The use of platelets in regenerative medicine and proposal for a new classiﬁcation system: guidance from the SSC of the ISTH
Harrison P, for the Subcommittee on Platelet Physiology. The use of platelets in regenerative medicine and proposal for a new classiﬁcation system: guidance from the SSC of the ISTH. J Thromb Haemost 2018; 16: 1895–900.
Summary. Autologous and single-donor allogenic platelet preparations are increasingly being used in many areas of regenerative medicine. However, there are few properly controlled randomized clinical trials, and the preparation, content and characteristics of platelet preparations are generally poorly deﬁned and controlled. The Platelet Physiology Subcommittee of the Scientiﬁc and Standardization Committee (SSC) of the ISTH formed a working party of experts with the aim of producing consensus recommendations for guidance on the use of platelets in regenerative medicine. Owing to a lack of investigations that provide deﬁnitive evidence for the efﬁcacy, deﬁnition and use of different platelet preparations in regenerative medicine, there were insufﬁcient data to develop evidence-based guidelines. Therefore, the RAND method was used, which obtains a formal consensus among experts particularly when scientiﬁc evidence is absent, scarce and/or heterogeneous. Using this approach, each expert scored as ‘appropriate’, ‘uncertain’ or ‘inappropriate’ a series of 45 statements about the practice of regenerative medicine with platelets, which included different sections on general aspects, platelet preparations, clinical trial design, and potential utility in different clinical scenarios. After presentation and public discussion at SSC meetings, the assessments were further reﬁned to produce ﬁnal consensus recommendations, which constitute the subject of the present report.
Correspondence: Paul Harrison, Institute of Inﬂammation and Ageing, University of Birmingham, Birmingham, B15 2TT, UK Tel: +44 121 371 3251 E-mail: email@example.com 1 See Appendix for a full list of contributors
Received: 2 May 2018 Manuscript handled by: N. Mutch Final decision: F. R. Rosendaal, 25 June 2018
Platelet-rich plasma (PRP), an autologous or allogeneic derivative of whole blood that contains a supraphysiological concentration of platelets, has gained increasing attention in both the scientiﬁc literature and the wider media for its potential application as a regenerative adjunctive therapy [1–5]. The platelet a-granules provide a vast array of growth factors and cytokines that are important in wound healing . These factors can promote local angiogenesis, stem cell homing, local cell migration, proliferation and differentiation, coupled with the deposition of matrix proteins, such as collagen, which all play a key role in enabling the restoration of normal tissue structure and function. The regenerative effect of PRP exerted by producing a local environment for tissue regeneration has been supported by in vitro and in vivo studies that suggest a positive inﬂuence on the migration and proliferation of a number of cell types. Although PRP therapy is becoming increasingly popular and widely employed, clinical use initially centered on its potential application in dental and maxillofacial surgery. The regenerative effects of PRP on bone, cartilage, skin, tendon and muscle have also attracted interest in orthopedic and plastic surgery, where restoration of poorly vascularized and damaged tissue is a critical determinant of successful clinical outcome [6–12]. Given the complexity of the material, the rudimentary knowledge of the mechanism of action of PRP, and the limitations of current studies, any future clinical trial should be carefully designed to accurately determine any clinical effects, and, importantly, should also use disease-speciﬁc outcome tools. Imprecision in effect size estimates from underpowered studies with PRP has also undoubtedly led to unreliable conclusions . The ﬁeld is also plagued with poor standardization and variability in the methods used to generate PRP, with variability in the terminology, purity, content and quality of products utilized [13,14]. Depending upon its method of preparation, PRP can also be deﬁned as platelet-enriched plasma, platelet-rich concentrate, platelet concentrate, leukocyte-rich PRP, platelet-rich ﬁbrin, PRP rich in growth factors, platelet-rich ﬁbrin matrix, autologous concentrated plasma, platelet gel, pure PRP, platelet releasate, etc. [15–19]. Platelet products therefore have varying concentrations of blood cells (platelets, leukocytes, and red blood cells), plasma, or ﬁbrinogen. Unsurprisingly, the content, purity and hence the biological properties of those products therefore vary widely and impact on their potential efﬁcacy. Several clinical trials have therefore been conducted without clear deﬁnition or quantiﬁcation of the PRP biological properties, leading to varying efﬁcacies and outcomes. There are also many types of commercial PRP preparation devices, resulting in further variation in product content . PRP regenerative properties are based on the production and release of bioactive proteins, including multiple growth and differentiation factors, upon platelet activation. However, the role of other cells that may be present in PRP, such as leukocytes and red blood cells, is still unclear and cannot be excluded. The presence of leukocytes may have a great impact on the biology of these preparations, not only because of their immune and antibacterial properties, but also because they are key players in tissue healing and local growth factor regulation . PRP also implies the application of autologous plasma along with platelet-derived proteins. Therefore, it promotes the development of a ﬁbrin scaffold at the desired location that can act as a temporary matrix to assist repair of the injured tissue. Deﬁning the potential role(s) of all of the bioactive factors present in any platelet preparation is therefore essential to understand the biological activities. Autologous PRP can be prepared in a laboratory, operating theater or a clinic room from anticoagulated blood collected at the time of the therapeutic application. Trisodium citrate is the most widely used anticoagulant, and has few negative effects on PRP preparation. Acid citrate dextrose (ACD) and citrate phosphate dextrose (CPD), including ACD with adenine (ACD-A) and CPD with adenine (CPD-A), are also effective anticoagulants. EDTA is not usually recommended for PRP preparation, as this causes platelet swelling and activation.
Traditionally, a relatively pure preparation and good yield of PRP can be easily obtained in a single step by centrifugation of anticoagulated blood at low force (170–200 9 g) for 10 min at room temperature . For clinical applications, there are essentially three main methods available that can rapidly provide a sterile PRP product: (i) gravitational centrifugation techniques; (ii) standard cell separators; and (iii) autologous selective ﬁltration technology (plateletpheresis). Standard cell separators and salvage devices generally operate on a full unit of blood. In general, they use continuous-ﬂow centrifuge bowl or continuous-ﬂow disk separation technology, coupled with hard and soft centrifugation steps. High g-force centrifugation is normally used to isolate the buffy coat layer containing platelets and leukocytes.
Differences in the g-force and centrifugation time used in the different preparation techniques will also result in signiﬁcant differences in yields, concentration, purity, viability and activation status of the isolated platelets, and will also affect eventual clinical efﬁcacy. PRP must also be activated for the platelets to release their a-granule contents, with the clot providing a scaffold to capture the secreted proteins and maintain their presence at the application site. This introduces another set of variables, including method of activation (thrombin, re-calciﬁcation, etc.), duration of activation, and whether activation should be performed prior to clinical application. Although platelets are popular and widely used, their use in regenerative medicine is clearly poorly standardized. Although some guidelines have been published, if this ﬁeld is to progress then standardization of the methods for the generation of the various platelet-rich preparations and evidence-based guidelines for future clinical trials are urgently required [23,24]. In January 2016, the Platelet Physiology Subcommittee of the ISTH formed a working party of 10 well-known experts in the ﬁeld of regenerative medicine with the aim of producing a series of consensus recommendations for standardizing the use of platelets in regenerative medicine. The working party then used a formal consensus method (RAND method) to develop its ﬁnal recommendations.
The RAND method – developed by the RAND corporation in the 1980s – obtains a consensus among expert groups about the appropriateness of healthcare interventions when scientiﬁc evidence is absent, scarce, or heterogeneous . A series of statements about platelets in regenerative medicine were therefore formulated, and each member of the working party then scored each one for appropriateness from 1 (completely inappropriate) to 9 (fully appropriate). Other panel members were blinded to individual scores, and the two extreme scores (highest and lowest) were then discarded. The median of the remaining eight scores were then calculated, and the statements were classiﬁed as either inappropriate (scores of 1– 3), uncertain (scores of 4–6), or appropriate (scores of 7– 9). The panel was also asked to give any further comments of relevance to this ﬁeld. Recommendations were then presented at ISTH Scientiﬁc and Standardization Committee (SSC) meetings, and it was agreed that these could form the basis of a consensus document. The recommendations of the working party on the use of platelets in regenerative medicine derive from 45 statements grouped into four main sections:
(i) general aspects; (ii) platelet preparation; (iii) clinical trial design; (iv) utility of platelets in different clinical scenarios. Overall, 26 statements were scored as appropriate (for 11 statements there was complete agreement, and there was also complete agreement for an additional 15 statements after removal of the two extreme scores), and 19 statements were judged to be uncertain. No statements were judged to be inappropriate. The term PRP used in the statements below encompasses all platelet and platelet-related products used for regenerative medicine.
Results: General aspects
1. It is uncertain whether PRP preparations (autologous or allogeneic) are clinically useful in tissue regenerative techniques (median score of 6; range of 5–8).
2. It is uncertain whether platelet gels or clots are clinically useful in regenerative medicine (median score of 6; range of 5–7).
3. It is uncertain whether platelet-rich ﬁbrin is clinically useful in regenerative medicine (median score of 6; range of 5–7).
4. Appropriate. The term PRP is confusing, too general, and incomplete (median score of 8.5; range of 7–9).
5. Appropriate. Autologous/allogeneic sterile PRP preparations are clinically safe (median score of 7.5; range of 5–8).
6. Appropriate. Clinical preparations of PRP are poorly standardized (median score of 8.5; range of 7–9; complete agreement by the entire panel).
7. Appropriate. The content, purity and biological properties of PRP all vary widely, and will impact on clinical efﬁcacy (median score of 8.5; range of 6–9).
8. Appropriate. Many trials or studies fail to fully deﬁne the content, purity and biological properties of platelet preparations (median score of 9; range of 8–9; complete agreement by the entire panel).
1. It is uncertain whether PRP should be prepared from citrate-anticoagulated whole blood (median score of 5.5; range of 3–7).
2. It is uncertain whether PRP should be prepared from ACD-anticoagulated whole blood (median score of 6.5; range of 4–8).
3. It is uncertain whether PRP should be prepared from CPD-anticoagulated whole blood (median score of 4.5; range of 2–7).
4. Appropriate. EDTA anticoagulant should not be used for PRP preparation (median score of 8; range of 6–9).
5. It is uncertain whether PRP should ideally be produced by low-g centrifugation for short periods (e.g. 170 9 g for 10 min) to maximize platelet yield and minimize cellular contamination (median score of 5; range of 5–8).
6. Appropriate. Platelet concentration, yield and recovery are dependent on the centrifugation protocol and collection methods utilized (median score of 8; range of 8–9; complete agreement by the entire panel).
7. It is uncertain whether PRP, once prepared, is stable for clinical use up to 6 h after preparation (median score of 6; range of 4–8).
8. It is uncertain whether PRP must be activated for platelets to release their granule contents before application (median score of 5; range of 4–8).
9. Appropriate. In some applications, collagen-rich tissues may also activate PRP and eliminate the need for a preapplication activation step (median score of 7; range of 5–8).
Utility in different clinical scenarios
1. It is uncertain whether PRP can be used to treat burn injuries (median score of 6.5; range of 5–7).
2. Appropriate. PRP can be used for general wound healing (median score of 7; range of 5–7).
3. It is uncertain whether PRP can be used to treat tendon injuries (median score of 5.5; range of 5–8).
4. It is uncertain whether PRP can be used for acute muscle injuries (median score of 5; range of 5–6).
5. It is uncertain whether PRP can be used for bone healing (median score of 6; range of 4–7).
6. It is uncertain whether PRP can be used for maxillofacial injuries (median score of 5.5; range of 4–8).
7. It is uncertain whether PRP can be used to treat sports injuries (median score of 5.5; range of 5–7).
8. It is uncertain whether PRP can be used to treat osteoarthritis (median score of 6.5; range of 5–8).
9. It is uncertain whether PRP can be used to prevent skin aging as a beauty therapy (median score of 4; range of 2–5).
All of these statements are also summarized in Table S1. Further general comments from the panel are also included in Data S1.
Clinical trial design
Clinical trials on the use of platelets in regenerative medicine should be adequately powered and controlled, with some essential design requirements, including the following:
1. Appropriate. Randomized placebo-controlled design (median score of 8; range of 7–9).
2. Appropriate. Sample size calculation based on clear predeﬁned endpoints (median score of 8.5; range of 7– 9; complete agreement by the entire panel).
3. Appropriate. Clear inclusion/exclusion criteria (median score of 9; range of 7–9; complete agreement by the entire panel).
4. Appropriate. A homogeneous study population (median score of 8; range of 7–9).
5. Appropriate. Standardized clinical assessments (median score of 9; range of 6–9; complete agreement by the entire panel).
6. Appropriate. Clinical endpoints objectively measured (median score of 9; range of 7–9).
7. Appropriate. Validated PRP production and delivery methods (median score of 9; range of 6–9).
8. Appropriate. Details of the applied treatment, including number of platelets, number of applications, duration of treatment. etc. (median score of 9; range of 7–9; complete agreement by the entire panel).
9. Appropriate. Robust clear clinical outcomes (median score of 9; range of 7–9).
10. Appropriate. Standardized post-treatment follow-up protocol (median score of 9; range of 7–9; complete agreement by the entire panel).
11 Appropriate. Full description of PRP preparation methodology (median score of 9; range of 8–9; complete agreement by the entire panel).
12. Appropriate. Details of the amount of autologous blood collected (median score of 9; range of 7–9).
13. Appropriate. Baseline number, volume and concentration of platelets utilized (median score of 8; range of 7–9; complete agreement by the entire panel).
14. It is uncertain whether the overall yield of platelets obtained during PRP preparation should be reported (median score of 6.5; range of 6–9).
15. Appropriate. The purity of the ﬁnal PRP preparation should be reported, i.e. how many red blood cells and white blood cells are contaminating the PRP and whether the preparation contains plasma (median score of 8; range of 7–9; complete agreement by the entire panel).
16. Appropriate. A measurement of PRP quality is required, i.e. platelet activation status prior to clinical use (to clarify whether platelets have already lost their granular content) (median score of 7; range of 5–9).
17. It is uncertain whether the measurement of the growth factor content of the PRP preparation used should be reported (median score of 6.5; range of 4–9).
18. Appropriate. The activation procedure, if used, should be reported (median score of 9; range of 7–9).
19. Appropriate. A new classiﬁcation system for PRP is required, taking into account all key variables (median score of 9; range of 7–9).
Autologous or allogeneic platelet administration remains an attractive and popular strategy in many clinical scenarios, given the cost-effective, minimally invasive and safe nature of this therapy . The statements presented here should contribute to improving the standardization and design of future clinical trials using platelets in regenerative medicine, and agree with some parts of recent recommendations [23,24]. Future trials should not only be appropriately controlled and adequately powered, but also take into account the content and quality control of the platelet preparations to ensure that clear correlations between the products and outcomes are established [23,24,26–28]. Further studies on the mechanism of platelet tissue regeneration and optimal platelet preparation may help to elucidate the best combination of bioactive factors to achieve maximal regenerative activity . It will be important for guidelines such as this one to also be reviewed and updated in the future, as the ﬁeld continually changes and evolves.
Proposed new classiﬁcation system for PRP and related products
The authors of this guideline suggest a new classiﬁcation system, which incorporates previous classiﬁcation systems, and includes the preparation method technique [16,18]. Activation is divided into three subcategories:
– I For PRP application without activator
– II For the use of PRP with activation
– III For use of frozen–thawed preparations
Platelet concentrates are subdivided into three categories (A, B, and C) based on the platelet count range in the samples. The three categories are: A Platelet count of < 900 9 103 lL–1 B Platelet count of 900–1700 9 103 lL–1 C Platelet count of > 1700 9 103 lL–1
The preparation methods are classiﬁed into three categories:
1 Gravitational centrifugation techniques 2 Standard cell separators 3 Autologous selective ﬁltration technology (plateletpheresis)
The modiﬁed classiﬁcation is summarized in Table 1. This includes the activation method if used, the total volume used, the frequency of dosing and subcategories of activation, and platelet concentration and preparation techniques, and would include the overall average counts and range (low–high) of platelets and red blood cells, and differential leukocyte counts (neutrophils, lymphocytes, and monocytes).
Paul Harrison is supported by the Scar Free Foundation. The authors would like to thank L. Schmeidler and C. Clark from the ISTH for their help with the distribution and grading of the RAND method described in this manuscript. The authors would also like to thank the ISTH platelet physiology SSC for support in organizing this study.
The authors state that they have no conﬂict of interest.
The Subcommittee on Platelet Physiology Working Party
J. Alsousou, Liverpool, UK; I. Andia, Barakaldo, Spain; T. Burnouf, Taipei City, Taiwan; D. Dohan Ehrenfest, Gwangju, South Korea; P. Everts, Fort Myers, USA; H. Langer, T€ubingen, Germany; J. Magalon, Marseille, France; R. Marck, Amsterdam, the Netherlands; P. Gresele, Perugia, Italy.
Additional supporting information may be found online in the Supporting Information section at the end of the article:
Table S1. A summary of the median scores and ranges of the working party for each of the 45 statements in the RAND survey about platelets in regenerative medicine. Each statement is classiﬁed as either inappropriate (scores 1–3), uncertain (4–6) or appropriate (7–9). The term PRP used in the statements encompasses all platelet and platelet-related products used for regenerative medicine. Data S1. General comments from the panel.
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