PRE-ANALYTICAL EFFECTS ON ESTABLISHED AND EMERGING BIOMARKERS OF CARDIOVASCULAR DISEASE (CVD)

Background: plasma renin activity (PRA), an established marker of CVD, is going to be replaced by plasma renin concentration (PRC). HMGB1 and TWEAK are emerging CVD biomarkers. We verified pre-analytical processing (sample type, temperature and duration of storage, centrifugation protocol) on PRA, DRA, HMGB1 and TWEAK. IL8 was positive control. Materials and methods: Blood from 10 donors was collected in serum (S), heparin, EDTA and citrate (Cit) tubes and kept at room temperature (RT) or refrigerated (COLD). For PRA and DRA a third series of EDTA tubes was chilled on ice (ICE). Samples were centrifuged one (1Centr) or two (2Centr) times after 30 minutes, 3 and 9 hours. Aliquots (-80°C) were analyzed within three months (immunometric assays). Results: PRA median CV at 3 and 9 hrs were: 8.16% and 7.68% (COLD), 2.18% and 5.23% (ICE), 18.22% and 17.25% (RT), being intra-assay CV 9%. DRA intra-assay CV was 3.2%; median CV at 3 and 9 hrs were: 1.94% and 4.87% (COLD), 3.32% and 6.07% (ICE), 1.94% and 2.96% (RT). S-HMGB1 was 10 fold lower than any other plasma type (F = 7.14, p = 0.002). Cit-HMGB1 median CVs at 3 and 9 hrs were lower than intra-assay CV (15%): 2.23% and 8.96% (COLD,1Centr), 8.64% and 5.24% (COLD,2Centr), 11.55% and 6.58% (RT,1Centr), 7.51% and 8.56% (RT,2Centr). At 3 hrs TWEAK median CV was lower than intra-assay CV (5%) for all pre-treatments. The median 9 hrs CV varied from a minimum of 6.36% (RT,2Centr) to a maximum of 11.37 (COLD,1Centr). S-IL8 at RT, not at COLD, progressively increased at 3 (28+8 pg/mL) (p = 0.0194) and 9 hrs (590+206 pg/mL) with respect to basal (7+3 pg/mL) (p = 0.0250). Conclusion: S-HMGB1 is not measurable, probably because it interacts with thrombomodulin; citrate plasma is recommended. TWEAK can be measured in any matrix. Refrigeration is not required for HMGB1, TWEAK and DRA.