Twenty-two dogs aged between 2 and 7-years old, weighing among 30 kg and 60 kg, with no history of receiving transfusion and not taking any medication were selected. The animals were privately owned. Dogs were individually submitted to physical examination, complete blood counts (CBC), blood chemistry profile, and infectious disease screening (Snap 4Dx Test, Idexx Laboratories Inc., Westbrook, USA; Vetcheck, Tecsa Lab, São Paulo, BRA). Included breeds were Golden Retriever (n = 12), German Shepherd (n = 4), Giant Schnauzer (n = 4) and English Mastiff (n = 2). All included dogs had updated vaccines and deworming and were considered healthy.
After clinical and laboratory approval, a total of 450 mL of whole blood (WB) were collected in bags using a triple-bag closed system (CPD/SAGM, JP Indústria Farmacêutica, São Paulo, BRA). Primary collection bags had sodium citrate as anticoagulant; and citrate, phosphate and dextrose (CPD) solution as an additive solution for red blood cells. Dogs were kept under gentle physical restraint and with no anesthesia for blood collection. After trichotomy and 70% alcohol asepsis, WB was collected by puncturing the jugular vein , using equipment with automatic control of homogenization, flow and donation volume (Biomixer 323, Ljungberg & Kögel AB, Helsingborg, SWE). After blood withdrawn, all included dogs received water and a tasty meal, and remained under observation for at least 30 min; subsequently they were released to their owners.
Platelet concentrates preparation using the platelet-rich plasma method
WB bags were allowed to stand at 22 °C for one hour after collection. The PRP method was applied to obtain the PC as follows: each WB bag was subjected to light centrifugation (1600 X g) for 6 min at 22 °C (Sorvall Legend RT + Centrifuge, Thermo Scientific, Waltham, USA). PRP was removed using a manual plasma extractor (ACS201, Terumo Medical of Brazil, São Paulo, BRA) and then subjected to a second centrifugation (3300 X g) for 8 min at 22 °C. Fluid weight and density (1.026 g/mL) were used to calculate the final volume of the bag . Excessive plasma was removed with the aid of a plasma extractor until 50–70 mL of residual plasma remained at the bottom of the bag with the sedimented platelets . PC were stored in polyvinyl chloride (PVC) bags plasticized with tri-2-ethyl-trimellitate (TOTM, JP Indústria Farmacêutica), which are specific for platelet storage and do not have anticoagulants nor additive solutions.
Storage and sampling of canine platelet concentrates units
PC bags were left undisturbed for one hour after preparation, and then were stored at 20–24 °C in a platelet environmental chamber (model CDCI 03, Indrel, São Paulo, BRA) under constant stirring (AP48L linear plate agitator, Presvac, BA, ARG). Aliquots were removed for analyses using a sampling site coupler previously sterilized with 70% alchool (Fenwal, Lake Zurich, USA) on days 1, 3, and 5 . All PC were kept out of store for sampling for a maximum of 60 s. On day 5 of storage, all 22 PC were submitted to aerobic and anaerobic microbiological culture in brain heart infusion (BHI) incubated at 37 °C for 10 days.
Immediately before aliquot sampling, platelet swirling was assessed. This technique consists in the visual evaluation of the PC against a light source to observe platelet movement. Swirling is classified according to a 0–3 scale, where 0 indicates no platelet swirling and 3 corresponds to platelets with very nice cloudy movements [27, 28]. Swirling was assessed in all PC bags by the same evaluator. Platelet count, PDW and MPV were determined using an automated hematology counter calibrated for canines (PocH-100iV Diff, Sysmex, Lincolnshire, USA). Residual leukocytes were counted in a Nageotte chamber (LO-Laboroptik GmbH, Bad-Homburg, DEU) on day 1 of storage (24 h after collection), as previously described .
Lactate and LDH concentrations were determined by dry chemistry (Vitros 250 Chemistry System, Jonhson & Jonhson, São Paulo, BRA). Glucose, HCO3, pO2 and pCO2 values were determined using a portable blood gas analyzer (CG8, i-Stat, Abbott Point of Care, Mississauga, CAN), according to the manufacturer’s instructions. On days 1, 3, and 5, ATP was extracted  and samples were frozen at − 80 °C until further analyses. Platelet ATP levels were determined by bioluminescence (Lite Luminescence ATP Detection Assay System, PerkinElmer, Waltham, USA), and samples were read in a multi-mode microplate reader (Spectramax M5, Molecular Devices Inc., San Jose, USA) in the same assay. The pH was measured using a calibrated pH meter (HI 99171, Hanna Instruments Inc., Woonsocket, USA) under controlled temperature (22 °C) and according to the manufacturer’s instructions. All samples were tested in duplicate.
Flow cytometry analyses
Platelet activation was determined according to CD62P (P-selectin) expression. About 500,000 platelets were incubated with titrated amounts of anti-human CD61 FITC (clone VI-PL2, BD Biosciences, San Jose, USA) and anti-human CD62P PE (clone AC1.2, BD Biosciences, San Jose, USA) monoclonal antibodies for 20 min at room temperature. Samples were resuspended in phosphate buffered saline (PBS) prior to acquisition. Percentages of platelets positive for CD61 and CD62P were recorded.
Assessment of Δψm was performed as described elsewhere . Briefly, platelets were incubated with MitoScreen JC-1 (BD Biosciences, San Jose, USA). Cells were pelleted by centrifugation and resuspended in MitoScreen buffer for acquisition. Relative degrees of mitochondrial polarization were quantified by measuring the red-shifted JC-1 aggregates, which are favored under conditions of high membrane potential, and green-shifted monomers, which tend to predominate under conditions of low membrane potential .
Phosphatidylserine exposure was assessed with Annexin V. About 500,000 platelets were incubated with ApoFlowEx® FITC Kit (Exbio, Praha, CZE) for 15 min at room temperature, according to the manufacturer’s instructions. Samples were washed once and resuspended in PBS before acquisition. The percentage of platelets binding to Annexin V was recorded.
CaspACE™ FITC-VAD-FMK (Promega, Madison, USA) was used to determine caspases involvement in apoptosis induction. About 5,000,000 platelets were incubated with 1 mM of CaspACE™ FITC-VAD-FMK for 20 min at 37 °C. Samples were washed once and resuspended in 1 mL of PBS before acquisition. Percentage of platelets presenting active caspase was recorded.
All flow cytometry experiments were performed on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, USA) using CellQuest™ Pro software, after daily quality control procedures. At least 30,000 events were acquired for each analysis. All flow cytometry data were analyzed using FCS Express 5 software (De Novo, Software, Ontario, CAN).
Platelet aggregation test
Platelet aggregation test was performed with light transmission aggregometry (Agreg Myr4 aggregometer, Qualiterm, Cesário Lange, BRA) using ADP (5 μM), collagen (5 μg/mL), and arachidonic acid (1 μM) as inducers.
Data analysis was carried out with ANOVA for repeated measurements, and means were compared with Duncan’s test. Correlations among the evaluated parameters were tested with Pearson’s correlation test. Results were considered significant at p < 0.05. All data were analyzed with GraphPad Prism 6.0 Software (GraphPad Software, La Jolla, USA).
This study was approved by the Ethics Committee on the Use of Animals of the Universidade Federal do Rio Grande do Sul (CEUA/UFRGS) under protocol #26102. The owners of all evaluated dogs provided written informed consent before inclusion in the study.