This study was approved by the Ethical Committee of the Universidad de Caldas.
Horses
Eighteen clinically normal Argentinean Creole horses (geldings) were used. The horses had a mean age of 12.5 (± standard deviation (s.d) 6.3) years old. All the horses were from the same farm, and the owner did know the nature of the study and authorized the blood extraction accordingly.
Blood collection and preparation of platelet concentrates
From each animal blood samples were collected in triplicate by jugular venipuncture and deposited randomly in tubes with either sodium citrate (SC) (12.35 mg sodium citrate and 2.21 mg citric acid [BD Vacutainer®, Becton Drive, Franklin Lakes, NJ, USA]) or acid citrate dextrose (ACD) solution A (ACD-A) (22.0 g/L trisodium citrate, 8.0 g/L citric acid and 24.5 g/L dextrose [BD Vacutainer®, Becton Drive, Franklin Lakes, NJ, USA]) or ACD solution B (ACD-B) (13.2 g/L trisodium citrate 4.8 g/L citric acid and 14.7 g/L dextrose [BD Vacutainer®, Becton Drive, Franklin Lakes, NJ, USA]).
Tubes with each anticoagulant were randomly processed for P-PRP production. The total whole blood used for P-PRP preparation using each anticoagulant varied between 110 and 140 mL. Briefly, after centrifugation at 120 g for 5 min, the first 50% of the top supernatant plasma fraction, adjacent to the buffy coat, was collected. This fraction was then centrifuged at 240 g for 5 min and the bottom quarter fraction was collected [16]. This fraction was considered to be P-PRP. The upper plasma fraction P-PRP was considered to be PPP (Figure 1). Plasma was obtained by centrifugation from each anticoagulated blood at 3500 g for 8 min. The time between blood collection and processing was approximately 1 h. All the samples were deposited and transported from the farm to the laboratory in an icebox.
Haematological analysis
Complete, automated haemograms (Celltac-α MEK 6450, Nihon Kodhen, Japan) were performed in duplicate for whole blood, P-PRPs and PPPs obtained from each anticoagulant. Platelet (PLT) counts, mean platelet volume (MPV fL), platelet distribution width (PDW %) and total leukocyte (WBC) counts were determined.
Activation of platelet concentrates
Four hundred μL of a 10% calcium gluconate (CG) solution (9.3 mg/mL) (Ropsohn Therapeutics Ltda®, Bogotá, Colombia) was added to 4 mL of P-PRP or PPP obtained with each anticoagulant to produce the P-PRGs and PPGs, respectively. P-PRGs and PPGs were incubated at 37°C for 3 h to stimulate GF release. Clots were mechanically released from the walls of the tubes and centrifuged at 3500 g for 8 min. The resulting supernatant was aliquoted, and frozen at −82°C for later determination of TGF-β1 and PDGF-BB concentrations.
Lysis of platelet concentrates
Samples of 4 mL of P-PRPs and PPPs obtained using each anticoagulant were incubated at 37°C for 15 min with 400 μL of a solution containing 0.5% of a non-ionic detergent (NID) (Triton® X100, Panreac Química, Barcelona, Spain). Platelet concentrates treated with NID were used as a positive control for GF release [11]. Lysates were processed in a similar fashion to supernatants from P-PRGs and PPGs.
Total protein determination
Total protein (TP) concentration from all the samples were determined using the biuret method (Proteína total (Biuret), BioSystems, Barcelona, Spain) [17], followed by spectrophotometric quantifications.
Determination of TGF-β1 and PDGF-BB concentrations by ELISA
The TGF-β1 and PDGF-BB concentrations from the supernatants and lysates of each blood component were determined in duplicate by a sandwich ELISA using commercially available antibodies against human TGF-β1 (Human TGF-β1, DY240E, R&D Systems, Inc., Minneapolis, MN USA) and PDGF-BB (Human PDGF-BB, DY220, R&D Systems, Inc.). Both ELISAs were performed according to the manufacturer’s instructions. Readings were performed at 450 nm. Both ELISAs were determined with human antibodies because there is a high homology of these growth factors between equines and humans [18,19]. Further, several equine PRP studies have validated these ELISA kits [6,14-16].
Statistical analysis
Data were analysed using commercial software (SPSS 18.0, IBM, Chicago, IL, USA). Data were initially assessed for normality (goodness of fit) by a Shapiro-Wilk test and a direct plot analysis of each evaluated variable. When the variables had a normal distribution (Shapiro-Wilk test, P > 0.05), they were presented as means (± s.d.) and evaluated by parametric tests (e.g., Student’s t-test for paired samples, and one way analysis of variance (ANOVA) and Tukey’s test (for post-hoc paired comparisons). Non-parametric variables (Shapiro-Wilk test, P <0.05) were presented as medians (interquartile range -IR-) and evaluated using a Kruskal-Wallis test followed, when necessary, by a Mann–Whitney U-test. A Wilcoxon test was used for non-related paired comparisons. All the variables were analysed for general and specific correlations using a Spearman (r
s) test. A P value ≤0.05 was considered to be significant for all tests.