Platelets have been studied extensively over recent decades for their ability to enhance cell proliferation, migration, and tissue regeneration. The clinical use of autologous platelets and their secreted growth factors (GFs) has shown promise as a therapeutic approach and has been applied in clinical fields such as traumatology, dentistry, and plastic surgery [[1], [2], [3], [4], [5], [6]]. Especially the application in osteoarthritis (OA) has been researched extensively [[7], [8], [9]]. More recently, their use has expanded to dermatology (e.g. skin rejuvenation) and sports medicine, where they are applied to treat chronic tendinopathies and ligament injuries [[10], [11], [12]]. Platelet-rich plasma (PRP) consists of a complex mixture of growth factors, cytokines, and cellular components that regulate key processes such as angiogenesis, inflammation, and extracellular matrix remodelling. These growth factors, including PDGF subtypes, VEGF and TGF-β, play crucial roles in tissue repair and regeneration [[13], [14], [15]]. Although the quality and effectiveness of these products are influenced by various factors during harvesting and processing—ranging from the choice of anticoagulant to the application method—no universal standards exist for these parameters in the use of PRP [16,17].

While many reviews address these aspects, most focus on select variables or specific fields of application; however, a comprehensive analysis of all factors affecting PRP has yet to be published [3,[18], [19], [20], [21], [22]]. Recent efforts have focused on developing classification systems (presented below), which aim to standardize the characterization of PRP based on platelet and leukocyte concentrations, anticoagulant use, and activation protocols.

This review aims to provide in-depth information on each step in processing PRP, highlighting differences between their forms and discussing factors that influence their quality. To foster a deeper understanding of the biochemical functions of platelets and their derivatives, we will describe their cellular and microscopic characteristics, evaluate the impact of different isolation, activation, and application methods, and outline key influencing factors, such as patient- and drug-related variables, offering a comprehensive analysis of all factors involved in producing platelet-rich products.

Our goal is to provide a practical framework for researchers and clinicians to optimize the use and avoid common pitfalls of this promising resource, and to contribute to its standardization in clinical practice.

Over the years, extensive research has led to the establishment of numerous naming conventions and classification systems for platelet-rich products. Due to the variability in the composition of platelet-rich products, which significantly impacts their biochemical function, the inconsistent terminology used to describe these products has contributed to the variability in reported outcomes [23]. Especially the term PRP has often been applied indiscriminately to several distinct platelet-rich products despite significant differences in their composition and corresponding biochemical effects. The use of overlapping and nonspecific terms presents a significant challenge in the field of platelet-rich products. Despite efforts towards establishing a standardized classification system (Harrison et al [16], the MARSPILL system [24], the DEPA classification by Magalon et al [25], and Mishra et al [26]), no single system has prevailed and been universally adopted in the literature. Classification systems for PRP typically include platelet and leukocyte concentrations and, in some cases, the recovery rate. These parameters are encoded using different letters or numerical codes, resulting in inconsistencies that can lead to the same product being classified differently across systems. For example, the MISHRA classification refers to platelet enrichment relative to baseline, whereas the MARSPILL system uses a different terminology for the same parameter, as reviewed by Magalon et al. (2021) [21]. For a more detailed comparison of classification systems and their differences, a comprehensive review by Acebes-Huerta et al. is recommended [27].

According to Everts and colleagues, an adequate classification system needs to include the biological product allocation, preparation technology, anticoagulant, platelet dosing, leukocyte content, RBC content, delivery form, fibrin matrix, activation methods, additives, and routes of administration [23]. In this review, we aim to provide comprehensive information to facilitate the evaluation of the quality of isolated PRP.

Therefore, we will define and clarify the key terms relevant to this review below.

Platelet-rich plasma (PRP): Platelet concentration of minimum 1 × 10⁶/µl [28], further divided by its composition (leukocyte-rich (LrPRP) and leukocyte-poor (LpPRP), red blood cell (RBC)-rich/poor PRP) or processing (activated/non-activated PRP) [16].

Platelet-rich fibrin (PRF): A fibrin matrix containing platelets and leukocytes. Whole blood collected without anticoagulant undergoes coagulation during centrifugation, resulting in the formation of a fibrin matrix that entraps platelets and leukocytes [27,29].

Platelet lysate (PL): A cell-free solution containing platelet-derived components, such as growth factors (GFs), obtained through platelet disruption followed by the removal of cellular remnants [2].

Plasma-free platelet lysate: Platelets isolated by centrifugation are resuspended in a HEPES buffer and further processed by filtration to remove cell debris [30].

A topic of ongoing debate is whether to use LrPRP or LpPRP, typically defined by leukocyte concentrations above or below baseline levels, respectively [24,26,31]. While leukocytes may contribute to inflammatory responses, they can also enhance GF concentrations, particularly TGF-β1 [32]. Clinical applications of LrPRP have demonstrated substantial benefits in randomised controlled clinical trials and preclinical studies [[33], [34], [35], [36]]. However, adverse effects—such as increased pain and swelling—have been reported in a meta-analysis of intra-articular LrPRP injections including 32 studies with an evidence level between 1 and 4 [37]. In vitro, Yin et al. reported superior effects of LpPRP on human bone marrow-derived mesenchymal stromal cells (MSCs), whereas LrPRP was found to induce an inflammatory response via the NF-κB pathway [38]. Xu and colleagues observed similar outcomes in cartilage regeneration [39]. Therefore, PRP composition should be tailored to the specific clinical scenario. For instance, LpPRP may be better suited for musculoskeletal applications, as LrPRP has been shown to have deleterious effects on synovial cells, inducing cell death and the production of proinflammatory mediators [38,40]. Conversely, LrPRP may be more effective in the treatment of large, infected wounds [41,42]. Everts et al. advocate for a more nuanced view of leukocyte content in PRP, noting that lymphocytes support tissue remodelling and secrete IGF-1, while macrophages and monocytes exert immunomodulatory effects [23].

Platelets, or thrombocytes, are anucleate cell fragments derived from megakaryocytes, typically measuring approximately 2 µm in diameter [43]. Platelets are involved in several physiological and pathological processes, such as haemostasis, wound healing and tissue regeneration, thrombosis, and tumor growth [[44], [45], [46], [47]]. Membrane-associated receptors, such as integrins and glycoproteins, play a crucial role in platelet adhesion, aggregation, and activation – key processes in haemostasis and wound healing [48]. Specialized integrins on the cell surface are essential for interactions with other cells, including leukocytes and endothelial cells [49]. Platelets also participate in a variety of processes within both the innate and adaptive immune systems, interacting with neutrophils and monocytes via toll-like receptor-mediated mechanisms [[50], [51], [52]]. Activated platelets, expressing P-selectin, can contribute to T-cell recruitment to injured blood vessel walls and influence their proliferation and function [53]. Platelet-derived cytokines and GF stimulate immune cells in various ways, possibly inducing both pro- and anti-inflammatory effects [[54], [55], [56], [57]]. Platelets have also been shown to interact with bacteria, either through direct binding or indirectly via inflammatory cytokines [58]. bacterial adhesion to platelets can trigger platelet activation through glycoproteins and Toll-like receptors. This activation promotes platelet aggregation, the release of antibacterial peptides, and interactions with innate immune cells such as neutrophils [59]. These interactions can enhance phagocytosis and strengthen the immune response, potentially contributing to the antibacterial properties of PRP [60,61].

Numerous signalling molecules are stored in the dense granules, α-granules, and lysosomes, which are part of the platelet structure, as well as released via extracellular vesicles generated by platelets (FIG. 1). Upon activation, platelets undergo a cytoskeletal rearrangement, changing shape and releasing their granule content [62,63]. This process is central to the function of platelet-rich products and their mechanisms of action.

Platelet-derived extracellular vesicles (PDEVs) are membrane vesicles encapsulated by phospholipid bilayers and secreted by platelets. They have been found to take part in coagulation via increased thrombin expression, as well as play distinct roles in cell communication and tissue repair [64,65]. PDEVs are categorized into platelet-derived exosomes (PDEs) and platelet-derived microparticles (PDMs), distinguished by their biogenesis, size, and content [65,66]. PDEs originate from early endosomes following membrane endocytosis and are secreted through the exocytosis of multivesicular bodies (matured endosomes). In contrast, PDMs are released directly via budding of the plasma membrane [67]. PDEs are smaller, ranging from 40–100 nm, whereas PDMs are significantly larger, measuring 0.1–1 µm [68,69].

PDEVs play an important role in coagulation [[70], [71], [72]], have been shown to increase angiogenesis [73,74], and mediate both pro- and anti-inflammatory signals [[75], [76], [77], [78], [79]]. PDEVs can induce regeneration and proliferation in many tissues and cell types [80], but have also been implicated in tumor progression [69,81,82]. PDEVs have also been proposed as markers of platelet activation, given their increased vesiculation in response to mechanical and chemical stimuli [[83], [84], [85]]. PDEVs have been explored for potential therapeutic applications and as alternatives to PRP due to their higher concentrations of GFs and smaller size compared to platelets. However, research into their clinical utility remains in its early stages [[86], [87], [88], [89]]. For a more in-depth overview on PDEVs, we recommend recent comprehensive reviews, e.g. by Anitua and colleagues [90] or Melki and colleagues [50].

Dense granules primarily contain adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin, and Ca2+-ions, which play roles in activating and recruiting additional platelets and initiating the release of α-granule contents [91,92]. Additionally, lysosomal granules house the proteinases cathepsins D and E [2], which are involved in cartilage turnover [93].

α-granules store GFs that regulate cell proliferation, differentiation, and gene expression across various cell types, including endothelial, immune, MSCs, chondrocytes, and osteoblasts [55,[94], [95], [96], [97]]. They also contain clotting factors (I, V, VIII), essential for haemostasis and thrombus formation, facilitating platelet interaction with the coagulation cascade to form a gel-like aggregate [98].

Many GFs function through overlapping pathways, binding distinct receptors, regulating each other, and activating multiple receptor types, making them most effective in combination [22]. For instance, PDGF-BB induces EGF transactivation in venous smooth muscle cells [99], while EGF and TGF-β1 synergistically enhance epithelial cell activity [100].

Common GFs associated with platelets are introduced below and listed in Table 1.

Before analyzing platelet-rich product processing, it is crucial to consider factors influencing PRP quality. Factors influencing PRP composition are summarized in Table 2.

The choice of phlebotomy method is one relevant factor, as both the blood draw site and needle type appear to influence platelet recovery and viability [128]. For example, Waters and Roberts suggest that blood samples from peripheral veins yield the highest platelet counts [129]. To minimize shear stress, Breddin and Harder advised against using a butterfly needle [130]. However, Mani and colleagues found that a 21-gauge butterfly needle does not affect platelet aggregation [131].

Patient-related factors such as age and sex should also be considered. Individuals over the age of 60 often exhibit thrombocytopenia, which may reduce the number of platelets that can be harvested [132]. Data on sex-related differences remains inconclusive in the literature [128,[133], [134], [135]]. However, some studies suggest higher cytokine and GF levels in male patients [136].

Patient hematocrit must also be considered, as higher WB dilution can result in reduced platelet counts [128]. Conversely, Piao et al. observed increasing platelet concentrations with decreasing leukocyte levels and recommended dilution prior to centrifugation [17]. Interestingly, Verboket and colleagues observed altered PRF composition in trauma patients, particularly regarding inflammatory cytokines and monocytes. The ratio of inflammatory to anti-inflammatory monocytes was significantly higher in trauma patients compared to healthy donors. Additionally, VEGF and IGF-1 levels were markedly lower in the PRF of trauma patients than in that of healthy donors. These findings suggest a pro-inflammatory effect of PRF derived from trauma or surgical patients, potentially influencing tissue healing [137]. However, further investigations on proinflammatory cytokine profiles will be needed to compliment these findings.

Medication-related factors must also be considered. PRP activation via thrombin receptor agonist peptide (TRAP) or thrombin can be influenced by a patient's use of direct oral anticoagulants, such as Dabigatran [138]. Additionally, ADP-induced platelet activation is inhibited by clopidogrel [91]. COX-2 inhibitors have been shown to have no effect on GF release from thrombin-activated PRP [139].

Considering all these influencing factors, it is evident that the production of a high-quality platelet-rich product must begin with a thorough evaluation of both patient-related and material-related variables. Researchers working with PRP should recognize and report these factors accordingly.