http://dx.doi.org/10.1590/1678-4162-12257 Arq. Bras. Med. Vet. Zootec., v.74, n.1, p.93-100, 2022 Cellular components and TGF-β1 content of a closed Tube system for Platelet Rich Plasma acquisition in horse] [Componentes celulares e TGF-β1 do plasma rico em plaquetas obtido por sistema fechado em equinos] P. Fantini1 , R. Jimenez-Aristizabal2 , A. Iborra2 , A.L.S. Miranda3 , R.P.A.Maranhão4 F.O.P. Leme4,5* , M.S. Palhares4,5 , M. Prades2 1Universitário UNA, Bom Despacho, MG, Brasil 2UniversitatAutònoma de Barcelona, Department of Animal Medicine and Surgery, Cerdanyola del Vallès, Spain 3Centro Universitário UNA, Contagem, MG, Brasil 4Universidade Federal de Minas Gerais, Escola de Veterinária, Belo Horizonte, MG, Brasil 5CAPES Foundation, Ministry of Education of Brazil, Brasilia, DF, Brasil ABSTRACT Platelet-rich plasma (PRP) has been proposed as an agent to accelerate the healing process and stimulate the regenerative capacity of tissues due to its abundance of growth factors. A large variety of kits and protocols are available to obtain PRP by different cell-separation systems. However, the lack of standardization may lead to inconsistent results. The aim of this study was to characterize cellular composition, platelet parameters using the ADVIA 120 flow cytometer, and TGF-β1 concentration from the PRP product obtained through a closed system, using simple centrifugation. Six clinically healthy horses were used in this study. The protocol in the closed system resulted in approximately 1.6-fold higher platelet and approximately 2.0-fold lower white blood cell concentrations in comparison with whole blood values. The evaluated system was efficient in concentrating platelets and in retrieving a small number of leukocytes, using a protocol of single centrifugation at low speed. Keywords: PRP, growth factors, platelet concentrate, concentration protocol RESUMO O plasma rico em plaquetas (PRP) tem sido proposto como um agente para acelerar o processo de cicatrização e estimular a capacidade regenerativa dos tecidos, devido à sua abundância de fatores de crescimento. Uma grande variedade de kits e de protocolos está disponível para obtenção de PRP, utilizando-se diferentes sistemas de separação de células. No entanto, a falta de padronização pode levar a resultados inconsistentes. O objetivo deste estudo foi caracterizar a composição celular, os parâmetros plaquetários pelo citômetro de fluxo ADVIA 120 e a concentração de TGF-β1 do PRP obtido em sistema fechado, por centrifugação simples. Seis cavalos clinicamente saudáveis foram usados neste estudo. O protocolo no sistema fechado resultou em concentrações de plaquetas aproximadamente 1,6 vez maior e concentrações de leucócitos aproximadamente 2,0 vezes menores em comparação com os valores do sangue total. O sistema avaliado foi eficiente na concentração de plaquetas e na recuperação de um pequeno número de leucócitos, utilizando um protocolo de centrifugação única em baixa velocidade. Palavras-chave: PRP, fatores de crescimento, concentrado plaquetário, protocolo de centrifugação INTRODUCTION capacity of tissues (Bosh et al., 2010). PRP  offers low-cost and easy acquisition of several Platelet-rich plasma (PRP) is an autologous components, such as fibrin, fibrinogen, platelet concentrate proposed to accelerate leukocytes, mesenchymal cells, and growth healing process and to stimulate the regenerative factors (GF), most of them found in the platelets *Corresponding author: fabiolapaesleme@vetufmg.edu.br Submitted: March 15, 2021. Accepted: September 28, 2021. Fantini et al. α-granules. GF are molecules of interest because example, infiltration of neutrophils causes of their properties, such as their ability in release of oxygen free radicals during modulating inflammation and tissue repair, inflammation and has been shown to play a production of chemotaxis, cell proliferation and central role in ischemic tissue injuries. differentiation, angiogenesis, neovascularization, Furthermore, there are studies that have shown and extracellular matrix deposition (Arguelles et their potential detrimental effects in tissue repair al., 2006; Everts et al., 2006). GF in the PRP (Zimmermann et al., 2003). include transforming growth factor beta-1 (TGF- β1), growth factor derived from platelets The wide size range of platelets, their rapid (PDGF), vascular endothelial growth factor shape changes, and tendency to clump in vitro (VEGF), and basic fibroblast growth factor make their counting less reliable in comparison (bFGF) (Weibrich et al., 2002). to other blood cells. Traditional cell counters which use impedance or light scattering PRP has been reported in several different contribute to relatively poor results. ADVIA applications in veterinary medicine including 120® (Bayer Corporation) an automated osteoarthritis (Carmona et al., 2007), skin lesions hematology analyzer measures simultaneously (Carter et al., 2003) and tendinitis (Arguelles et the volume and refractive index or density of al., 2008; Maia et al., 2009; Bosch et al., 2010). platelets, using a cell-by-cell with two angles of However, the results are controversial because of laser light scatter. This methodology is more the lack of standardization in the preparation reliable in obtaining accurate platelet counts and processes, such as time and centrifugation allows better discrimination between platelets of velocity, as well as the ideal anticoagulant all sizes and other particles (Kunicka et al., needed to maintain platelet parameters. All those 2000). ADVIA 120® allows a more accurate factors lead to a PRP product of variable volume, platelet count than conventional impedance molecular composition and different platelet, automated hematology analyzers and provides leukocyte and other cell type counts (Dohan and new parameters, such as mean platelet Choukroun, 2007; Dohan et al., 2009; Lei et al., component (MPC), mean platelet mass (MPM), 2009; Maia et al., 2009). mean platelet volume (MPV), plateletcrit (PCT), platelet distribution width (PDW), platelet mass Leukocyte function in platelet concentrates distribution width (PMDW), large platelets and remains controversial, considered to influence aggregates. the clinical outcome of the PRP therapy (Sundman et al., 2011). The presence of MPC has been recently proposed as a valid leukocytes can provide a local environment at screening parameter for assessing platelet the site of PRP application with increased activation. This parameter represents the average immunomodulatory capability that may aid in refractive platelet index and is quickly and easily preventing or controlling infection. It is also measured by flow cytometry. A linear possible that WBC enhances PRP growth factor relationship has been shown between the density concentration through its own release of GF or of platelets and their degree of activation. This by stimulating platelets to do so (Castillo et al., relationship is reduced when platelets 2011). Carmona et al. (2011) stated that WBC degranulate, indicating activation (Ifran et al., can act directly in accelerating tissue repair on 2005). The MPC has showed values inversely cartilage explants, due to its immunomodulatory proportional to the expression of P-Selectin in function, thus optimizing GF action. According dogs (Moritz et al., 2003, 2005) and cats to Zimmermann et al. (2001), WBC (Zelmanovic and Hetherington, 1998). In a study concentration in PRP is accounted for one-third investigating morphological and phenotypic to one-half of the variance of GF concentration. properties of the equine platelet activation, Conversely, WBC release catabolic cytokines, focused on the expression of CD62 (P-Selectin) matrix metalloproteinases (MMPs) and and mediated by platelets-leucocytes interaction, Interleukin-β (Clutterbuck et al., 2010) that may was observed that 20% of the resting platelets negatively interfere with tissue recovery by had constitutive expression of P-Selectin. This increasing local inflammation. Therefore, WBC might be due to earlier leukocyte stimuli and might not be a desired component in the PRP complicates the evaluation of the activation using samples to me used (Castillo et al., 2011). As an this specific marker (Lalko et al., 2003). Segura 94 Arq. Bras. Med. Vet. Zootec., v.74, n.1, p.93-100, 2022 Cellular components… et al. (2007), evaluating foals and adult horses MATERIAL AND METHODS with disseminated intravascular coagulation (pathologic condition that activates platelets), This study was approved by the Ethical observed that MPC may be used to detect committee in animal research (CEUA/UFMG n. activated platelets in sick animals but stated that 033/11) and by the Institutional Committee for further studies are necessary. Care and Use of Animals of the Autonomous University of Barcelona, Spain. Blood analysis In a human PRP study authors concluded that the was performed in the Service of Hematology quality of PRP is closely related to the type of Department, at the School of Veterinary anticoagulant used (Pignatelli et al., 1996), Medicine located in the Universitat Autònoma de because avoids spontaneous activation of Barcelona. platelets (Macey et al., 2002). The microstructure of platelets collected with For this study, six clinically healthy horses of heparin, citrate, acid citrate dextrose (ACD) and different breeds were used, five females and one citrate-theophylline-adenosine-dipyridamole male, with ages ranging from six to 14 years. (CTAD) was observed. ACD was proved to be better than heparin and sodium citrate (SC) to Whole blood was aseptically extracted by jugular produce better quality PRP, maintaining the venipuncture using a vacuum blood collection integrity of platelet structures and preventing the system with a 21G butterfly catheter (Venofix®, platelet spontaneous activation (Lei et al., 2009). Melsungen, Germany) and were processed Giraldo et al. (2015), on the other hand, showed within 15 minutes after blood collection. To no difference between ACD-A, ACD-B and SC perform hemogram, blood was collected in an regarding platelet count and GF’s release. EDTA vacuum tube (Aquisel ®, Barcelona, Miranda et al. (2018), while studying the effect Spain). of SC and ACD on equids’ PRP procurement, concluded that SC was better in concentrating Blood samples were collected in four syringes of platelets using a single-spin protocol when 10mL capacity, containing 1mL of an compared to ACD. No correlation between the anticoagulant: two containing ACD (trisodium numbers of platelet concentrates and citrate – 22g/L, citric acid – 8g/L and dextrose regenerative effect was demonstrated thus far, 24.5g/L) (Venosafe, Leuven, Belgium) and two but the key to successful PRP therapy is containing sodium citrate- SC (BD, Madrid, associated with the use of live platelets that are Spain). Once collected, samples were transferred not activated during sampling and centrifugation to a 20 mL Proteal® tube according to processes (Marx, 2004; Carmona and Lopez, manufacturer instructions. A 0.22 uM-filtered 2011). needle was first placed in one of the injection ports of the embolus (Fig. 1A), aiming to allow In the present investigation, a new method and the internal-external pressure equilibrium. Then, protocol to obtain PRP in horses using simple the blood sample was introduced in the tube centrifugation was evaluated. Two using a second needle port of the embolus (Fig. anticoagulants (ACD and SC) were compared 1B). using the same speed centrifugation protocol. The hypothesis is that the ACD is better for One tube from each anticoagulant (ACD and CS) equine platelets than SC because it has a higher were centrifuged at 133g for 8 minutes and the source of carbon (dextrose), thus providing a other tube from each anticoagulant at 360g for 8 substrate for platelet metabolism. It is also minutes. The centrifuge allows a slow assumed that low speed and single spin deceleration for 2 minutes, to prevent re-mixed centrifugation cause less platelet activation and cells. After centrifugation, the lower 5 mL platelet recovery can be of approximately twice plasmatic fraction was recovered using the the value of that in whole blood, recovering only Push.Out® system, a closed mechanism that a few leukocytes. allows the selection of specific blood centrifuged fractions directly using luer-lock syringes (Fig. 1C). Arq. Bras. Med. Vet. Zootec., v.74, n.1, p.93-100, 2022 95 Fantini et al. Once the PRP was selected, a 0.5 mL aliquot was The supernatant was collected and immediately obtained for platelet analysis by flow cytometry frozen for later determination of TGF-β1 in the (ADVIA 120® -Bayer Corporation). Another 1.0 Servei di Cultius Cellulars, Producció mL sample was collected for platelet activation d'Anticossos i Citometria (SCAC) in Instituti de evaluation. The remaining PRP was centrifuged Biotecnologia I de Biomedicina of the at 9000 rpm for 10 minutes to pellet the platelets. Universitat Autònoma de Barcelona. 1A 1B 1C Figure 1. Closed system used in the preparation of platelet rich plasma. 1A: insertion of a 0.22uM filter needle in the embolus port; 1B: transference of blood into the Proteal® tube; 1C: extraction of PRP by coupling a syringe and using the Push.Out® system. A complete blood count of each sample was activation of the binding protein joined by performed using the ADVIA 120® analyzer disulfide bonds, thus forming TGF-β1 complex (Bayer Lab, NY, USA). RBC, WBC, Platelet inactive secreting mechanism. This activation of count (PLT), plateletcrit (PCT), mean platelet TGF-β1 was performed in vitro by adding 100µL volume (MPV), mean platelet component of activation buffer [2.5N acetic acid (Ref. (MPC), platelet distribution width (PDW), mean 010,520, Probus, Spain)], 10M urea (Ref. U- platelet mass (MPM), platelet mass distribution 5128, Sigma, Barcelona, Spain) added to the width (PMDW) and large platelets and same volume of plasma, and then mixed and aggregates were measured. Calculation of incubated at room temperature for 10 min. The centrifugation efficiency (Ce) was based on the pH of the sample was neutralized with 100µL of formula: Ce = Platelet final neutralization buffer [2.7M NaOH (Ref. concentration/platelet initial concentration. 141687.1211 Panreac, Spain)]/1M HEPES (ref. H3375, Sigma-Aldrich, Barcelona, Spain). For The quantification of TGF-β1 in plasma was the test, different dilutions of the activated performed using a sandwich ELISA, using samples with dilution buffer (1.4% bovine serum commercial antibodies from R & D Systems albumin, Ref 10735078001, Roche, Spain), (DuoSet ref. DY240, R&D Systems). Mouse 0.05% Tween20 (Ref. 170-6531, Bio-Rad, anti-human TGF-β1 was used as capture Spain) in PBS were used. antibody and biotinylated chicken anti-human Growth factor concentration efficiency (GFCe) TGF-β1 as a detection antibody. Standards for was established by the formula: GFC= growth human recombinant TGF-β1 were employed for factor final concentration/growth factor initial the calibration curve as previous studies have concentration. demonstrated and validated the cross-reactivity of these antibodies with the equine specific All data were analyzed by a commercial software antigen (Arguelles et al., 2006). (SPSS Inc., Illinois, USA). Data are expressed as mean ± standard deviation. Statistical Quantification of TGF-β1 by ELISA requires a comparisons were analyzed using analysis of previous release of the amino-terminal variance (ANOVA) and post-hoc comparisons propeptides (latency-associated peptides) and were performed using the Tukey test to 96 Arq. Bras. Med. Vet. Zootec., v.74, n.1, p.93-100, 2022 Cellular components… determine differences between media that was observed an increase of 1.59 times baseline present a normal distribution, including variables platelet count. that showed normal distribution after log transformation (logX+1). For variables that were There was no significant difference (p˃0.05) not normally distributed, even after log between the protocols for MPV, MPC and transformation, Kruskal-Wallis analysis and clumps, but both centrifugations’ protocols post-hoc comparisons between each groups using differed (p˂0.05) from whole blood (EDTA the Wilcoxon test were performed. Statistical samples). significance was accepted as p values <0.05. The concentration of leukocytes decreased as the RESULTS centrifugation speed increased. PRP and whole blood cell count are presented in Table 1. A higher platelet concentration was observed when using ACD at 133g for 8 minutes. TGF-β1 analysis showed no significant Regardless of the anticoagulant employed in this differences between protocols (p˃0.05). protocol, its parameters were significantly However, the lower speed protocols showed different in comparison to the 360g protocol. It higher TGF-β1 concentration, as well as platelet count. TGF-β1 values are presented in Table 2. Table 1. Average related values (X) and standard deviation (SD) of hematologic variables in samples of whole blood and PRP obtained from six healthy horses using different centrifugation protocols and anticoagulants VARIABLES EDTA 133g/8’ CS 133g/8’ ACD 360g/8’ CS 360g/8’ ACD X SD X SD X SD X SD X SD PLT (103/µL) 165.50BC 65.52 245.00AB 45.27 262.50A 44.84 131.67C 48.88 155.83C 54.91 MPV (fL) 8.10B 0.94 12.27A 1.88 11.47A 0.98 11.55A 1.79 11.62A 1.342 MPC (g/dL) 23.53A 3.47 18.05B 2.34 18.15B 1.25 17.62B 2.41 17.47B 1.73 PCDW (g/dL) 8.45 1.242 7.87 0.60 7.58 0.279 7.75 0.59 7.40 0.49 MPM (pg) 1.86A 0.085 1.81AB 0.082 1.80AB 0.085 1.66C 0.05 1.68BC 0.05 PMDW (pg) (103/µL) 0.63A 0.04 0.57AB 0.034 0.56BC 0.043 0.51C 0.03 0.51C 0.03 Large PLT (103/µL) 11.50B 7.74 30.00A 14.792 27.33AB 12.64 12.33B 5.43 14.17AB 4.96 Clumps 1327.17A 1620.87 38.0B 14.46 41.67B 8.48 115.33B 196.65 41.0B 23.07 RBC frag (106/µL) 7.71A 0.89 0.04B 0.01 0.04B 0.01 0.02C 0.01 0.02C 0.01 WBC (103/µL) 7.77A 2.19 0.09B 0.09 0.07B 0.04 0.01C 0 0.02C 0.01 Neut. (103/µL) 5.3A 2.26 0.02B 0.01 0.02B 0.01 0.01C 0 0.01BC 0.01 EDTA- ethylenediaminetetraacetic acid; SC, sodium citrate, ACD, acid citrate dextrose, PLT, platelet; MPV, mean platelet volume, MPC; mean platelet component concentration; PCDW; platelet component distribution width; MPM mean platelet mass; PMDW platelet mass distribution width; RBC-frag, red blood cell fragments; WBC, white blood cell; NEUT, neutrophil; Means within rows with different letters are significant different (p<0.05). Table 2. Average values and standard deviation of transforming growth factor (TGF) -β1 in platelet rich plasma from six healthy horses using different centrifugation protocols and anticoagulants. Variable 133g/8’ SC 133g/8’ ACD 360g/8’ SC 360g/8’ ACD X SD X SD X SD X SD PLT 245.0AB 45.3 262.5A 44.8 131.7BC 48.9 155.8BC 54.9 TGF-β1 (ng/mL) 3942.9 1762.3 3911.5 1810.9 2864.2 744.0 2372.4 1492.8 TP (mg/mL) 370.4 258.0 477.9 117.1 394.0 139.3 319.2 238.9 TGF-β1 (ng/mL TP) 9.7 4.4 14.0 4.7 13.8 3.2 10.9 5.6 SC, sodium citrate, ACD, acid citrate dextrose, PLT, platelet; TGF-β, transforming growth factor beta; TP, total protein; CFC: Growth factor concentration efficiency. Means within rows with different letters are significant different (p<0.05). DISCUSSION (Wasterlain et al., 2012). A PRP containing less than a 2-fold increase in platelet concentration More than 16 platelet separation systems are can be obtained by a simple centrifugation currently available and produce different types of protocol which allows to collect all the platelet-rich concentrates, significantly different supernatant plasma (Tamimi et al., 2007). In the in the relative amounts of platelets, leukocytes, system evaluated in this study, approximately a erythrocytes, and anabolic and catabolic GF 1.6-time platelet concentration - when compared Arq. Bras. Med. Vet. Zootec., v.74, n.1, p.93-100, 2022 97 Fantini et al. to whole blood - was achieved, using the 133g In a study evaluating PRP anabolic and catabolic for 8 minutes protocol, thus supporting one of activities, with different PRP cellular our hypothesis that single-spin centrifugation can compositions, it has been suggested that higher concentrate a good platelets count. The main platelet concentrations (obtained from double goal is to capture only platelets using a slower centrifugation), caused a pro-inflammatory and shorter centrifugation spin regime. This effect, resulting in a diminished biosynthesis of yields platelet concentration typically around 2-3 extracellular matrix (Kisiday et al., 2012). Based times from the baseline whole blood levels on this data, the authors suggested that a simple (DeLong et al., 2011). centrifugation method is more appropriate to obtain PRP to be used in intra-articular therapies. According to Mazzocca et al. (2012) the “more The kit tested on this present study significantly is better” theory for the use of higher platelet decreased the WBC count, assuming that PRP is concentration cannot be supported, since the appropriate for intra-articular use, especially in optimal concentration depends on the cell type in ACD samples that rescued less WBC. Arguelles which proliferation is desired. The protocols et al. (2006) also observed low levels of WBC, using double centrifugation can concentrate more using simple centrifugation, and hypothesized platelets (Nagata et al., 2010), however, with a that PRP’s different compositions may lead to its simple centrifugation system we had equivalent use in different tissues, aiming their several results (Arguelles et al., 2006). Simple characteristics on healing processes. centrifugation could concentrate platelets 1.4- time, whereas using double centrifugation, a 1.7- The ideal number of platelets to be injected in a time concentration was achieved. Kisiday et al. lesion have not yet been defined and may vary (2012) evaluated the effect of PRP obtained with according to the tissue type and number of single and double spin centrifugation on anabolic receptors available for GF’s - without hyper and catabolic activity in meniscus cartilage saturation - thus inducing a positive response on explants and suggested a careful use of the cellular proliferation, assured by absence of a double spin PRP protocol, because of the higher negative feedback. However, only a few concentrations of white blood cells. comparative studies on healing effects based on the PRP platelet concentration have been done, Since the regenerative effect of PRP depends on especially in veterinary medicine (DeLong et al., the level of GF released, the integrity of platelets 2011). is crucial. If the platelets are activated during the PRP production process, GF in platelets will be TGF-β1 values obtained by Arguelles et al. released into plasma (most of which is discarded (2006), who performed prior platelet activation, during preparation), which will consequently were higher on both samples from single and decrease the local therapeutic efficacy of PRP dual spin centrifugation. There was no difference (Arguelles et al., 2006). Therefore, less platelet (p˃0.05) in TGF-β obtained between the activation and more GF retention in platelets are protocols here evaluated. However, Dugrillon et the key factors to improve the potency of al. (2002) reported that the number of platelets is therapeutic PRP effects. Suggested parameters not always proportional to the amount of GF and that may be related to evaluation of platelet more attention should be given to GF quality activation (Mazzocca et al., 2012), like MPC and than to the concentration of platelet in the PRP. MPV, did not differed between tested protocols Platelets can be premature activated by excessive on the present study, which can be explained by manipulation and pipetting, speed versus time of action of the anticoagulant used, although centrifugation, and use of glass instruments, increase in large platelets, as activation evidence which can lead to the prior release of GF and was most common seen in sodium citrate determine a less tissue effect (Schmitz et al., samples. Even if one can consider the small 1998). number of samples, clumps were more observed on EDTA samples. This information together CONCLUSION evidences the superiority of ACD to SC as anticoagulant in the maintenance of platelet Simple closed centrifugation at lower speed integrity. protocol is more efficient in concentrating platelets as well as retrieving a small number of 98 Arq. Bras. Med. Vet. Zootec., v.74, n.1, p.93-100, 2022 Cellular components… leukocytes. 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