Stefan Siebert

MRI versus CT-based thrombolysis treatment within and beyond the 3 h time window after stroke onset: a cohort study

BACKGROUND Thrombolytic treatment with recombinant tissue plasminogen activator (rtPA) is approved for use within 3 h after stroke onset. Thus only a small percentage of patients can benefit. Meta-analyses and more recent studies suggest a benefit for a subset of patients beyond 3 h. We assessed the safety and efficacy of an MRI-based selection protocol for stroke treatment within and beyond 3 h compared with standard CT-based treatment. METHODS We assessed clinical outcome and incidence of symptomatic intracerebral haemorrhage (ICH) in 400 consecutive patients treated with intravenous rtPA. Patients eligible for thrombolysis within 3 h were selected by CT or MRI and beyond 3 h only by MRI. 18 patients were excluded from analysis because of violation of that algorithm. The remaining 382 patients were divided into three groups: CT-based treatment within 3 h (n=209); MRI-based treatment within 3 h (n=103); and MRI-based treatment beyond 3 h (n=70). FINDINGS Patients in group 3 (MRI > 3 h) had a similar 90 day outcome to those in the other two groups (48% were independent in the CT < or = 3 h group, 51% in the MRI < or = 3 h group, and 56% in group 3), but without an increased risk for symptomatic ICH (9%, 1%, 6%) or mortality (21%, 13%, 11%). MRI-selected patients overall had a significantly lower risk than CT-selected patients for symptomatic ICH (3% vs 9%; p=0.013) and mortality (12% vs 21%; p=0.021). Time to treatment did not affect outcomes in univariate and multivariate analyses. INTERPRETATION Our data suggest that beyond 3 h and maybe even within 3 h, patient selection is more important than time to treatment for a good outcome. Furthermore, MRI-based thrombolysis, irrespective of the time window, shows an improved safety profile while being at least as effective as standard CT-based treatment within 3 h.

TIME-RESOLVED DETECTION AND IDENTIFICATION OF SINGLE ANALYTE MOLECULES IN MICROCAPILLARIES BY TIME-CORRELATED SINGLE-PHOTON COUNTING (TCSPC)

A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the...

Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser

Abstract We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).

EFFICIENT DNA SEQUENCING WITH A PULSED SEMICONDUCTOR LASER AND A NEW FLUORESCENT DYE SET

Abstract A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5′-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.

When do we really need coronary calcium scoring prior to contrast-enhanced coronary computed tomography angiography? Analysis by age, gender and coronary risk factors.

Aims To investigate the value of coronary calcium scoring (CCS) as a filter scan prior to coronary computed tomography angiography (CCTA). Methods and Results Between February 2008 and April 2011, 732 consecutive patients underwent clinically indicated CCTA. During this ‘control phase’, CCS was performed in all patients. In patients with CCS≥800, CCTA was not performed. During a subsequent ‘CCTA phase’ (May 2011–May 2012) another 200 consecutive patients underwent CCTA, and CCS was performed only in patients with increased probability for severe calcification according to age, gender and atherogenic risk factors. In patients where CCS was not performed, calcium scoring was performed in contrast-enhanced CCTA images. Significant associations were noted between CCS and age (r = 0.30, p<0.001) and coronary risk factors (χ2 = 37.9; HR = 2.2; 95%CI = 1.7–2.9, p<0.001). Based on these associations, a ≤3% pre-test probability for CCS≥800 was observed for males <61 yrs. and females <79 yrs. According to these criteria, CCS was not performed in 106 of 200 (53%) patients during the ‘CCTA phase’, including 47 (42%) males and 59 (67%) females. This resulted in absolute radiation saving of ∼1 mSv in 75% of patients younger than 60 yrs. Of 106 patients where CCS was not performed, estimated calcium scoring was indeed <800 in 101 (95%) cases. Non-diagnostic image quality due to calcification was similar between the ‘control phase’ and the ‘CCTA’ group (0.25% versus 0.40%, p = NS). Conclusion The value of CCS as a filter for identification of a high calcium score is limited in younger patients with intermediate risk profile. Omitting CCS in such patients can contribute to further dose reduction with cardiac CT studies.

Diode laser based time-resolved detection and identification of individual mononucleotide molecules in aqueous solution

We applied a short-pulse diode laser emitting at 637 nm with a repetition rate of 30 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual labeled mononucleotide molecules in water. A newly synthesized oxazine dye and the commercially available carbocyanine dye Cy5 were used as fluorescent labels. Multichannel scalar traces, the fluorescence autocorrelation function and fluorescence decay times determined by time- correlated single-photon counting have been measured simultaneously. The time-resolved signals of the two mononucleotides were analyzed and identified by a maximum likelihood estimator. The results showed out that 60 detected photons per transit of a single molecule are sufficient to distinguish two labeled mononucleotides in water with a misclassification of less than 10 percent via their characteristic fluorescence lifetimes of 1.07 +/- 0.27 ns and 1.89 +/- 0.34 ns.

Finding the Conformation of Organic Molecules with Genetic Algorithms

Finding the optimal conformation for fluorescent labeled nucleosides is difficult for standard GAs. We investigated the structure of molecule and of the resulting search space itself and concluded that the problem is of the “needle in the haystack” type.

Conservatively Treated Incidental Aneurysm of the Distal Left Main Coronary Artery: Detection by Coronary Angiography and Noninvasive Followup Using Coronary Computed Tomography Angiography

Coronary artery aneurysms are relatively rare and commonly associated with significant coronary artery disease (CAD), inflammatory diseases (Kawasaki syndrome, infection), or iatrogenic complications. Herein, we report an unusual case of an incidental coronary aneurysm of the left main artery in a patient without specific clinical symptoms of myocardial ischemia or systemic inflammation and without angiographically significant CAD. Angiographic images are provided, acquired during cardiac catheterization, as well as coronary computed tomography angiography (CCTA) images obtained at 1 year of followup.

Giant Dilatation of the Right Coronary Aortic Bulb with Compression of the Right Ventricular Outflow Tract Mimicking a Ventricular Septal Defect: Diagnostic workup Using Echocardiography, Heart Catheterization, and Cardiac Computed Tomography

Annuloaortic ectasia is a relatively rare diagnosis. Herein, we report an unusual case of an annuloaortic ectasia with asymmetric dilatation of the right coronary bulb mimicking a membranous ventricular septal defect (VSD) with Eisenmenger reaction by transthoracic echocardiography. Aortic angiography showed a dilated aortic root and moderate aortic regurgitation. Right cardiac catheterization, on the other hand, exhibited normal pulmonary artery blood pressure and normal pulmonary resistance, whereas normal venous gas values were measured throughout the caval vein and the right atrium, excluding relevant left-right shunting. Further diagnostic workup by cardiac computed tomography angiography (CCTA) unambiguously illustrated the asymmetric geometry of the ectatic aortic cusp and root causing compression of the right heart and of the right ventricular (RV) outflow tract. After review of echocardiographic acquisitions, the blood flow detected between the left and right ventricles (mimicking VSD) was interpreted as turbulent inflow from the left ventricle into the ectatic right coronary cusp. Furthermore, elevated pulmonary artery blood pressure measured by echocardiography was attributed to “functional pulmonary stenosis” due to compression of the RV outflow tract by the aorta, as demonstrated by CCTA.

Ultrasensitive Detection and Identification of Biomolecules with Diode Lasers - From Dyes to DNA

The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biological applications with absorption and emission beyond 600 nm. We applied the pattern recognition technique taken from information theory to ultrasensitive fluorescence detection and identification of dye molecules. Using pulsed diode lasers emitting at 630-640 nm in combination with new efficient rhodamine and oxazine dyes four-dye one-lane DNA sequencing in capillary gelelectrophoresis with time-resolved fluorescence detection is demonstrated.