Vacuette polypropylene tube

Peripheral blood samples were collected from the antecubital vein into a single VACUETTE polypropylene tube containing a serum separator and clot activator (Greiner-Bio One GmbH, Kremsmunster, Austria). Fasting PMF patients and controls were allowed to rest for at least 15 min before carrying out blood withdrawal, with the procedure performed between 8.00 and 9.00 a.m. To separate serum, blood withdrawals were kept for 30 min at room temperature and then centrifuged at 1890× g for 10 min. Serum samples were transferred into a new tube, and an aliquot of 500 μL was used for the subsequent organic solvent deproteinization [26 (link)]. Briefly, proteins were removed by the addition of 1 mL of ice-cold far UV, HPLC-grade acetonitrile to 0.5 mL of serum. After vigorous vortexing for 90 s, samples were centrifuged at 20,890× g for 10 min at 4 °C, supernatants were collected and transferred to a new tube, supplemented with 3 mL chloroform, vigorously vortexed for 120 s, and again centrifuged at 20,890× g for 10 min at 4 °C. The upper aqueous phase was collected and again extracted with chloroform to remove the organic solvent (acetonitrile). The resulting aqueous phase, free of proteins, was ready for the high-performance liquid chromatographic (HPLC) analyses of hydrophilic, low-molecular-weight metabolites.

Blood samples were taken for metabolic biomarker analysis immediately prior to the first ILB^® injection at day 0 (pre-treatment: Pre) and again at day 36 (one week post-treatment: Post). Peripheral venous blood samples were collected from both patients and controls after at least 15 min of complete rest, using the standard tourniquet procedure, from the antecubital vein into a single VACUETTE^® polypropylene tube containing serum separator and clot activator (Greiner-Bio One GmbH, Kremsmunster, Austria). After 30 min at room temperature, blood withdrawals were centrifuged at 1890× g for 10 min and the resulting serum samples saved at temperatures not higher than −20 °C until analysis.

Peripheral venous blood samples were collected, from both controls and patients, using the standard tourniquet procedure from the antecubital vein, into a single VACUETTE polypropylene tube containing a serum separator and clot activator (Greiner-Bio One GmbH, Kremsmunster, Austria). The blood withdrawals were carried out between 8.00 and 9.00 am and after at least 15 min rest before blood collection. After 30 min at room temperature, the blood withdrawals were centrifuged at 1890× g for 10 min and the resulting serum samples were collected and proteins removed by adding 1 mL of ice-cold far UV, HPLC-grade acetonitrile to 0.5 mL of serum, as previously described [22 (link),23 (link)]. After vortexing for 90 s and centrifugation at 20,890× g for 10 min at 4 °C, supernatants were collected, supplemented with 3 mL chloroform, vigorously mixed for 120 s, and again centrifuged at 20,890× g for 10 min at 4 °C. The upper aqueous phase was collected and again extracted with chloroform to remove the organic solvent (acetonitrile), thus leaving protein-free aqueous serum extracts that were suitable for high performance liquid chromatographic (HPLC) analyses of metabolites. The upper aqueous phases, containing water-soluble low-molecular weight metabolites, including those under evaluation, were diluted three times with HPLC-grade water before the metabolite analyses.