Classification of Platelet Concentrates (Platelet-Rich Plasma — PRP, Platelet-Rich Fibrin — PRF) for Topical and Infiltrative Use in Orthopedic and Sports Medicine

January 15, 2018

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The development of platelet concentrates for surgical use, often referred to collectively by the abbreviations PRP (Platelet-Rich Plasma) or PRF (Platelet-Rich Fibrin), is an important and current interdisciplinary field of research spanning many basic and clinical disciplines. These products are frequently associated with such keywords as “growth factors,” “regenerative medicine,” “stem cells,” and other fashionable buzzwords.

When considering these products, as with many others, it is important to ask three good questions:

1. What are platelet concentrates for topical and infiltrative use?
2. Why do we use them?
3. What are the results after 30 years of use?

Platelet concentrates for topical and infiltrative use are primarily blood preparations obtained after various forms of processing a whole-blood sample, mainly by centrifugation.

The purpose of processing is to separate the blood components, discard elements considered unsuitable (mainly red blood cells, which are heavy and easily separated), and collect and concentrate the elements that may be used for therapeutic applications (fibrinogen/fibrin, platelets, growth factors, leukocytes and other forms of circulating cells) in a liquid plasma solution.

In short, all these products, regardless of the technique used, are extracts of tissue circulating in the blood. They are tissues themselves, not pharmaceutical drugs.

These preparations are used at a surgical or injured site to stimulate, improve and accelerate healing. In all wounds, blood coagulation, with the formation of a fibrin/platelet clot and matrix, is the initial stage of the natural healing process. The use of platelet concentrates was developed to reinforce this natural process, much like fibrin glues, which have been used for more than 40 years as surgical adjuncts to improve healing.

Over time, this concept of optimizing healing evolved into the more complex concept of tissue regeneration supported by the growth factors and cells contained in these preparations. Initially regarded as surgical adjuncts, PRP/PRF became popular and prominent tools in the new strategies of regenerative medicine.

Based on the international scientific literature on this subject and the evolution of clinical trends, it is difficult to determine which products are genuinely useful. Many authors have noted that published experimental results are difficult to classify and interpret, clinical outcomes are ambiguous or at least contradictory, and the relevance of their use remains debatable when the literature, overall practical experience and practical considerations (such as the high cost of most of these methods) are taken into account.

The reasons for this unsatisfactory situation have been identified in many recent discussions and conferences:

• Many different techniques are available for preparing platelet concentrates for surgical use (commercial systems or custom systems developed for particular experimental studies), resulting in very different final preparations.
• There has been no appropriate terminology for classifying and describing the many different variations of platelet concentrates.
• There is considerable confusion between techniques, together with a lack of precise characterization of the products tested in most articles on the subject. This has resulted in a vast literature comprising thousands of articles that form a “blind library of knowledge.”

The answers to the three initial questions illustrate the current situation very well. Several years ago, the need for clarification, terminology and classification was highlighted, but this work is still ongoing. To understand the essence of these methods, it is necessary to recall their actual history.

A Long History and Considerable Confusion

The history of these preparations is often incorrectly associated with the first article by Marx et al. in 1998 on growth factors in Platelet-Rich Plasma (PRP). In fact, the history of these methods began much earlier, with Matras’s research on fibrin glues used to improve wound healing in a rat model in 1970. This work led to fibrin glues that are still used today, such as Tisseel (Baxter, Deerfield, Illinois, USA).

In these technologies, the fibrin matrix is the main blood component used to stimulate healing, together with several other associated matrix proteins. Consequently, the range of blood components that can be used is somewhat limited.

Nevertheless, the fibrin matrix is the final product of the coagulation reaction cascades and the first healing matrix. The use of such glues therefore represents one of the first biological surgical adjuncts developed in the modern era.

A few years later (1975–1979), several research papers proposed an improved concept involving the use of blood extracts called “platelet-fibrinogen-thrombin mixtures” or “platelet-gelatin-gel foam.” In this new concept, fibrin glues contained a significant concentration of platelets in the final preparation. The idea was to reinforce the fibrin gel while also combining the healing properties of platelets with those of fibrin.

This modification of fibrin glues made it possible to prepare more natural products by appropriately combining a greater number of natural blood components. These methods were the first platelet-rich plasma gels in the sense in which we understand them today. The new strategies emphasized the role of platelets in the fibrin gel and reported excellent preliminary results in ophthalmology, neurosurgery and general surgery.

This approach was subsequently confirmed under other names, such as “platelet-derived wound-healing factors” (PDWHF), and was successfully tested for the treatment of skin ulcers, following the principles developed by Matras with fibrin glues 15 years earlier.

These methods continued to develop slowly until the papers by Whitman in 1997 and, in particular, Marx et al. in 1998. These publications marked the beginning of the enthusiasm for these methods in oral and maxillofacial surgery and for the concept of platelet growth factors in regenerative medicine.

From that point onward, the number of publications and commercially available systems increased rapidly, creating the situation of widespread confusion described above. Yet the products themselves were not fundamentally or conceptually different from those used in earlier studies. All these products were subsequently called Platelet-Rich Plasma (PRP), regardless of their contents or architecture, and this lack of terminology persisted for many years.

At the same time, another form of platelet concentrate was developed in France and named Platelet-Rich Fibrin (PRF), reflecting the strong polymerization of the fibrin gel in the preparation. This method was so clearly different from other PRP products that it was called a second-generation platelet concentrate. However, this expression is probably inadequate, given the long history of the evolution of platelet concentrates. Today, it is considered simply another family of products.

This evolution in terminology was nevertheless very important, because for the first time one product was clearly distinguished from the others, justifying the use of completely different terminology.

The second major evolution in terminology appeared only in recent years, when several authors, particularly the groups of Dohan Ehrenfest, Everts and Bielecki, pointed out that these platelet concentrates are also associated with various forms of circulating cells, especially leukocytes. The need for more detailed study of the cell population was proposed in several articles and has now become one of the most important subjects of debate in this field, particularly in sports medicine.

The history of these products illustrates the changing trends that have driven research over many years. Initially, interest focused on the fibrin matrix alone as a healing material; priority was then given to the healing properties of platelets; subsequently, attention turned to the effects of growth factors (circulating and platelet-derived) on tissue regeneration. Finally, the role of circulating cells became the new frontier.

Which of all these elements should be considered the most important? In light of our general knowledge of coagulation and healing—and a certain amount of common sense—it is now considered that all these elements are important and must be appropriately combined to achieve the best clinical results.

Fibrin, platelets, slowly released growth factors, leukocytes and other cells are all key active participants in the natural healing process. Together, they form a kind of engineered tissue extracted from the tissue circulating in the blood. This complex combination is the key to optimal performance.

For this reason, the L-PRF clot, consisting of leukocyte- and platelet-rich fibrin, has often been described as an “optimized blood clot” that can be surgically handled and used. This expression is, more or less, accurate for all well-designed platelet products.

Current General Classification

Following discussions about the content and role of the various components of these preparations, the first classification was proposed in 2009. It is now widely cited as a milestone in the clarification of terminology. This classification is actually very simple and divides products according to at least two key parameters: the presence of cellular content (mainly leukocytes) and the fibrin architecture. This distinction made it possible to define four main families into which the products can be grouped.

1. Pure Platelet-Rich Plasma (P-PRP) — or leukocyte-poor platelet-rich plasma — consists of preparations without leukocytes and with a low-density fibrin network after activation. By definition, all products in this family can be used as liquid solutions or in the form of an activated gel. They can therefore be injected (for example, in sports medicine) or applied to a skin wound or suture during gel formation, in a manner similar to the use of fibrin glues.

There are many preparation methods, particularly those using cell separators (continuous-flow plasmapheresis) from hematology laboratories, as proposed by many authors. However, this method is too cumbersome to be used frequently and easily in everyday practice.

One widely promoted P-PRP method is known under the commercial name PRGF (Plasma Rich in Growth Factors, or growth-factor-rich preparations, also known as EndoRet; Biotechnology Institute, BTI, a dental implant company, Vitoria, Spain). It has been tested in many clinical studies, particularly in sports medicine. The main drawbacks of the technique are its lack of ergonomics and the need for approximate pipetting steps during preparation.

The literature on this technique remains very difficult to evaluate because most of the articles were produced by the company promoting it. Another P-PRP technique has been widely promoted for skin ulcers and is known commercially as Vivostat PRF (Platelet-Rich Fibrin, Vivostat A/S, Allerød, Denmark). This name may cause confusion because, according to the terminology, the method is not PRF but is clearly a P-PRP product.

2. Leukocyte- and Platelet-Rich Plasma (L-PRP) consists of preparations containing leukocytes and a low-density fibrin network after activation. Like P-PRP, all products in this family can be used as liquid solutions or in the form of an activated gel. They can therefore be injected (for example, in sports medicine) or applied to a skin wound or suture during gel formation, in a manner similar to the use of fibrin glues.

This family includes the largest number of commercial and experimental systems, with many interesting results in general surgery, orthopedic medicine and sports medicine.

In recent years, many automated protocols have been developed that require specific kits and allow blood samples to be processed minimally while ensuring maximum standardization of the preparations. Examples include Harvest Smart-PreP (Harvest Technologies, Plymouth, MA, USA) and Biomet GPS III (Biomet Inc., Warsaw, Indiana, USA). Other kits involve more extensive processing, such as Plateltex (Prague, Czech Republic) and Regen PRP (RegenLab, Le Mont-sur-Lausanne, Switzerland).

3. Pure Platelet-Rich Fibrin (P-PRF) — or leukocyte-poor, platelet-rich fibrin — consists of preparations without leukocytes and with a high-density fibrin network. By definition, these products exist only in a strongly activated gel form and cannot be injected or used like traditional fibrin glues. However, because of their strong fibrin matrix, they can be handled as a genuine solid material for other applications.

There is only one product in this family, commercially known as Fibrinet PRFM (Platelet-Rich Fibrin Matrix; Cascade Medical, Wayne, New Jersey, USA), also marketed for orthopedic applications by Vertical Spine (Wall, New Jersey, USA). The main disadvantages of this method remain its cost and relative complexity compared with other forms of PRF, particularly L-PRF (Leukocyte- and Platelet-Rich Fibrin).

4. Leukocyte- and Platelet-Rich Fibrin (L-PRF) consists of preparations containing leukocytes and a high-density fibrin network. By definition, these products exist only in a strongly activated gel form and cannot be injected or used like traditional fibrin glues. However, because of their strong fibrin matrix, they can be handled as a genuine solid material for other applications.

The technique was initially developed and evaluated as an open-access method based on the concept of single-step blood centrifugation without an anticoagulant and without a blood activator. The preparation is completely natural, and this remains its key distinction from all other product families. At present, the only FDA-cleared L-PRF system using certified materials is marketed under the name Intra-Spin L-PRF (Intra-Lock Inc., Boca Raton, Florida, USA).

The technique is very simple, fast and inexpensive, and makes it possible to produce a large number of fibrin clots and membranes in a very short time, particularly with the use of a compression preparation box. It is currently the main method in oral and maxillofacial surgery, especially because L-PRF membranes and clots can be combined very easily with modern surgical techniques.

Some applications of this technique have been proposed in sports medicine and orthopedics, with interesting results. However, these applications remain experimental because a way must be found to use the clots in each specific surgical procedure (in particular, how to keep the membranes/clots in an appropriate position), whereas PRP products are often simply injected as pharmaceutical preparations.

This classification system was widely cited, defended and confirmed by a multidisciplinary consensus conference published in 2012. POSEIDO (Periodontology, Orthopedic Surgery, Esthetic and Implant Dentistry Organization) considered it a guideline for all publications on this subject in 2013.

This terminology and classification are now regarded as a basis for consensus in many fields, particularly oral and maxillofacial disciplines. However, further developments of varying importance may be required in the future, depending on the clinical field.

From Terminology to Biological Mechanisms

The two parameters selected to define the four product families are obvious and logical, and there is consensus about their importance. Any researcher working with these products can immediately notice the substantial differences between the four families. These differences should also be identified and quantified using biological and clinical parameters.

Each product family has certain basic characteristics, but every individual product also has its own identity. Part of this identity has been defined and investigated as its biological signature, in terms of the amount and duration of the slow release of growth factors. Several studies have attempted to evaluate and compare the biological signatures of these materials.

The in-vitro behavior of L-PRF membranes and P-PRP gels was compared by evaluating the slow release of growth factors and matrix molecules.

These two gel families were placed in a culture medium for seven days. The slow release of three key growth factors—transforming growth factor beta 1 (TGF-β1), platelet-derived growth factor AB (PDGF-AB) and vascular endothelial growth factor (VEGF)—and three key coagulation and matrix proteins—thrombospondin 1 (TSP1), fibronectin and vitronectin—was quantified at seven experimental time points: 20 minutes, 1 hour, 4 hours, 24 hours (day 1), 72 hours (day 3), 120 hours (day 5) and 168 hours (day 7).

These studies showed that the products had two very different profiles. The L-PRF membrane remained solid and intact after seven days and continuously released large amounts of growth factors, a significant proportion of which was produced by the cell population within the membrane. In contrast, the P-PRP gel released most of its growth factors during the first few hours and completely dissolved in the medium after three days, even after maximum fibrin polymerization.

These studies confirmed earlier work on differences in fibrin structure between PRF families (natural polymerization involving intrinsic growth-factor enzymes) and PRP gel families (artificially induced polymerization involving the addition of external growth factors, resulting in their immediate release and use/degradation). These differences formed the basis of the classification system.

These studies identified very different biological signatures and mechanisms among the various product families. However, many other differences can be found within the families themselves, particularly when changes in cell populations and their preservation are considered.

From Terminology to General Clinical Implications

The classification of products and identification of their numerous differences have also made it possible to understand that each product family has its own characteristics and specific potential clinical applications. A general overview of the literature on this subject allows several preliminary statements to be made:

• The L-PRF family meets the requirements of oral and maxillofacial surgery because L-PRF clots and membranes have a volume and shape that can be easily combined with most surgical techniques, either as a biomaterial for filling and interposition or as a protective healing membrane. These membranes are also strong and provide the slow release of many growth factors over extended periods.

Finally, they are easy and inexpensive to prepare in large quantities, which makes them particularly suitable for everyday clinical practice.

• The PRF family as a whole can be used in other disciplines, with interesting results, particularly in the treatment of skin wound ulcers. However, these products exist only in a highly polymerized, activated form. Some applications have been described in orthopedics and sports medicine, but PRF products cannot be used, for example, as injectable products in sports medicine.
• The various PRP families are not well suited to routine oral applications because they are complex, expensive and have mixed clinical relevance. However, they are interesting alternatives to fibrin glues in many other surgical procedures, particularly for improving skin wound healing. Using PRP in gel form at a surgical site makes it an appropriate surgical adjunct in many situations, although its precise effects compared with fibrin glues remain largely debated.
• Liquid PRP solutions also have the advantage of remaining fluid and can therefore be used as injections in various sports medicine or orthopedic applications. In this regenerative-medicine strategy, platelet suspensions are injected in the same way as other pharmaceutical preparations. However, the results of this method remain largely debated in the literature, probably because of the large number of different protocols.

These initial statements provide a general overview of the current situation. While there is no longer much debate about which methods should be used in oral and maxillofacial surgery (currently, the discussion is largely settled because of the cost and complexity of alternatives and their lack of genuine advantages compared with the L-PRF method), the situation is much more confusing in other fields. In sports medicine, in particular, there is considerable debate about the choice of an appropriate technique, especially with regard to the exact cellular content of platelet injection suspensions.

Some groups have expressed the view that the presence of leukocytes may have a negative effect on therapeutic outcomes because of the potential risk of stimulating an inflammatory process after injection into an injured area. Conversely, other groups have insisted that a certain population of leukocytes is necessary in injectable PRP in order to increase growth-factor production, the release of pain-relieving mediators and natural anti-infective activity.

In general, many leukocytes, particularly lymphocytes, play a key role in regulating the healing and inflammatory processes, and there are no compelling reasons (or published results) to exclude them.

Leukocytes are not merely inflammatory cells: they also exert antinociceptive effects through various chemokines, anti-inflammatory cytokines (IL-4, IL-10 and IL-13) and opioid peptides (β-endorphins, metenkephalins and dynorphin A). They may therefore contribute to clinically significant inhibition of pathological pain. During inflammation, these cytokines counteract the action of pro-inflammatory mediators that are naturally released during the early stages of inflammation.

The current, not-yet-published consensus on this issue is that leukocytes are probably beneficial, but this depends on which leukocytes are present (lymphocytes, monocytes or granulocytes), in what quantity, and in what condition. The centrifugation process may gently activate them, pathologically stimulate an inflammatory state, or destroy white blood cells. This remains one of the most important points under discussion.

Other Classifications Proposed in Sports Medicine

The classification described above is the only system that covers all forms of platelet concentrates for surgical use. However, two other classification systems have been proposed in recent years. They are limited because they refer only to Platelet-Rich Plasma products and to sports medicine.

1. Mishra et al. proposed a classification specifically for use in sports medicine, taking into account platelet and leukocyte concentrations. This classification establishes four PRP types according to the presence or absence of leukocytes and whether or not the PRP is activated. Under this proposal, type 1 PRP is an L-PRP solution, type 2 PRP is an L-PRP gel, type 3 PRP is a P-PRP solution, and type 4 PRP is a P-PRP gel.

This classification therefore follows the same concept as the general classification published in 2009, but it is more limited (PRP only) and less intuitive (the PRP types are less self-explanatory than the clear terminology). The only new parameter in this classification is the assessment of platelet concentration: type A PRP has a platelet concentration five times or more than that in blood, whereas type B PRP has a concentration less than five times that in blood.

This last parameter is controversial because the concept of platelet concentration was largely abandoned in previous years for a logical reason: platelet concentration depends only on the volume of liquid serum used to keep the platelets in suspension. The amount of serum varies considerably according to the protocol and intended application and does not affect the expected effect.

The concept of the absolute platelet count would be more logical, even though most publications have failed to identify a clear and reproducible effect of this parameter on clinical outcomes. From this perspective, the fivefold threshold has no consensus-based meaning or justification.

2. Another system, called PAW (Platelets, Activation, White cells), has been proposed to organize and compare results in the literature. It emphasizes the platelet count (absolute number), the platelet activation method and the presence of white blood cells. This system is again limited to PRP families and is in fact very similar to the proposal by Mishra et al.

Leukocytes and activation (liquid or gel) are already clearly identified parameters. The question of platelet quantity remains a significant subject of debate because no publication has been able to determine what the optimal platelet quantity would be, or even whether such a concept truly exists for complex multicomponent materials such as platelet concentrates.

Both proposals are interesting, but they are not supported by sufficient evidence and do not actually provide a basis for updating the current terminology and classification as defined in 2009.

Perspectives for the Evolution of Classification

The terminology and classification introduced in 2009 represent an important step, but they are probably still incomplete, given the number of parameters involved in characterizing such complex products.

From a biological point of view, characterizing the presence of cells (such as leukocytes) is a critical step. However, many other parameters must be taken into account, including the platelet collection rate/quantity, leukocyte collection rate/quantity, detailed cellular composition, and the preservation of cells (their shape and level of stress) during collection and centrifugation. Activation of the cellular contents during or after centrifugation is also important for the biology of these products.

Other practical parameters should also be considered because they directly affect the feasibility of using these methods in everyday clinical practice. These include centrifuge size, preparation time, cost and ergonomics, the final volume of the product, and its physical form (liquid, soft gel or solid gel material). Finally, as was clearly stated in the second classification paper published in 2009, all these parameters must be considered together.

This integrated approach is still far from evident in the current literature, although some improvement can be observed in the characterization of the products being tested.

Classification and terminology will evolve in the coming years, and these developments are expected to focus on the precise cellular content of the L-PRP and L-PRF families.

Most publications on growth factors and platelet concentrations have demonstrated the relative importance of these parameters because of substantial inter-individual variations and the short-lived effects of these factors. Platelets are activated and remain active for only a very short period, while the released growth factors are consumed locally or dissolved in the bloodstream within minutes or hours after their release.

It is expected that an explanation for the mixed clinical results described in the literature will be found in the cell populations and activation characteristics of these products. Platelet concentrates for surgical use should be considered as an integration of all blood elements within a coherent healing platform, including the fibrin matrix, platelets, mediators and cells, all working together to achieve a clear and reproducible clinical outcome.

Many types of cells are present in these preparations. The precise leukocyte formula is an important parameter: the lymphocyte population is very diverse and does not have the same effects as monocytes and granulocytes. In addition, many other cells, such as circulating stem cells, may be found in platelet concentrates and should not be overlooked.

Finally, it is still unclear how the existing classification and terminology can be significantly improved. However, many different aspects may evolve in the future. In the meantime, it is very important for all authors in this field to describe the products they test precisely, in order to make a genuine and substantial contribution to the literature on this simple yet complex subject.