Over the past decade, platelet-rich plasma has gained increased attention in orthopaedic sports medicine. Several investigators have advocated the use of platelet-rich plasma in the management of bone, muscle, tendon, and cartilage injury1-3. Platelets contain growth factors in their alpha-granules, such as transforming growth factor-beta (TGF-β), fibroblast growth factor-2 (FGF-2), platelet-derived growth factors (PDGF-AB), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF-A), which are thought to produce beneficial effects on the healing process. The ultimate goal of platelet-rich plasma treatment is to concentrate these growth factors and reintroduce them to a site of injury1.
Despite the growing popularity of platelet-rich plasma treatment, little is known regarding the specifics of plasma preparations or the devices used in their production1-3. Platelet-rich plasma is a generic term referring to any sample of autologous plasma with platelet concentrations above baseline blood values2. Defining platelet-rich plasma preparations according to platelet count can be difficult, as consensus on adequate concentration is lacking. Platelet concentrations of 200 × 103 platelets/μL up to 1000 × 103 platelets/μL are considered therapeutic for tissue-healing, whereas much higher counts appear to be biologically unfavorable1,4-6. Describing platelet-rich plasma according to the method of production may be more appropriate as either one or two-step centrifugation processes are used to fraction whole blood and concentrate the platelets1,3,7.
Currently, there are several commercial devices available for the preparation of platelet-rich plasma. While their common purpose is to fraction whole blood into its composite parts to allow isolation of plasma with elevated levels of platelets, each device functions differently. Specifically, these devices and their protocols differ in their method of isolation (one or two-step centrifugation), type and operation of the collecting tube, speed of the centrifuge, and other processes of production, which result in plasma preparations with varying volumes, platelet numbers, quantities of growth factors, and concentrations of residual white and red blood cells1,3,7,8.
Variability in the cellular composition of platelet-rich plasma preparations can create methodological challenges for investigators1,3,7. Results obtained from both in vitro and in vivo studies utilizing platelet-rich plasma can be difficult to interpret if cell type and quantity of the plasma preparations are inconsistent. Despite its importance, research to quantify differences in separation methods is limited. Recently, Castillo et al. compared cell concentrations in platelet-rich plasma preparations from three different single-step separation methods (MTF Cascade, Arteriocyte Magellan, and Biomet GPS III)9. While concentrations of platelets, red blood cells, fibrinogen levels, and active TGF-β1 remained consistent, substantial differences in the amount of VEGF-A and PDGFs as well as the amount and concentration of white blood cells were observed9. Considering the concentration-dependent function of white blood cells to be either beneficial or harmful in platelet-rich plasma treatments, it is critical to define the differences in the cellular characteristics of platelet preparations10-12.
To elucidate differences between methods of procurement of platelet-rich plasma, laboratory investigation conducted in a clinically applicable manner is needed. Historically, basic-science investigation has utilized automatic cell separation to produce platelet-rich plasma1. While valid, this method does not reflect a clinical environment in which centrifuge devices are used and platelet-rich plasma treatment is sometimes administered in repeated doses over a given period of time. These conditions raise questions over the consistency of platelet preparations with regard to methods of separation and within-individual consistency of preparations with repeated blood draws1.
Our hypotheses were that (1) despite variability in specific methods, so-called one-step separation methods will result in comparable preparations of platelet-rich plasma with regard to platelet, growth factor, and red and white blood-cell concentrations, and (2) inconsistent blood cell counts and concentrations will be seen with each system following repeated blood draws. Two major study goals developed for these hypotheses were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in one-step (clinically used commercial devices) and two-step separation systems (literature-based platelet-rich plasma method) and (2) to determine the influence of three separate blood draws in terms of within-subject differences on the resulting components of platelet-rich plasma.
Subjects
Blood samples were obtained from eight healthy subjects (two female and six male subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) as part of an investigative study examining various properties of platelet-rich plasma. Institutional review board approval was obtained. Inclusion criteria included healthy subjects between the ages of eighteen and sixty-five years without known blood dyscrasia. Exclusion criteria included a medical history of blood-derived illness or any medication known to affect platelet or bone marrow function or concentration for a minimum of two weeks prior to testing.
Platelet-Rich Plasma Preparation
Approximately 125 mL of peripheral blood was drawn from each subject at three different time points (zero, fourteen, and thirty days) to allow sufficient platelet recovery. A 60-mL syringe prefilled with 5 mL of acid citrate dextrose (ACD-A) was used for the standardized blood draw. ACD-A binds calcium and prevents blood clotting with no known interference to platelet function. Blood was then transferred directly to each of the three different separation systems. As separation methods, a single-spin method that would be expected to result in platelet-rich plasma (PRP) with a lower platelet and white blood-cell number (PRPLP), an alternative system expected to result in a high amount of platelets and high number of white blood cells (PRPHP), and a double-spin method (PRPDS) were chosen to represent an overall survey of the techniques clinically available.
PRPLP
The Arthrex ACP Double Syringe (Arthrex, Naples, Florida) was used for production of autologous conditioned plasma. Ten milliliters of blood was filled into the double syringe to produce 3 mL of PRPLP. Syringes were centrifuged at 1500 rpm for five minutes. This separated the erythrocytes from the remaining plasma components. The top portion of plasma was drawn up with use of the inner syringe without disruption of the erythrocyte layer.
PRPHP
The GPS III Platelet Concentrate System (Biomet, Warsaw, Indiana) was used to produce approximately 3 mL of PRPHP out of 27 mL of blood. The tubes were centrifuged for fifteen minutes at 3200 rpm according to the manufacturer's protocol. With the specific construction of the tubes, it was possible to draw the portion of platelet-rich plasma into a 3-mL syringe according to the manufacturer's instructions.
PRPDS
A literature-based double-spin method was utilized to fractionate whole blood13. After a first centrifugation of 1500 rpm for five minutes, the top layer of plasma was separated and centrifuged a second time (twenty minutes at 6300 rpm). Finally, half of the superficial plasma layer was removed, and the platelet pellet was suspended in the remaining half of the plasma volume.
Platelet Concentration and Number of Blood Cells
A 1-mL sample of each platelet-rich plasma preparation and each native blood specimen were analyzed by the clinical core laboratory at the University of Connecticut Health Center. The platelet concentration, number of red blood cells, and white blood-cell differentiation was determined by blood cell count (Gen-S System 2 Hematology Analyzer; Coulter, Miami, Florida)14. Linearity is 10 – 1000 × 103/μL for platelet count, 0.3 – 7.0 × 106/μL for red blood-cell count, and 0.1 – 100 × 103/μL for white blood-cell count, respectively (data provided by the University of Connecticut Health Center blood laboratory).
Growth Factor Concentration
Enzyme-linked immunosorbent assay (ELISA) in duplicate aliquots with the Quantikine Human Immunoassay kits (R&D Systems, Minneapolis, Minnesota) were used to quantify the growth factor concentration of each platelet-rich plasma preparation and native blood sample. Samples for each were immediately frozen (–20°) to preserve growth factor integrity, stored for less than twenty-eight days, and thawed on ice (one freeze-thaw cycle) before the ELISA assays were performed. Due to the high costs of the multiple ELISA assays, only quantification for all products of all subjects at the time point of the first blood draw was feasible. All assays had in common the employment of the quantitative sandwich enzyme immunoassay technique.
The growth factors EGF, FGF-2, HGF, IGF-1, PDGF-AB, TGF-β1, and VEGF-A were chosen for analysis because of their specific roles in tissue-healing and regeneration1,7. Human EGF was measured with a 3.5 to 4.5-hour solid phase ELISA containing Escherichia coli-derived recombinant human EGF and antibodies raised against the recombinant factor. The FGF basic (FGF-2) kit contained recombinant human FGF-2 and antibodies raised against the recombinant factor (4.5-hour solid phase). The 4.25 to 4.5-hour HGF ELISA contains Sf 21-expressed recombinant human pro-HGF and antibodies raised against the recombinant factor. The Quantikine Human IGF-1 Immunoassay is a 3.5-hour solid-phase ELISA containing Escherichia coli-expressed recombinant human IGF-1. PDGF-AB was determined by a 4.5-hour solid-phase ELISA containing Escherichia coli-expressed recombinant human PDGF-AB. The TGF-β1 assay contained recombinant human TGF-β1 expressed by CHO (Chinese hamster ovary) cells and used a 4.5-hour solid-phase ELISA. Finally, the human VEGF kit contained Sf 21-expressed recombinant human VEGF165 and antibodies raised against the recombinant protein to measure VEGF165 in a 4.5-hour solid-phase ELISA.
Statistical Analysis
Data were analyzed with SPSS software (version 15.0; SPSS, Chicago, Illinois). The Kolmogorov-Smirnov and Shapiro-Wilk tests were performed for each variable to identify non-normal distributions. Since none of the variables showed normative distribution (p ≤ 0.05), the nonparametric Kruskal-Wallis test was used to compare group means. Comparisons with a significant difference in means were followed by post hoc tests (Tamhane T2). A p value of ≤0.05 was used to determine significance. For comparison of the variability of repetitive blood draws, means and standard deviations were determined for each subject and overall. Additionally, Cronbach α was calculated as a measure of reliability with the intraclass correlation measurement for two-way random average measurements (α ≥ 0.7 was regarded as reliable). This was used as a measure of the internal consistency and/or reliability of a repetitive measurement in multiple subjects.
Source of Funding
The University of Connecticut Health Center-New England Musculoskeletal Institute has received direct funding and material support for this study by Arthrex (Naples, Florida). The company had no influence on study design, data collection, or interpretation of the results.
Platelet Concentration
With regard to the total number of platelets, all separation systems produced a significantly increased platelet number compared with native blood (142.7 ± 44.40 × 103/μL). The PRPHP (873.8 ± 207.82 × 103/μL) also showed a significantly higher number of platelets compared with PRPLP (378.3 ± 58.64 × 103/μL) or PRPDS (447.7 ± 183.7 × 103/μL) (p ≤ 0.05). No significant difference in platelet number was seen when PRPLP was compared with PRPDS (p = 0.52) (Fig. 1).
Red Blood-Cell Concentration
Overall, the highest level of red blood cells was in native blood (4.1 ± 0.4 × 106/μL). This was significantly different from all of the other separations. The PRPLP (0.2 ± 0.1 ×106/μL) and the PRPDS (0.02 ± 0.04 × 106/μL) were not significantly different compared with each other. However, both the PRPLP and the PRPDS group had significantly fewer red blood cells compared with the PRPHP group (1.0 ± 1.4 × 106/μL).
White Blood-Cell Concentration
There were significantly different amounts of white blood cells in all four separations compared with each other (p ≤ 0.05). The PRPHP (20.5 ± 6.7 × 103/μL) showed the highest amount, whereas the PRPLP (0.6 ± 0.3 × 103/μL) system showed the fewest amounts of white blood cells. The PRPDS contained fewer white blood cells (1.7 ± 1.8 × 103/μL) than native blood (5.6 ± 1.7 × 103/μL) (Fig. 2).
White Blood-Cell Distribution
The distribution of the white blood cells according to separation methods is shown in Table I. All cell types showed significant differences compared with each other (p ≤ 0.05), except for PRPLP and PRPDS, which showed no significant difference between any cell types. No significant differences were observed when numbers of eosinophils isolated from the PRPHP were compared with whole blood.
Influence of Draw Repetition on Platelet Count
Table II and Figures 3-A, 3-B, and 3-C show the intrasubject and intersubject variability of platelet concentrations according to different blood draws.
Growth Factors
Results for each growth factor are shown in Table III. The single-spin method (PRPLP) had a significantly higher concentration of the factors HGF, IGF-1 and PDGF-AB in comparison with the double-spin method (PRPDS), whereas the PRPHP separation produced significantly more growth factors compared with the other separations, with the exception of VEGF-A (p ≤ 0.05). The PRPLP separation method released significantly more HGF, IGF-1, and PDGF-AB compared with the PRPDS separation (p ≤ 0.05).
Note: The authors thank Steven Delaronde, MPH, MSW, for his statistical advice.