ones, has its limitations and only covers the PRP families. In general, it is very similar to the classification proposed by Mishra et al. The question of the platelet quantity remains the subject of fierce debate, since there is not a single publication that could determine what would be an optimal platelet quantity, or even if the concept exists with complex multi-component materials such as platelet concentrates.

Unfortunately, none of these classifications is supported by sufficient empirical evidence. Also, the authors did not take into account the final volume of the preparation, the presence or absence of red blood cells in PRP, and the doses of platelets in the final volume of the obtained PRP. It is not surprising that these systems have not received unanimous recognition.

In 2016, Magalon et al. developed the DEPA classification (Dose, Efficiency, Purity, Activation), which considered the number of platelets obtained using various systems, the purity of the obtained preparation, and the activation of platelets before the injection of the drug. The DEPA classification is based on four different elements:

1. The dose of injected platelets, which is calculated by multiplying the concentration of platelets in PRP by the obtained volume of PRP. The injected dose (measured in billions or millions of platelets) can be regarded as: (a) very high (> 5 billion); (b) high (from 3 to 5 billion); (c) medium (1 to 3 billion); and (d) low (< 1 billion).

2. The efficiency of the device or system used to obtain PRP. The platelet capture efficiency is determined by the percentage of platelets recovered in the PRP from the whole blood. Four categories were identified: (a) high device efficiency (platelet recovery rate > 90%); (b) medium device efficiency (platelet recovery rate from 70 to 90%); (c) low device efficiency (platelet recovery rate from 30 to 70%); and (d) poor device efficiency (platelet recovery rate < 30%).

3. The purity of the obtained PRP: this correlates with the relative content of platelets, leukocytes, and red blood cells in the obtained PRP. It is categorised as (a) very pure PRP (the percentage of platelets in PRP compared to red blood cells and white blood cells, is > 90%); (b) pure PRP (percentage of platelets in the PRP compared to RBC and white blood cells, is from 70 to 90%); (c) heterogeneous PRP (platelet percentage in PRP compared to red blood cells and white blood cells, is from 30 to 70%); and (d) whole blood PRP (platelet percentage in PRP compared to red blood cells and white blood cells, is < 30%).

4. Activation process: whether any exogenous clotting factor is added to activate platelets (for example, autologous thrombin or calcium chloride).

This last classification seems to be complete and richer than the previous ones. However, a physician is unlikely to be able to determine the number of plasma constituent elements independently, so that either an additional medical device must be installed on each medical device intended for preparation of PRP, or the classification system becomes inapplicable by default.