Point-of-Care: Roadmap for Analytical Characterization and Validation of a High-Sensitivity Cardiac Troponin I Assay in Plasma and Whole Blood Matrices.

BACKGROUND: High-sensitivity cardiac troponin (hs-cTn) assays enable more precise use of traditional diagnostic strategies and earlier rule-out/rule-in at 0/1 h or 0/2 h after presentation of acute myocardial infarction (AMI). Availability of hs-cTn measurements at point-of-care (POC) can improve timely management of AMI patients. A roadmap for regulatory and analytical validation is exemplified with studies with the Atellica VTLi hs-cTnI at POC. METHODS: High-sensitivity performance was assessed with AACC/IFCC expert recommendations. Clinical Laboratory Standards Institute protocols were used for characterizing limit of blank, limit of detection (LoD), limit of quantitation (LoQ), 10% CV, precision, linearity, and analytic specificity with several reagent lots. Bland-Altman, Passing-Bablok, and hematocrit bias plots compared hs-cTnI measurement in lithium-heparin plasma (PL) and whole blood (WB) matrices. RESULTS: LoB was 0.55 ng/L; LoD and LoQ were 1.24 ng/L and 2.1 ng/Lm for PL and 1.60 ng/L and 3.7 ng/L for WB, respectively. The male 99th percentile is 27 ng/L, and female 99th percentile upper reference limit is 18 ng/L; 10% CVs were 6.7 ng/L for PL and 8.9 ng/L for WB. Also ≥50% of hs-cTnI values for healthy cohorts exceeded the LoD, confirming high-sensitivity performance. Linearity spanned from LoQ to 1250 ng/L. Specificity was >90% for 40 potential interferences; no hook effect was detected. WB and PL correlation was WB = 1.02*plasma + 0.3 ng/L (r = 0.996, n = 152). No hs-cTnI association with hematocrit was detected (R2 = 0.003). CONCLUSION: This analytical roadmap showed high-sensitivity performance, good analytic characteristics, and excellent PL and WB agreement for the Atellica VTLi hs-cTnI POC system. Essential clinical performance studies in patients by intended POC users may now commence.

Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation.

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.

TGF-beta inhibits Ang II-induced MAPK p44/42 signaling in vascular smooth muscle cells by Ang II type 1 receptor downregulation.

Vascular changes in diabetes are characterized by reduced vasoconstriction and vascular remodeling. Previously, we demonstrated that TGF-beta1 impairs Ang II-induced contraction through reduced calcium mobilization. However, the effect of TGF-beta1 on Ang II-induced vascular remodeling is unknown. Therefore, we investigated the effect of TGF-beta1 on Ang II-induced activation of the MAPK p44/42 pathway in cultured rat aortic smooth muscle cells (RASMC). Activation of MAPK p44/42 was determined with a phospho-specific antibody. Angiotensin type 1 receptor (AT(1)) and AT(1) mRNA levels were measured by [(3)H]candesartan-binding and real-time PCR, respectively. AT(1) gene transcription activity was assessed using AT(1) promoter-reporter constructs and by a nuclear runoff assay. In TGF-beta1-pretreated cells, Ang II-induced phosphorylation of MAPK p44/42 was inhibited by 29 and 46% for p42 and p44, respectively, and AT(1) density was reduced by 31%. Furthermore, pretreatment with TGF-beta1 resulted in a 64% reduction in AT(1) mRNA levels and decreased AT(1) mRNA transcription rate by 42%. Pretreatment with TGF-beta1 blocked Ang II-induced proliferation of RASMC, while stimulating Ang II-induced upregulation of plasminogen activator inhibitor-1. In conclusion, TGF-beta1 attenuates Ang II-mediated MAPK p44/42 kinase signaling in RASMC through downregulation of AT(1) levels, which is mainly caused by the inhibition of transcription of the AT(1) gene.

Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells.

Ultrasound and microbubbles targeted gene delivery (UMTGD) is a promising technique for local gene delivery. As the endothelium is a primary target for systemic UMTGD, this study aimed at establishing the optimal parameters of UMTGD to primary endothelial cells. For this, an in vitro ultrasound (US) setup was employed in which individual UMTGD parameters were systematically optimized. The criteria for the final optimized protocol were: (1) relative high reporter gene expression levels, restricted to the US exposed area and (2) induction of not more than 5% cell death. US frequency and timing of medium replacement had a strong effect on UMTGD efficiency. Furthermore, US intensity, DNA concentration and total duration of US all affected UMTGD efficiency. Optimal targeted gene delivery to primary endothelial cells can be accomplished with Sonovue microbubbles, using 20 microg/ml plasmid DNA, a 1 MHz US exposure of Ispta 0.10 W/cm(2) for 30 s with immediate medium change after UMTGD. This optimized protocol resulted in both an increase in the number of transfected cells (more than three fold) and increased levels of transgene expression per cell (170%).

Ultrasound and microbubble-targeted delivery of small interfering RNA into primary endothelial cells is more effective than delivery of plasmid DNA.

Ultrasound and microbubble-targeted delivery (UMTD) is a promising non-viral technique for genetic-based therapy. We found that UMTD of small interfering RNA (siRNA) is more effective than delivery of plasmid DNA (pDNA). UMTD (1 MHz, 0.22 MPa) of fluorescently labeled siRNA resulted in 97.9 ± 1.5% transfected cells, with siRNA localized homogenously in the cytoplasm directly after ultrasound exposure. UMTD of fluorescently labeled pDNA resulted in only 43.0 ± 4.2% transfected cells, with localization mainly in vesicular structures, co-localizing with endocytosis markers clathrin and caveolin. Delivery of siRNA against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) effectively decreased protein levels to 24.3 ± 7.9% of non-treated controls (p < 0.01). In contrast, 24 h after delivery of pDNA encoding GAPDH, no increase in protein levels was detected. Transfection efficiency, verified with red fluorescently labeled pDNA encoding enhanced green fluorescent protein, revealed that of the transfected cells, only 2.0 ± 0.7% expressed the transgene. In conclusion, the difference in localization between siRNA and pDNA after UMTD is an important determinant of the effectiveness of these genetic-based technologies.