William R. Parrish

Normal platelet function in platelet concentrates requires non-platelet cells: a comparative in vitro evaluation of leucocyte-rich (type 1a) and leucocyte-poor (type 3b) platelet concentrates

Background Therapeutic success of platelet-rich plasma (PRP) may vary based on the composition and preparation method. The objective of this study was to evaluate the cellular components of platelet concentrates produced by a leucocyte-rich (LR-PRP) and a leucocyte-poor PRP systems (LP-PRP). Methods Parameters evaluated included platelet recovery, platelet concentration, red blood cell (RBC) and white blood cell (WBC) composition, platelet growth factor release and stimulation of human tendon cell proliferation in vitro. Results Platelet recoveries were 52% for LP-PRP and 89% for LR-PRP. LR-PRP demonstrated greater reproducibility with a 4.2% coefficient of variation (CV) compared with 19.4% for LP-PRP (p<0.001). LR-PRP demonstrated a greater increase in platelet concentration (7.9-fold) than LP-PRP (2.2-fold; p<0.001). LP-PRP showed 5.0-fold reductions in WBCs, while LR-PRP showed a 4.0-fold increase (p<0.001). LP-PRP reduced RBCs to a haematocrit of 0.25, while LR-PRP reduced haematocrit to 11.8. LP-PRP did not coagulate robustly on reactivation with CaCl2, and released significantly lower levels of epidermal growth factor (EGF) and transforming growth factor β1 (TGF-β1) than whole blood (p<0.03). LP-PRP also did not stimulate tendon cell proliferation greater than whole blood. In contrast, LR-PRP showed increases in each growth factor on activation with CaCl2 (p<0.01) and stimulated greater proliferation (p<0.05) compared with whole blood. Forced activation of LP-PRP with exogenous thrombin rescued the coagulation deficiency and induced greater growth factor release than comparable whole blood (p<0.03). Conclusions These data suggest that non-platelet cellular components in platelet concentrates are important for proper platelet function, including thrombin generation, growth factor release and clot retraction.

Efficacy of platelet-rich plasma injections for symptomatic tendinopathy: systematic review and meta-analysis of randomised injection-controlled trials

Aim To determine the efficacy of platelet-rich plasma (PRP) injections for symptomatic tendinopathy. Design Systematic review of randomised, injection-controlled trials with meta-analysis. Data sources Systematic searches of MEDLINE and EMBASE, supplemented by manual searches. Eligibility criteria for selecting studies Randomised controlled trials with 3 months minimum follow-up that evaluated pain reduction with PRP versus control (saline, local anaesthetic, corticosteroid) injections in patients with symptomatic tendinopathy. Results A total of 16 randomised controlled trials (18 groups) of PRP versus control were included. Median sample size was 35 patients, a study size that would require an effect size ≥1.0 to achieve statistical significance. PRP was more efficacious than control in reducing tendinopathy pain, with an effect size of 0.47 (95% CI 0.22 to 0.72, p<0.001), signifying a moderate treatment effect. Heterogeneity among studies was moderate (I2=67%, p<0.001). In subgroup analysis and meta-regression, studies with a higher proportion of female patients were associated with greater treatment benefits with PRP. Conclusions Injection of PRP is more efficacious than control injections in patients with symptomatic tendinopathy.

Effect of Leukocyte Concentration on the Efficacy of PRP in the Treatment of Knee OA: Letter to the Editor.

Dear Editor: We read with interest the network meta-analysis by Riboh and colleagues titled ‘‘Effect of Leukocyte Concentration on the Efficacy of Platelet-Rich Plasma in the Treatment of Knee Osteoarthritis,’’ which compared intra-articular injection of leukocyte-rich platelet-rich plasma (LR-PRP), leukocyte-poor platelet-rich plasma (LP-PRP), and hyaluronic acid (HA) for treatment of knee osteoarthritis (OA). While the authors should be applauded for the considerable effort involved in the conduct of this research, the methodology and conclusions are questionable and warrant further clarification. The primary concern relates to the paucity of available data. The number of studies reporting Western Ontario and McMaster Universities Arthritis Index (WOMAC) outcome scores were 3 for HA, 1 for LR-PRP, 3 for LP-PRP, and 1 for placebo. Similarly, International Knee Documentation Committee (IKDC) scores were provided in 2 HA studies, 3 LR-PRP studies, 2 LP-PRP studies, and 1 placebo study. Lastly, adverse reactions were included for 2 LR-PRP studies, 3 LP-PRP studies, 4 HA studies, and 1 placebo study. Our view is that no valid conclusions can be derived from a network meta-analysis with such limited data. A second concern relates to the method of analyzing WOMAC and IKDC data. For each outcome, the reported statistic was the absolute change from baseline to final follow-up. Yet mean baseline scores in the included studies ranged from 39 to 80 for WOMAC and from 41 to 56 for IKDC. Given these differences in baseline symptom severity among studies, a given absolute WOMAC improvement from a baseline score of 39 has a vastly different interpretation than the same absolute improvement from a baseline of 80. In fact, the likely reason that LP-PRP was deemed most efficacious was because the study with the greatest WOMAC improvement also had an unusually high baseline WOMAC score. A third concern relates to study selection criteria and heterogeneity in patient characteristics. Each of the 3 LR-PRP studies that reported IKDC scores were performed by the same group using the same protocol involving repeat injections with devitalized freeze-thawed PRP, which is not characteristic of clinical practice. Comparing outcomes from studies using freshly isolated LP-PRP comprising living cells with studies that used freezethawed LR-PRP containing dead cells is not appropriate. In addition, the study by Patel et al that compared 1 versus 2 PRP injections in bilateral OA patients and the study by Kon et al that compared lowversus high-molecularweight HA should have been excluded based on the authors’ study selection criteria (exclusion 3). An example of the deleterious synergistic effects of these concerns relative to WOMAC scores is demonstrated in Table 1, which shows extremely limited data and tremendous differences in baseline symptom severity such that reporting absolute improvement as the outcome of interest is flawed and pooling data among studies is questionable. The main findings of this network metaanalysis are dominated by outcomes where no or limited direct evidence is available, which suggests high risk of bias. Inclusion of nonrandomized trials in this systematic review compounds this risk. Finally, since all surface under the cumulative ranking curve (SUCRA) data were derived from the pooled meta-analysis data that suffer from these same data limitations, the SUCRA data should be considered unreliable. In summary, a more appropriate conclusion to this manuscript would have been that conduct of a network metaanalysis is inappropriate and no reasonable conclusions can be drawn regarding the effect of leukocyte concentration on the safety and efficacy of PRP in knee osteoarthritis.

Comparison of the Acute Inflammatory Response of 2 Commercial PRP Systems: Letter to the Editor

Dear Editor: We read the article by Dragoo and colleagues titled ‘‘Comparison of the Acute Inflammatory Response of Two Commercial Platelet-Rich Plasma Systems in Healthy Rabbit Tendons’’ with great interest. The authors conducted the work, which was supported by the manufacturer of the leukocyte-poor platelet-rich plasma (LP-PRP) device, under the premise that leukocyte-rich PRP (LRPRP) would incite a greater acute inflammatory response to the detriment of the treated tissue than LP-PRP, based on the results of in vitro studies. However there is an everexpanding body of primary clinical literature that does not support these findings, and to the contrary, indicates an anti-inflammatory and pain relieving therapeutic effect of LR-PRP in tendinopathy (reviewed in Fitzpatrick et al) and in osteoarthritic patients (reviewed in Chang et al, Khoshbin et al, and Laudy et al). Much of the primary literature comprising these systematic reviews was available at the time that this article was published, however those findings were not considered by Dragoo et al when conceiving their study, nor was this body of evidence adequately addressed in their discussion. In fact, the results of more recent studies and systematic reviews clearly indicate that LR-PRP gives a greater patient benefit in tendinopathy as well as in osteoarthritis than does LP-PRP. The objective of the rabbit study by Dragoo and colleagues was to evaluate the inflammatory effect of LRPRP versus LP-PRP after intratendinous patellar tendon injection. The outcome measures employed were based on standard tissue histopathological analysis using only hematoxylin and eosin (H&E) staining at 5 and 14 days after injection. Therefore, the entire study relied on the assumption that leukocyte detection in extravascular tissues 5 days after injection is indicative of an acute inflammatory reaction leading to the recruitment of leukocytes from the circulation into the injected tissue. The 5-day time point was chosen based on a typical rabbit leukocyte lifespan of less than 5 days, supposing that at this time point only recruited leukocytes would be present in the injected tissue. Literature from in vitro studies does indeed support a short lifespan of isolated granulocytes, however it has also been well established that a multitude of plateletand leukocyte-derived growth factors, chemokines, and inflammatory mediators increase the granulocyte lifespan several fold (reviewed in Kolaczkowska and Kubes as well as Mantovani et al), leading to a neutrophil halflife of over 5 days. In addition, the major leukocytes in rabbit blood are mononuclear cells, not granulocytes, representing approximately 70% of the total cells. These mononuclear leukocytes have a lifespan of at least 2 weeks. Therefore, there is an inherent and obvious problem with the assumption that leukocyte detection in extravascular tissues 5 days after injection is indicative of an acute inflammatory reaction. Standard H&E staining cannot distinguish between activated leukocytes that may have been recruited to the injection site from the circulation (properly interpreted as an inflammatory reaction) and the naı̈ve resting leukocytes that were concentrated into the LR-PRP (Table 3 of Dragoo et al) and introduced to the tissue via injection. Because the authors did not validate the disappearance of injected leukocytes 5 days after injection or differentially label cells to distinguish between recruited and injected populations, it was not established that any of the leukocytes observed in the LR-PRP– injected tissue were actually recruited from the circulation to the injection site. This deficiency leaves any conclusions with respect to inflammatory reactions due to LR-PRP factually baseless and invalid. Thus, the only conclusion that can be made from the standard histopathology in this case is that LR-PRP containing concentrated leukocytes was indeed injected into the tissue. To the contrary, because LP-PRP showed a lack of leukocytes in the tissue at 5 days after injection, the finding of a large leukocyte infiltration at day 14 does indeed signify that a robust, albeit not acute, inflammatory reaction occurred. However, since the sterile saline control showed a similar robust inflammatory reaction on day 14, no specific conclusions regarding inflammatory effects of LP-PRP could be valid either. As other authors have not reported similar effects of saline injections into rabbit tendon tissue, it is possible that the particular model and/or injection paradigm used in the Dragoo et al study is inappropriate for studying inflammatory reactions. The lack of leukocytes in the LP-PRP–injected tissue at 5 days after injection is also of great interest because it is well established that acute leukocyte infiltration into damaged tissue is driven primarily by chemokine and cytokine release from activated platelets. This observation would indicate either that platelets in LP-PRP are completely deficient in activation and subsequent degranulation in vivo, as has been shown on in vitro studies, and are therefore incapable of initiating an acute inflammatory reaction required for activating a healing response, or that the tissue samples analyzed for histology were biased against demonstrating leukocyte infiltration that may be perceived as detrimental to the tissue. The authors also biased their statistical analyses by counting white blood cells twice in the tendon scores presented in Tables 4 and 5 of their article; once as individual parameters, and again as the total white blood cell count. It would be of interest to see the statistical analyses once this error is corrected. There is also no specific staining included in the study to determine vascularity, fiber structure, or the degree of fibrosis. Given the apparent pathological state of the tissue as presented in the The American Journal of Sports Medicine, Vol. 44, No. 12 2016 The Author(s)

Re: Use of Platelet-Rich Plasma in Intra-Articular Knee Injections for Osteoarthritis

We read with great interest the systematic review by Dr. Lai and colleagues in the June 2015 issue [1], which assessed the use of platelet-rich plasma (PRP) in intraarticular knee injections for osteoarthritis (OA). Althoughwe agree with the authors’ main conclusion that PRP intra-articular injections of the knee may be an effective alternative treatment for knee osteoarthritis, we believe that the authors have not adequately or objectively acknowledged the growing body of evidence on the safety and efficacy of PRP formulations that contain leukocytes (white blood cells [WBCs]) and/or red blood cells (RBCs). For example, the authors state in their conclusions, “Future studies also need to evaluate the impact of WBCs and RBCs. Recent in vitro work suggests that PRP formulations that are leukocyte-rich or contain RBCsmaybe cytotoxic in cultured synoviocytes. However, further human subject studies are needed to determine if these findings are present in vivo.” The authors base this statement regarding potential concern for the safety of leukocyte-rich PRP on the results of a single in vitro study [2]. However, Lai and colleagues cite several recent clinical studies using PRP that contains WBCs and/or RBCs with positive findings that specifically support the safety and efficacy of these formulations in patients with knee osteoarthritis [3-5]. Strikingly, the body of current human clinical data shows a stark absence of any significant adverse events or safety signals that would suggest in vivo cytotoxicity [1,6-8], and it is surprising that Lai and colleagues do not acknowledge this in their article. Indeed, the authors present the in vitro synoviocyte study by Braun and colleagues [2]without bringing to the attention of the reader that this in vitro study contains important methodological flaws that limit interpretability and the applicability of the findings to the clinical setting. For example, Braun and colleagues treated approximately 1.3 10 cultured synoviocytes with greater than 100