Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.     

Laser activated fluorescence measurements and morphological features: an in vivo study of clearance time of fluorescein isothiocyanate tagged cell markers

Fourteen BALB/c mice were divided into two groups. One group served as the control and the second group was injected with a squamous cell carcinoma cell line to the tongue. After tumor development (1-4 weeks), mice were injected with a FITC conjugated CD3 marker to their tongues. Immediately after the marker injection, the clearance of the marker was measured using a laser spectroscopy system. The markers were excited by an argon laser at 488 nm and the fluorescence signal was measured as a function of time. A biopsy was taken from every mouse after the procedure and the excised tissue was histologically evaluated. Analysis of clearance times revealed a second order exponential decay for both groups with a slower pace of signal clearance for the sick mice.     

Evaluation of platelet response to different clopidogrel dosing regimens in patients with acute coronary syndrome in clinical practice

High-post clopidogrel platelet reactivity in acute coronary syndrome (ACS) patients is associated with adverse outcomes and may be related to clopidogrel dosing. Clinical studies evaluating different clopidogrel doses have resulted in conflicting conclusions. Clopidogrel dosing regimens have evolved over time, enabling us to evaluate platelet reactivity in real-life ACS patients undergoing percutaneous coronary intervention and treated with three different clopidogrel doses. Platelet reactivity was assessed with light transmitted aggregometry on the third day post clopidogrel loading in 404 consecutive ACS patients. Of them, 198 were treated with a standard regimen (300?mg loading, 75?mg/day maintenance dose), 95 with a high loading regimen (600?mg loading, 75?mg/day maintenance dose) and 111 with a high loading/high maintenance regimen (600?mg loading, 150?mg/day maintenance). Compared with the standard regimen, the high loading regimen resulted in significantly lower mean platelet reactivity to adenosine diphosphate (ADP) with a lower proportion of patients exhibiting clopidogrel non-responsiveness (11% vs. 28%, p?=?0.004). Compared with the high loading regimen, the high loading/high maintenance regimen resulted in significantly lower mean platelet reactivity to ADP, but without a further drop in the number of non-responders (8.1% vs. 11%, p?=?0.16). In conclusion, greater overall inhibition can be achieved with higher loading and maintenance doses in ACS patients. However, despite high clopidogrel doses, a sizable proportion of patients remained “resistant” to the effects of clopidogrel.     

An Er:YAG laser endoscopic fiber delivery system for lithotripsy of salivary stones

BACKGROUND AND OBJECTIVES: Endoscopic applications of Erbium:YAG lasers are still very limited due to lack of appropriate fiber delivery capabilities. Recent reports on potential advantages of this laser for lithotripsy of ureteral stones prompted us to develop an Er:YAG fiber delivery system for endoscopic lithotripsy of salivary stones. We report on the development of this system and its clinical use on 17 patients. STUDY DESIGN/MATERIALS AND METHODS: Ho:YAG and Er:YAG laser fragmentation performances were initially compared. Optimal laser parameters for lithotripsy of salivary stones were then established ex vivo using a commercial dental Er:YAG laser (Lumenis Opusdent 20). Metal hollow waveguides optimized for Er:YAG laser transmission were end sealed with a polished sapphire rod of 0.63 mm diameter and designed to adapt to the Opusdent laser and to a Storz sialoendoscope. The system was tested ex vivo for durability and clinical compatibility at input energies up to 700 mJ, 10-20 Hz. Following Helsinki approval the system was clinically tested on 17 patients with sialolithiasis. RESULTS: Lithotripsy threshold was around 80 mJ/pulse (26 J/cm2) while efficient fragmentation, with microscopic fragments, was observed at an output energy range of 150-300 mJ/pulse. At 10 Hz, fragmentation rates of about 1.8 mm3/second were achieved enabling lithotripsy of a 6 mm stone in about 2 minutes. Front surface damage to the sapphire rod occurred but did not contribute to significant loss in fragmentation efficiency. Of the 21 stones treated clinically, 5 were fully fragmented, 7 were prepared for extraction by mini forceps, and 9 were released from surrounding soft tissues for subsequent removal. Fifteen of the 18 treated glands returned to normal function without any symptoms. CONCLUSIONS: The Er:YAG endoscopic delivery system described is a clinically viable and cost-effective device for a range of hard and soft tissue wet field applications accessible through rigid or semi-rigid endoscopes. Further improvements in the waveguide may allow access also through fully flexible endoscopes.     

Layered peptide arrays: high-throughput antibody screening of clinical samples

High-throughput methods to detect and quantify antibodies in sera and other patient specimens have use for many clinical and laboratory studies, including those associated with cancer detection, microbial exposures, and autoimmune diseases. We developed a new technique, termed layered peptide array (LPA), to serve as a screening tool to detect antibodies in a highly multiplexed format. We demonstrate here that a prototype LPA was capable of producing approximately 5000 measurements per experiment and appeared to be scalable to higher throughput levels. Sera and saliva from Sj?gren's syndrome patients served as a test set to examine antibody titers in clinical samples. The LPA platform exhibited both a high sensitivity (100%) and high specificity (94%) for correctly identifying SSB antigen-positive samples. The multiplex capability of the platform was also confirmed when serum and saliva samples were analyzed for antibody reactivity to several peptides, including Sj?gren's syndrome antigens A and B. The data indicate that LPA analysis will be a useful method for a number of screening applications.     

Functional optical detection based on pH dependent fluorescence lifetime

BACKGROUND AND OBJECTIVES: Detection of possible alterations of physiological parameters (e.g., pH and temperature), resulting from malignant transformation of initially healthy tissue, can be a powerful diagnostic tool for earlier cancer detection. Such variations can be observed by comparing these parameters with those of healthy tissue surrounding the abnormality. Time-resolved spectroscopy of specifically targeted fluorescent labeled antibodies can be sensitive to such variations and provide a high resolution functional image of the region of interest. The goal of this study was to establish a forward experimental setup for calibration of the lifetime dependencies of near-IR fluorescent dyes on physiological parameters, and to develop analytical solutions, taking into account the effects of light propagation in turbid media (e.g., tissue), that was able to extract an original lifetime fluorescence signal from time-of-flight intensity distributions, measured in vivo from a deeply embedded live organ for further analysis. STUDY DESIGN/MATERIALS AND METHODS: Tissue-like phantoms with embedded fluorescent dyes and background optical properties simulating those of live tissues were designed and created. Fluorescence decay curves were measured for different fluorophore positions, and pH values. Those measurements were made with a system based on a time-correlated single photon counting (TCSPC) instrument and a tunable femtosecond Ti-Sapphire system built by our group. RESULTS: Decay curves were recorded for fluorophore depths of up to 5 mm and source-detector separation of 7 mm. It was shown that a forward model, based on the random walk theory, adequately described the experimental data. Measured pH dependencies of the fluorescence lifetime were characterized for two different dyes. CONCLUSIONS: Good correlation between experimental data and predictions of the theoretical model allows the use of close-form analytical solutions to separate the effects of photon time delays due to multiple scattering in tissues from the original intensity fluorescence time decay curve, determined by the fluorophore itself and its immediate surroundings. It is the latter dependence that can be diagnostically important. Experimentally obtained scaling between lifetime and a parameter of interest can be used in vivo to obtain a map of physiological parameter changes which can serve as a base for an in vivo specific diagnostic system.     

Theoretical and experimental investigation of the thermal effects within body cavities during transendoscopical CO2 laser-based surgery

BACKGROUND AND OBJECTIVES: The recent development of flexible hollow waveguides for MID-IR lasers may be utilized transendoscopically to ablate selectively neoplastic, superficial tissues within body cavities. Study goals are to investigate theoretically and experimentally heat distribution and thermal response of cavity lining, during CO2 laser minimally invasive surgery (MIS), and to thermally optimize the procedure under practical conditions. STUDY DESIGN/MATERIALS AND METHODS: Mathematical model was developed to predict temperature distribution along cavity lining. Experimental setup was built, including all the necessary components for a fully feedback-controlled MIS, i.e., laser generator, gas insufflating system, surgical suction, and infrared imaging feedback mechanism, all controlled by central PC-based program. Thermal images of cavity lining were recorded and analyzed throughout varying conditions. RESULTS: Thermal gradients along the cavity lining, during and after the laser irradiation, were obtained mathematically and experimentally. Diverse modes of heat dispersions were observed, as well as the relative contributions of user-controlled parameters to the maximal heat of cavity lining. The software-controlled setup has demonstrated the capacity to instantly manage varying conditions, by which it automatically protects cavity lining from getting overheated. CONCLUSIONS: Analytical predictions and experimental measurements were highly correlated. The software-controlled system may serve a powerful tool to control thermal side effects during MIS within body cavities.     

Subjects' Dissimilarity in the Analysis of Laser–Tissue Dose–Response Experiments: A Potential Cause for Puzzling Results

The in vivo dose–response laser-tissue experiment is usually performed on several subjects, each exposed several times at different tissue sites. The collected data are then unified into a single statistical batch and analysed under the tacit assumption that the tolerances of all the subjects are similar. However, if this assumption is incorrect the data unification may lead to very biased results. This study reanalyses a raw data set measured by the US Army Medical Research Detachment Walter Reed Army Institute of Research (USAMRD-WRAIR), which was used to study the influence of the laser wavelength on the energy threshold of retinal injury. The USAMRD-WRAIR report reveals a significant variation of threshold with small changes in wavelength. Despite an extensive study, performed by the USAMRD-WRAIR researchers, which included possible lasers variations, many aspects of experimental technique and biological absorption properties of the eye, a cause for the threshold variation was not found. Our current results suggest that unaccounted specimen's dissimilarity might be the cause for this unclear threshold variations. Paper received 4 September 2000; accepted after revision 27 March 2001.     

Flexible waveguides for IR laser radiation and surgery applications

Flexible plastic waveguides were developed to deliver IR radiation, especially at 10.6 μm, which is the CO₂ laser radiation wavelength. The waveguide is made from teflon tube with the inner wall coated with a metal layer and a dielectric over-layer. The internal diameter (ID) is 1.0 mm, length 1.0–1.2 m, and the distal tip decreases moderately to ID ∼0.6 mm. The distal part on the last 10 centimeters is coated externally with a metal layer. Maximum power that can be delivered at the outlet is ∼30 W and 10.6 × 10³ W/cm². This type of waveguide was used in several medical operations to evaluate its cutting characteristics and the resistances to heat reflection from the tissue while operating in orifices containing liquid substances. © 1994 Wiley-Liss, Inc.     

A new thermography-based approach to early detection of cancer utilizing magnetic nanoparticles theory simulation and in vitro validation

This work describes the utilization of tumor-specific magnetic nanoparticles together with an alternating magnetic field as a means to thermally mark a tumor so as to detect it using a thermal imaging system. Experiments were conducted using an in vitro tissue model, an inductive heating system, and an infrared camera. The thermal images, recorded by the infrared camera during the experiments, were analyzed using an algorithm that was developed as part of this work. The results show that small tumor phantoms (diameter of 0.5 mm) that were embedded under the surface of the tissue phantom (up to 14 mm below the surface) can be detected and located, indicating that the proposed method could potentially offer considerable advantages over conventional thermography and other methods for cancer early detection. Nevertheless, several issues should be clarified in future studies before the method can be offered for clinical use.From the Clinical EditorTumor-specific magnetic nanoparticles exposed to an alternating magnetic field provide a method to thermally mark a tumor for detection using thermal imaging systems. In-vitro tissue model experiments demonstrated that tumor phantoms of 0.5mm up to 14mm below the surface can be detected and located, indicating that the proposed method could offer considerable advantages over conventional thermography.