Liquid biopsy - Performance of the PAXgene® Blood ccfDNA Tubes for the isolation and characterization of cell-free plasma DNA from tumor patients.

BACKGROUND: In most research laboratories the use of EDTA tubes for the isolation of plasma DNA from tumor patients is standard. Unfortunately these tubes do not allow for an extended storage of samples before processing and prevent EDTA tubes from being shipped at ambient temperature. The aim of our study was to compare the quantity and quality of plasma DNA isolated from EDTA and PAXgene® Blood ccfDNA Tubes in different downstream applications. METHODS: We enrolled 29 patients in our study. Blood samples were drawn into EDTA and PAXgene® Blood ccfDNA Tubes and were processed on day 0 and day 7 after storage at ambient temperature. The plasma DNA from 10 patients was isolated manually. For the DNA isolation from the plasma of 19 additional patients we used the automated QIAsymphony system. The total amount DNA from all samples was measured with a quantitative real-time PCR assay. In addition the amount of methylated mSHOX2 plasma DNA was determined. RESULTS: While the 7day storage lead to an increased amount of total DNA in almost all EDTA tubes, this effect was only seen in very few PAXgene® Blood ccfDNA Tubes. The stabilization solution which prevents the lysis of blood cells had no effect on the method for quantification of methylated sequences in these samples. CONCLUSION: The quantity and quality of plasma DNA from both types of blood draw tubes are comparable. DNA from PAXgene® Blood ccfDNA was successfully used for PCR-based quantification of total amount of cell-free DNA and for methylation analysis as well.

Comparison of outcome in 1809 patients treated with drug-eluting stents or bare-metal stents in a real-world setting.

PURPOSE: The aim of this study was to determine the long-term safety of drug-eluting stent (DES) versus bare metal stent (BMS) implantation in a "real-world" setting. PATIENTS AND METHODS: A total of 1809 patients who were treated with implantation of either BMS or DES were assessed. Kaplan-Meier and multivariate Cox regression analyses concerning primary endpoint of cardiac mortality were performed. RESULTS: A total of 609 patients received DES. Mean age was 66.2 ± 11.3 years, 69.4% were male, and 1517 (83.8%) were treated for acute coronary syndrome (unstable angina 510 [28.2%], non-ST-elevation myocardial infarction [NSTEMI] 506 [28.0%], and ST-elevation myocardial infarction [STEMI] 501 [27.7%]). Mean follow-up was 34 ± 15 months. During follow-up, 268 patients died of cardiac causes (DES 42 [7.3%]; BMS 226 [19.6%]; P < 0.001). Univariate Kaplan-Meier analysis showed an advantage of DES over BMS concerning the primary end-point (P < 0.001). When adjusting for classic risk factors and additional factors that affect the progression of coronary heart disease (CHD), DES was not found to be superior to BMS (hazard ratio 0.996, 95% confidence interval 0.455-2.182, P = 0.993). Severely impaired renal function was an independent predictor for cardiac mortality after stent implantation. CONCLUSION: Treatment with DES is safe in the long term, also in patients presenting with STEMI. However, in multivariate analyses it is not superior to BMS treatment.

Comparison of unfractionated heparin, low-molecular-weight heparin, low-dose and high-dose rivaroxaban in preventing thrombus formation on mechanical heart valves: results of an in vitro study.

Thromboembolism and bleeding after mechanical heart valve replacement are still unsolved problems, particularly for patients requiring anticoagulative bridging therapy. The aim of this study was to investigate whether rivaroxaban, a new oral selective and direct coagulation factor Xa inhibitor, is as effective as enoxaparin and unfractionated heparin (UFH) in preventing thrombus formation on mechanical heart valves using an in vitro system. Blood from healthy male donors was anticoagulated with either UFH, enoxaparin, rivaroxaban at 300 ng/ml, (n = 10 each), or rivaroxaban at 30 ng/ml (n = 3). Mechanical aortic valve prostheses were placed into the in vitro testing system THIA II and exposed to the anticoagulant blood mixtures at a pulsatile flow for 60 min. Overall thrombus weight, coagulation parameters, and electron microscopic features of thrombus formation on the valve surface were quantified as endpoints. The mean thrombus weights were 163 ± 64 mg for group 1 (UFH), 341 ± 63 mg for the group 2 (enoxaparin), 238 ± 83 mg for group 3 (rivaroxaban 300 ng/ml) and 1.739 ± 16 mg for group 4 (rivaroxaban 30 ng/ml). Whereas high-dosed rivaroxaban showed no significant differences compared to UFH or enoxaparin, low-dosed rivaroxaban generated a massive thrombus generation, thus differing significantly from all other treatment groups regarding the thrombus weight. We hypothesize that high-dose rivaroxaban is a competitive oral available alternative to UFH and LMWH's, that might be a worthwhile alternative for patients in need of anticoagulative bridging therapy. Prospective studies have to evaluate if rivaroxaban might even overcome the limitations of OAC in patients after implantation of artificial heart valves.

Comparison of bivalirudin, enoxaparin, and unfractionated heparin in preventing cardiac catheter thrombosis. Results of an in-vitro study.

Bivalirudin, a direct thrombin inhibitor binds specifically and reversibly to both fibrin-bound and unbound thrombin. Bivalirudin is approved for use as an anticoagulant in patients undergoing percutaneous coronary intervention. The OASIS-5 trial presented a significant increase in cardiac catheter thrombosis for the pentasaccharid fondaparinux compared to enoxaparin. Catheter thrombosis has never been reported in any trial using bivalirudin. Our study compared the development of catheter thrombosis for bivalirudin, enoxaparin, and unfractionated heparin in a controlled in-vitro environment. Ten healthy male volunteers were pretreated with aspirin 500 mg 2 hours before venesection of 50 ml of blood. The seven groups of anticoagulant combinations tested were: UFH, UFH + eptifibatide, enoxaparin, enoxaparin + eptifibatide, bivalirudin bolus, bivalirudin + eptifibatide, bivalirudin bolus + continuous infusion. The blood/anticoagulant mix continuously circulated through a cardiac guiding catheter for 60 minutes or until the catheter became blocked with thrombus. Thrombus development was assessed by weighing each catheter before and after the procedure. Electron microscopy was used to quantify the degree of erythrocyte, platelet and fibrin deposition. Following anticoagulation with bolus dose bivalirudin, the catheter was invariably occluded with thrombus after 33 minutes of circulation. However, a continuous infusion of Bivalirudin prevented the development of occlusive catheter thrombosis. In the bolus bivalirudin group the mean thrombus weight was significantly greater than in all other groups (p-value < 0.01 in all analyses). Bivalirudin given as a bolus was not sufficient to prevent cardiac catheter thrombosis in our in-vitro study. However, a continuous infusion of bivalirudin had similar anti-thrombotic efficacy compared to other treatment strategies.

In-vitro comparison of fondaparinux, unfractionated heparin, and enoxaparin in preventing cardiac catheter-associated thrombus.

BACKGROUND: The Organization to Assess Strategies in Acute Ischemic Syndromes trials showed that fondaparinux (fonda) is at least as effective and safe as unfractionated heparin (UFH) and enoxaparin (enoxa) in acute coronary syndromes. Unexpectedly, there was an increase in catheter-related thrombus formation during percutaneous coronary interventions in fonda-treated patients. METHODS: Ten healthy male volunteers were pretreated with aspirin 500 mg 2 h before venesection of 50 ml of blood. Eight groups of anticoagulant (combinations) were tested and volunteers donated blood eight times, thus, acting as their own controls. The groups were UFH, UFH+eptifibatide, enoxa, enoxa+eptifibatide, fonda, fonda+eptifibatide, fonda+(half-therapeutic) UFH, and fonda+eptifibatide+(half-therapeutic) UFH. The blood/anticoagulant mix was kept at 37 degrees C and continuously circulated through a guiding catheter for 60 min or until the catheter became blocked with thrombus. Thrombus development was assessed by weighing each catheter before and after the procedure. Electron microscopy of the catheter lining was used to quantify the degree of erythrocyte and fibrin deposition. RESULTS: Despite fonda anticoagulation, catheters were invariably occluded by thrombus before the 60 min perfusion period had elapsed. Thrombotic catheter occlusion occurred even with higher fonda concentrations and combined fonda/eptifibatide use. All other combinations (including fonda and half-therapeutic UFH) ensured catheter patency for 60 min. Furthermore, thrombus weight and the cell/fibrinogen counts were significantly increased in fonda and fonda+eptifibatide compared with other treatment groups. CONCLUSION: Treatment with fonda, even in combination with eptifibatide, was not sufficient to prevent cardiac catheter thrombus development in our in-vitro study. However, the combination of fonda with 'half' therapeutic dosages of UFH were as efficient as other treatment strategies in preventing thrombus formation.