Vacuette

Venous blood was collected from 119 volunteers after informed consent was obtained in accordance with the Declaration of Helsinki. Blood was collected on K₂-EDTA (Vacuette®, Greiner Bio, Kremsmünster, Austria) for platelet count determination in whole blood. All measurements were performed within 3 h after blood collection to ensure platelet count stability [13 ].
Furthermore, whole blood from 74 out of 119 volunteers was also collected on 1:6 (v/v) acid citrate dextrose (ACDA; Vacuette®, Greiner Bio, Kremsmünster, Austria) and 3.2%(w/v) trisodium citrate (Vacuette®, Greiner Bio, Kremsmünster, Austria) for PRP sample preparations. PRP was prepared from ACDA whole blood by centrifuging it at 200 x g for 10 min and then further concentrated by an extra centrifugation step at 900 x g for 10 min to obtain the desired platelet concentration (3600 × 10³/μl). The sodium citrate anticoagulated plasma was used to prepare platelet poor plasma (PPP) by double centrifugation (2750 x g for 5 min and 10,000 x g for 10 min). Combining PRP and PPP, a series of different concentrations of validation samples were prepared with the platelet count between 250 and 3600 × 10³/μl.

Blood samples were collected from a jugular vein between 9:00 am and 11:00 am, in June and October. All samples were taken by the same operator. Two aliquots of blood were collected from each animal, one was put in a tube for serum isolation (evacuated tubes; Z serum clot activator, Vacuette^®, Greiner Bio-one, Kremsmünster, Austria) and another one was put in tubes with EDTA (evacuated tubes, K3-EDTA, Vacuette^®, Greiner Bio-one, Kremsmünster, Austria), used to evaluate hematological parameters and to isolate plasma. Both groups of tubes were immediately refrigerated at 4°C.

Blood samples for all laboratory analyses were collected from fasted dogs. Samples for determination of haematological parameters were analysed within one hour after collection. Serum tubes (Vaccuette; Greiner Bio-One, Kremsmünster, Austria) stood for 30 min at room temperature before centrifugation for 10 min at 1300×g at room temperature. The serum used for CRP, TNF-α, and IL-6 measurements was frozen at − 80 °C and analysed in batch at the end of the study. Serum biochemical parameters were measured on the day of collection. EDTA tubes (Vacuette; Greiner Bio-One, Kremsmunster, Austria) were used for the collection of samples for measurement of plasma NT-proBNP and MDA concentrations. These tubes were centrifuged for 15 min at 1500×g at 4 °C and obtained plasma samples immediately frozen at − 80 °C until analysis. Concentrations of NT-proBNP and MDA were measured in batch at the end of the study. Blood samples for determination of whole blood GPX activity were collected into tubes containing the anticoagulant lithium heparin (Vacuette; Greiner Bio-One, Kremsmunster, Austria). Aliquots of heparinized whole blood were prepared and immediately frozen at − 80 °C until analysed in batch.

Peripheral blood was collected by venipuncture from Portuguese healthy adult volunteers with an age range between 25–40 (7 female and 4 male donors), not taking antibiotics or anti-inflammatory medication within the previous 14 days. Blood was drawn using one of the following anticoagulant tubes: K₃EDTA (Vacuette, Greiner Bio-one, Kremsmünster, Austria), sodium citrate (Vacuette, Greiner Bio-one, Kremsmünster, Austria) or lithium heparin (Becton Dickinson, NJ, USA). Blood was collected under a protocol approved by the Institutional Review Board of the University of Minho (SECVS 002/2014 (ADENDA)), which is in strict accordance with the Declaration of Helsinki and Oviedo Convention. All donors gave written informed consent to have blood taken.

For platelet isolation, venous blood was drawn into 3.5 ml CTAD (0.129 mM trisodium citrate, 15 mM theophylline, 3.7 mM adenosine, 0.198 mM dipyridamole) tubes (Vacuette, Greiner-Bio One, Kremsmünster, Austria) to avoid post-sampling platelet activation. Whole blood was centrifuged at 120 × g for 20 min at room temperature (RT) with the centrifugation brake off to avoid contamination with other blood cells.
Afterward, platelet-rich plasma was transferred into a fresh tube containing prostacyclin I2 (0.8 µM) to avoid platelet aggregation and degranulation during the following washing process. Platelets were then pelleted by centrifugation (3000 × g, 3 min), washed twice in phosphate-buffered saline containing prostacyclin I₂ (0.8 µM) and finally pelleted, shock-frozen in liquid nitrogen and stored at −80 °C. To obtain platelet-poor plasma, venous blood was drawn into 3.5 ml sodium citrate (0.129 mM citrate) vacuum tubes (Vacuette, Greiner-Bio One, Kremsmünster, Austria), centrifuged at 2500 × g for 15 min at 15 °C and stored at −80 °C. All platelet and plasma samples were further processed within 6 months to avoid stability problems.

Murine whole blood samples for rotational thromboelastometry (ROTEM® delta, Tem International, Basel, Switzerland) was collected from the left ventricle into a 2 ml syringe as described above for platelet preparation, to a final concentration of 3.2% liquid sodium citrate (VACUETTE, Greiner Bio-One International GmbH, Kremsmunster, Austria). After 7 minutes incubation at 37 °C 300 µL of citrated whole blood was analyzed after manufacturer’s instructions with STARTEM, INTEM, EXTEM and FIBTEM liquid reagents (Tem International, Basel, Switzerland). The measuring time was increased to 90 minutes to obtain higher MCF values, but no other parameters were changed.
For whole blood aggregation analysis on the Multiplate® analyzer (Roche Diagnostics Ltd., Rotkreuz, Switzerland) 2 ml Lithium Heparin blood collection tubes (VACUETTE, Greiner Bio-One International GmbH, Kremsmunster, Austria) were used. After a minimum of 30 minutes resting period in room temperature, 300 µL of whole blood was analyzed after manufacturer’s instructions with ADPtest (Roche Diagnostics Ltd., Rotkreuz, Switzerland).