Platelet function and constituents of platelet rich plasma.

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Int J Sports Med. 2013 Jan;34(1):74-80. doi: 10.1055/s-0032-1316319. Epub 2012 Aug 14. …

Int J Sports Med. 2013 Jan;34(1):74-80. doi: 10.1055/s-0032-1316319. Epub 2012 Aug 14.
Platelet function and constituents of platelet rich plasma.
Pelletier MH, Malhotra A, Brighton T, Walsh WR, Lindeman R.

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  • 1. 74 Clinical Sciences Platelet Function and Constituents of Platelet Rich Plasma Affiliations Key words ▶ ● platelet rich plasma ▶ ● blood ▶ ● autologous ▶ ● therapy ▶ ● p-selectin ▶ ● viable M. H. Pelletier1, A. Malhotra1, T. Brighton2, W. R. Walsh1, R. Lindeman2 1 2 Surgical & Orthopaedic Research Labs, University of New South Wales, Randwick, Australia Department of Haematology, Prince of Wales Hospital, Randwick, Australia Abstract ▼ Platelet Rich Plasma (PRP) therapies require blood to be processed prior to application, however, the full assessment of the output of platelet sequestration devices is lacking. In this study the products of the Autologous Fluid Concentrator (Circle BiologicsTM, Minneapolis, MN) and the Gravitational Platelet Separation System (GPS, Biomet, Warsaw, IN, USA) were evaluated in terms of platelet viability and PRP constituents. The AFC and GPS produced 6.4 ( ± 1.0) ml and 6.3 ( ± 0.4) ml of PRP, with platelet recovery of 46.4 % ( ± 14.7 %) and 59.8 % ( ± 24.2 %) producing fold increases of platelets of 4.19 ( ± 1.62) Introduction ▼ accepted after revision May 10, 2012 Bibliography DOI 10.1055/s-0032-1316319 Published online: August 14, 2012 Int J Sports Med 2013; 34: 74–80 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0172-4622 Correspondence Dr. Matthew H Pelletier, PhD Surgical & Orthopaedic Research Labs University of New South Wales Level 1 Clinical Sciences Bld 2031 Randwick Australia Tel.: +61/293/822 687 Fax: +61/293/822 660 Clinical use of Platelet Rich Plasma (PRP) has been reported since the early 1990s for oral and maxillofacial surgery as a source of growth factors to increase the rate and degree of bone formation at early time points [31]. Since then PRP has found an increasing use in a variety of clinical applications. Primarily PRP has been used to support hard and soft tissue healing and formation [5, 9, 35, 44], but has also extended to other uses such as on nervous tissue [10, 42], in facial plastic and cosmetic surgery [6, 28, 39], burns [36] and chronic leg ulcers [46]. The autologous nature of PRP also alleviates concerns of transmissible diseases or immunogenic reactions [34]. The potential clinical benefit of PRP relies on the maintenance of intact platelet α-granules. Upon activation, platelets degranulate and secrete an array of mitogenic and chemotactic growth factors, which include platelet derived growth factor (PDGF-αα, PDGF-αβ, PDGF-ββ), transforming growth factor (TGF-β1, TGF-β2), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), interleukin-1 (IL-1), and epi- Pelletier MH et al. Platelet Function and Constituents … Int J Sports Med 2013; 34: 74–80 and 5.19 ( ± 1.62), respectively. Fibrinogen concentration was increased above baseline PPP produced with the AFC. pH was lower for both of the processed samples than for whole blood. White Blood Cell count was increased around 5 fold. Functional tests showed preserved viability with both devices. This represents essential knowledge that every treating physician should have before they can confidently administer PRP therapy produced by any method. These are the first published results of platelet function for the GPS system and the first performance results of the AFC system. The PRP produced is classified according to broad classifications as LeukocytePRP (L-PRP) for both devices. dermal growth factor (EGF). These secreted proteins have important roles in healing and tissue regeneration [1, 5, 27, 40]. Although there are many proteins that may promote wound healing in PRP, such as fibrinogen, the basic premise for PRP remains the delivery of a soup of plateletderived proteins at concentrations above baseline [13–15, 50]. PRP can be generated by centrifugation of anticoagulated whole blood and collected with pipette or syringe, but interest in PRP therapies has led to the development of several commercial systems aimed at improving ergonomics, decreasing variability, and improving platelet recovery in a closed system. While these devices do produce PRP with a concentration of platelets above circulating baseline blood levels [29], the product varies between devices, leading Ehrenfest et al. [11] to propose a classification to highlight their properties. Specifically, the classification takes account of differences in production including whether or not the buffy coat (which contains a near 7-fold increase in leukocytes [7]) is collected, fibrin and fibrinogen characteristics, collection efficiency, as well as practical consid- Downloaded by: UNSW Library. Copyrighted material. Authors
  • 2. Clinical Sciences 75 Fig. 1 Allocation of collected blood. Whole Blood in 2 x 30ml syringes loaded w (ACD-A) 10 % for each device 1 ml: Whole Blood (Heparin syringe) 4 ml: Whole Blood (EDTA vacutainer) 4.5 ml: Whole Blood (Li-Heparin vacutainer) 3.5 ml: Whole Blood (Citrate vacutainer) AFC or GPS Processing pH Full Blood Count Biochemistry Fibrinogen PPS; GPS c-PPP; AFC 0h p-selectin resting 0h p-selectin ADP 0h Hypotonic Stress 0h Aggregation 4h p-selectin resting 4h p-selectin ADP 4h Hypotonic Stress 4h Aggregation pH Fibrinogen Full Blood Count 0h p-selectin resting 0h p-selectin ADP 0h Hypotonic Stress 0h Aggregation erations such as size, weight of the centrifuge, and ease of use. The development of this classification will hopefully resolve variable and sometimes conflicting results regarding PRP therapies in the literature [19, 28, 30, 37, 38, 44, 47]. Here we present a first evaluation of the Autologous Fluid Concentrator (AFC) (Circle BiologicsTM, Minneapolis, MN), a platelet sequestration device which has recently been granted approval by the Food and Drug Administration for use in the United States, and a paired comparison of an established device (GPS, Biomet, Warsaw, IN, USA). Materials and Methods ▼ Both devices yield PRP, with the GPS system additionally producing Platelet Poor Plasma (PPP) and the AFC offering the option of PPP or Concentrated Platelet Poor Plasma (c-PPP). An evaluation of c-PPP, PRP, and whole blood included blood count, platelet count, pH, white cell count, platelet recovery, and fibrinogen concentration. Platelet function testing on obtained PRP was performed in a smaller subset of samples at 0 and 4 h to investigate platelet reactivity and short-term stability. These tests included assessments of p-selectin levels while resting and upon activation with Adenosine Diphosphate (ADP), reaction to hypotonic shock and agonistinduced light transmission aggregometry. 4h p-selectin resting 4h p-selectin ADP 4h Hypotonic Stress 4h Aggregation tested in this study utilised a plasma concentration chamber at the rear and a chamber that, when viewed from the front, resembled an hourglass. Inside the hourglass chamber there is a collection tube with a moveable collection area. The GPS system uses a buoy of specific density within a cylindrical chamber ▶ (● Fig. 2). Blood and ACD-A filled syringes were emptied into the devices which were centrifuged at 3 200RPM for 15 min at room temperature. Following centrifugation the plasma, red cell and buffy ▶ coat layers were clearly evident (● Fig. 3). PPP was drawn off with syringes connected to respective ports and was further processed by 3 filter passes for the AFC resulting in (c-PPP). PRP was drawn off with a moveable collection window between 2 O-ring seals with the AFC and via a separate port with the GPS device. For the collection of PRP, the selector valve was turned back to “draw”, the collection window was moved so that the top seal was level with the top of the buffy coat and all available fluid was drawn off using a 10 ml syringe connected to port B. A portion of both PRP, PPP and, where processed, c-PPP was placed in capped test tubes for FBC (n = 59) and fibrinogen concentration (n = 59) and a Li Heparin syringe for pH (n = 59). A portion of PRP was also placed in a capped tube for tests of platelet function (n = 12 for each test). Blood assays Subjects This study conforms to ethical standards in sport and exercise research as described by Harriss, et al. [21]. Volunteers were recruited at the University of New South Wales and Prince of Wales Hospital following approval of the Human Research Ethics Committee. A total of 64 subjects were recruited for the study. Volunteers were excluded if taking anticoagulant, anti-platelet, or anti-inflammatory medication, or aspirin. Subjects were deidentified by assigning a number relating to all subsequent blood test results. Blood collection Blood was collected from the medial cubital vein with a 19 G needle by trained phlebotomists. Blood was collected in a 4 ml EDTA Vacutainer for Full Blood Count (FBC), a 4.5 ml Li-Heparin Vacutainer for standard biochemistry, a 3.5 ml Citrate Vacutainer for fibrinogen concentration, 1 ml heparin syringe for pH, and for each device 2 × 30 ml syringes preloaded with acid-citrate-dex▶ trose formula A (ACD-A) anticoagulant, 10 % (● Fig. 1). Platelet sequestration The centrifuge and AFC devices arrived in sterile packages ready for use. The AFC is a 2 chamber modular system. The devices All blood assays were performed at the South Eastern Area Laboratory Services (SEALS) which is accredited by the National Association of Testing Authorities (NATA), Australia’s government-endorsed, international laboratory accreditation body. 59 matched samples of whole blood, PRP and PPP were evaluated for pH, fibrinogen concentration and FBC. pH was measured on an ABL800 FLEX Radiometer Blood Gas Analyser (Radiometer, Copenhagen). Full Blood Count was performed on a Roche Sysmex Model XE-2 100 (Sysmex Corp., Kobe, Japan). Biochemistry assays were performed on a Beckman Coulter UniCel DXC880 blood analyzer (Beckman Coulter, USA). Fibrinogen assays were performed on an STA R Evolution Coagulation Analyzer (Stago, USA). Platelet recovery was calculated from platelet counts as 100 % *(platelet count of PRP * volume of collected)/(Platelet count of whole blood * volume of whole blood). Platelet concentration factor was calculated as Platelet count of PRP/whole blood platelet count. Surface P-selectin expression (CD62p antigen), as a measure of platelet activation, was assessed by flow cytometry in resting and activated state samples. Approximately 1 ml of PRP from each device was set aside for p-selectin assays. Phosphate buffered saline (2.92 μl) or ADP agonist (40 μM) was added to 70 ml subsamples of PRP. These samples were incubated for 15 min at Pelletier MH et al. Platelet Function and Constituents … Int J Sports Med 2013; 34: 74–80 Downloaded by: UNSW Library. Copyrighted material. pH Fibrinogen Full Blood Count PRP
  • 3. 76 Clinical Sciences Downloaded by: UNSW Library. Copyrighted material. Fig. 2 Left to Right; Circle Biologics and Biomet devices, before use (top row) and after centrifugation (bottom row). Fig. 3 Close up of buffy coat interface, Circle Biologics (Left) and Biomet (Right). Black arrows indicate the PPP portion, the White arrows indicate the buffy coat, and the double white arrows indicate the red cell pack. 37 °C. 4 × 5 μl aliquots were taken and incubated with CD41aPerCP antibody and either 1μl CD62-PE or 1 μl mouse IgG1-PE (isotope control) (DB Biosciences, San Jose, CA). These samples were incubated in the dark at room temperature for 20 min. 600 ml of Ringer’s solution was added following incubation. Samples were analyzed in a BD FACS Canto II flow cytometer (Becton, Dickinson and Company, USA). For determination of % p-selectin expression, forward and light scatter and fluorescence were acquired using logarithmic scale. A total of 20 000 platelet events were gated. The isotope matched control antibody was used to set threshold for CD62P positivity. Hypotonic shock response (HSR) measures the platelet’s ability to recover its normal volume after swelling when exposed to a hypotonic environment. HSR is an optical method and PRP required an additional soft spin (900RPM/10 min) to remove intervening red cells for output from the AFC device. The resulting PRP was removed from the red cells with a pipette and platelet concentrations adjusted to < 500 × 109/ml and allowed to rest Pelletier MH et al. Platelet Function and Constituents … Int J Sports Med 2013; 34: 74–80
  • 4. Clinical Sciences 77 AFC GPS Whole Bld fibrinogen (g/L) pH platelet concentration ( × 10^9/L) WBC ( × 10^9/L) c-PPP PRP PPP PRP 2.96 (0.73) 7.34 (0.03) 222 (45.7) 6.36 (1.42) 3.30 (0.73)+* 7.13 (0.03)+* 18.7 (12.7)+* 0.04 (0.135)+ 2.97 (0.65) 7.02 (0.06)+* 926 (378)+* 32.2 (11.7)+* 2.95 (0.65)* 7.11 (0.04)+* 11.0 (6.2)+* 0.01 (0.027)+ 2.94 (0.64) 7.08 (0.05)+* 1149 (497.7)+* 31.0 (8.9)+* Table 1 Blood Assays. Data summary for whole and processed blood samples showing mean (standard deviation). *difference compared to whole blood value, + difference between devices, p < 0.05 AFC 0 h HSR ( %) aggregation ( %) p-selectin resting ( %) p-selectin activated ( %) AFC 4 h GPS 0 h GPS 4 h 54.0 (5.0) 95.2 (7.3) 7.7 (4.2) 25.4 (8.1)* 54.1 (6.6) 96.5 (7.1) 6.4 (3.0)+ 24.3 (9.2)*+ 50.6 (6.8) 97.4 (3.4) 12.4 (6.5) 26.5 (6.1)*^ 55.6 (6.1) 98.5 (2.7) 9.8 (2.5)+ 31.9 (3.6)*+^ Table 2 Platelet Function Testing. Data summary for platelet function assays showing mean (standard deviation). for 30 min. 6 × 150 μl of PRP from each device was transferred into the wells of a microtitre plate. 3 × 150 μl of saline was added to PRP samples and the microtitre plate was placed in a BioTeck EL808 Ultra Microplate Reader (BioTek, Instruments, Inc., Winooski, VT). 150 μl of H2O were added with a micropipette to the remaining 3 samples simultaneously, the lid was closed and reading commenced. Readings of light transmission was performed at 405 nm for 10 min at 15 s intervals. Results were recorded with this protocol via Gen5 Data Analysis Software (Generation 5, Toronto, Canada). Light transmission (T) was converted to optical density according to OD = − log10 T or T = lO − OD. The optical density (OD) of the saline samples acted as the baseline for measurements. The difference in the peak OD and the baseline divided by the difference in OD at 10 min and baseline produced a percent recovery. Aggregation was performed following preparation to remove red cells as above. 240 μl samples of PRP, PPP and c-PPP were tested in a 4 station AggRAM aggregometer (Helena Laboratories, Gateshead, UK). All samples were stirred with magnetic stir bars at 600 rpm. Following a quality check, the OD was measured in the matched samples of PPP, which acted as a control. The PRP was then placed in the aggregometer. Continuous reading of OD continued for 10 min while 10 ul of collagen aggregant was added to achieve a final concentration of 20 μg/ml and final volume of 250 μl. Results of all tests were compared with paired t-tests using Tukey’s criteria and a significance level of 0.05. Results ▼ Of the 64 blood samples collected for the study, none were excluded based on irregular biochemistry results, one was lost to human error, and post processing assays were precluded by insufficient volume in 4 samples resulting in a sample size of n = 59 for fibrinogen, cell count and pH tests and n = 12 for all platelet function assays. 54.1 ( ± 0.2) ml (AFC) and 55.7 ( ± 1.5) ml (GPS) of whole blood plus ACD-A was loaded into the device and 6.4 ( ± 1.0) ml for the AFC and 6.3 ( ± 0.4) ml for the GPS of PRP was collected. Platelet recovery from whole blood samples for the AFC and GPS was 46.4 % ( ± 14.7 %) and 59.8 % ( ± 24.2 %), respectively, representing concentration factors (fold increase) of 4.19 ( ± 1.62) and 5.19 ( ± 1.62). Fibrinogen concentration was increased above baseline for c-PPP (AFC only) but not PRP. pH was lower for both of the processed samples than for whole ▶ blood. Results are summarized in ● Table 1. ▶ The results of platelet function assays are summarized in ● Table 2. HSR tests at 0 h and 4 h showed platelet recovery of around 54 % with no differences detected between time points or device. Tests of the aggregation of PRP revealed total aggregation at 10 min was above 95 % at both time points with no differences found between time or device. Upon in vitro activation with collagen there was a significant increase in surface expression of CD62p (P selectin) confirming the collected platelets are functional and capable of activation at 0 and 4 h for both devices. Discussion ▼ The current study demonstrates that both the AFC and GPS devices produces platelet and leukocyte enriched plasma in a closed system with functional viable platelets in volumes suitable for clinical use. Although platelet concentration has been previously reported, platelet viability has not. Additionally the AFC performance has not been reported. Platelet function Damage to platelets during processing can lead to the premature release and subsequent loss of growth factors [1]. Viable platelets exhibit several behaviours when activated. Some of these behaviours can be monitored such as shape change, aggregation and surface marker exposure. Platelet aggregation is considered the gold standard in evaluating platelet function [41]. Mean platelet aggregation above 95 % in this study represents the retention of activity. Likewise HSR has been indicated as a very sensitive marker for viability [22], and predictor of in vivo survival [23]. During these tests platelet shape recovery was apparent. Granular release is closely related to p-selectin expression [18]. Identifying p-selectin on the surface of activated platelets allows them to be quantitatively assessed as a percentage of all platelets present. While absolute numbers of p-selectin assays are shown to vary a great deal between laboratories [12], it is a useful measure to determine activation for matched samples within a study centre. The results of the current study showed that platelets could be activated beyond the resting state, indicating preserved function. The broad classification of PRP encompasses a range of constituents at varying concentrations. The basic definition dictates that it contains a platelet concentration that is increased above circulating levels. Marx et al. [29] describes PRP as a platelet concentration in a 5 mL volume with 1 000 000 platelets/μL Pelletier MH et al. Platelet Function and Constituents … Int J Sports Med 2013; 34: 74–80 Downloaded by: UNSW Library. Copyrighted material. *difference between resting value, + difference between devices, ^ difference between time points
  • 5. 78 Clinical Sciences Fibrin While platelet concentration and growth factor potential are obviously essential aspects of PRP, fibrin (the activated form of fibrinogen) is thought to directly induce angiogenesis by providing a matrix scaffold supporting cell migration and providing chemotactic activity. The binding of growth factors such as PDGF and VEGF to fibrin also supports wound healing [8, 25]. The fibrinogen concentration could therefore be an important feature not always addressed when comparing different PRP products. Passing the PPP repeated through the filter increased the concentration of fibrinogen, with each successive pass decreasing the overall volume as water was removed. Our conservative processing with the AFC showed an increased concentration compared to whole blood. This process can be repeated any desired number of times further increasing fibrinogen concentration producing a product that could be used as a scaffold and when added to PRP may alter the velocity of aggregation [24], however, this was not examined in the current study. pH Although there was a difference between the whole blood pH and that of the processed samples, all groups were greater than 6.9 which is well above the level of 6.2 that would indicate a loss of platelet function [22, 33]. The increase in the acidity is likely due to the addition of citrate to the blood from the ACD-A. Leukocytes Platelets have a known direct and significant role in immunity and host defence against pathogenic microorganisms [4, 45]. Moojen, Everts et al. [32] demonstrated the antimicrobial potential of PRP in vitro. In addition to platelets, the PRP product collected from the buffy coat layer has been reported to contain around a 7-fold increase in leukocytes [7]. The output of both the devices produced around a 5 fold increase above circulating levels. The collection of leukocytes with the PRP product is an area of uncertainty. Anitua et al. [2] studied a concentrated platelet product, Preparation Rich in Growth Factors (PRGF), which avoids leukocyte content with the intent of avoiding the proinflammatory effects of leukocytes. An increase in leukocytes, combined with the view of platelets themselves as innate inflammatory cells with acute host defence functions [16], would suggest the PRP product may also be useful against postoperative infections. The inclusion of leukocytes during processing may also have beneficial effects. Neutrophils have been suggested to produce additional VEGF [48], a protein known to promote angiogenesis [2, 50]. Autologous Conditioned Serum (ACS) is created by incubating whole blood with glass beads, and has been shown to increase the concentration of relevant growth factors considerably without unwanted side effects in human and animal tests [49, 50]. A portion of the increase in growth factor concentration may be attributable to the inclusion of leukocytes during the activation process. Understanding whether or not to include, and the extent to which WBCs are concentrated in the PRP is likely to be relevant for future studies. It has been suggested that PRP products be described as Leukocyte Platelet Rich Plasma (L-PRP), and Pure Platelet Rich Plasma (P-PRP) to differentiate between the 2 PRP variations [11]. Classification It is commonly understood that not all PRPs are produced equal [30], and as such, PRP from different methods and devices need to be well characterised in order for the final product to have the anticipated effect. According to the specifications laid out by Ehrenfest et al. [11] the product of the ACP device is classified as L-PRP as it contains leukocytes, just as the GPS output is. The volume collected would be classified as small ( < 25 % blood volume) except that it can be varied by adding fibrin-rich PPP in the case of the AFC. Platelet and Leukocyte concentration were good (40–80 % of all available) and platelets were healthy after collection. The systems we tested could be activated with any agonist so fibrin is delivered unaltered, however the ability to concentrate the fibrin in the PPP with the option to add this to PRP may alter the classification. One aspect that should be noted regarding the output of the AFC is that the output can be purposefully modified by the user by changing the height of the collection window while drawing. The protocol used in the present study collected a portion of the red cell pack, the buffy coat and the plasma portion resulting from a single spin. The PRP included some red cells which, while biologically may have little impact in common use, but may be of interest for future studies depending on the application. It is possible that the protocol could be altered to allow collections excluding red cells, and possibly the leukocyte layer, although this was not studied in the present study. The location of the drawing window also dictates that a full blood draw be used with the device so that the buffy coat layer is in the drawing area. Pelletier MH et al. Platelet Function and Constituents … Int J Sports Med 2013; 34: 74–80 Downloaded by: UNSW Library. Copyrighted material. (approximately a 4–5 fold increase). Gociman et al. [17] summarized the reported platelet enrichments of 11 platelet-concentrating devices. These results showed fold increases ranging from 1.34 to 6.07. In Gociman’s study the GPS produced a 4.86 fold increase on platelet count, which aligns well with the 5.19 increase seen here. The AFC was capable of increasing platelet concentrations by a factor of 4.19 which places it roughly in the middle of Gociman’s results. While many devices report fold increases of 3–7 fold [3, 6, 43], it is important to note that highly concentrated platelet preparations may have an inhibitory effect on healing [20, 47]. The devices were capable of recovering 46.4 % (AFC) and 59.8 % (GPS) of all available platelets which is in line with previous publications. Leitner [26] compared 4 devices, and reported results of 17 % for the Vivostat PRF Preparation Kit (Vivosolution A/S, Birkeroed, Denmark), 70 % for the Harvest SmartPReP 2 APC 60 Process (Harvest Technologies Corp., Plymouth MA, USA) 67 % for the PCCS Platelet Concentrate Collection System (3i PCCS, 3i Implant Innovations, Palm Beach Gardens FL, USA) and 66 % for the Fibrinet Autologous Fibrin & Platelet System (Cascade Medical Enterprises Ltd, Plymouth, UK). Everts et al. [14] reported results for the Autologous Growth Factor Filter (Interpore Cross, Irvine CA, USA) at 32 %, the Electa Cell-Separator (Sorin Group, Irandola, Italy) at 48 % but reports a lower value for the GPS Platelet Separation System (Biomet Biologincs, Inc., Warsaw IN) at 36 %. Although the ideal concentration of platelets within PRP remains unknown, the effect of PRP is likely to be linked closely to platelet concentration. Leitner et al. [26] has shown that PDGF release is closely related to platelet count. Likewise Everts et al. [14] demonstrated the need for devices to maintain platelet viability during preparation to maximise growth factor release upon activation when required. Regardless of the device or method used, platelet recovery and viability are 2 important factors when evaluating the PRP product.
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