There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of
infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained
microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of
immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use
of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the
antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational
modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after
washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be
quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been
successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of
rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining
results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this
technology in the field: first, simplifying assay operation through the automation of flow control using power-free
means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding
capacity, in order to move towards portable battery powered electronics.
To assess vascular responses of the human hand to inspiratory gasps and hand cooling, two imaging "remote
sensing" instruments were utilized: 1) a high-resolution infrared (IR) imaging camera and 2) a full-field laser
perfusion imager (FLPI). Data analysis was performed on the data sets collected simultaneously from both
instruments.
A non-localized drop of both FLPI and IR signals was observed at ~0.5-2.0 min after gasp onset. Spontaneous
oscillations, much below the human cardiac and respiratory frequencies, were observed with both imagers. The
dominant oscillations for both imaging modalities centered around 0.01Hz. Spectral frequencies, their power, and
the duration of temperature oscillations (bursts) for different hand areas changed in time and were spatially
heterogeneous. The highest spatial correlation between the two data sets was found between the mean IR
derivative image and the mean original FLPI image for the baseline conditions. Heterogeneous images of the
human hand were consistently detected non-invasively by both instruments. After cooling, a temperature
elevation of ~0.5ºC was seen as a spotted pattern mainly in the thenar and hypothenar areas. A generalized
increase in perfusion over the same areas was observed in FLPI images.
Both IR and FLPI imagers sensitively identify vasoconstrictor responses induced by inspiratory gasp and hand
cooling maneuvers. The specificity to physiological changes and high imaging rate for both instruments, coupled
with the current ease of use of optical cameras in clinical settings, make the described combination of two
instruments an ideal imaging approach to studying the dynamics of thermal and perfusion heterogeneity in human
skin.
The skin of athymic nude mice is irradiated with a single dose of x-ray irradiation that initiated fibrosis. Digital photographs of the irradiated mice are taken by illuminating the mouse skin with linearly polarized probe light of 650 nm. The specific pattern of the surface distribution of the degree of polarization enables the detection of initial skin fibrosis structures that were not visually apparent. Data processing of the raw spatial distributions of the degree of polarization based on Fourier filtering of the high-frequency noise improves subjective perception of the revealed structure in the images. In addition, Pearson correlation analysis provides information about skin structural size and directionality.
Anisotropy of mouse and human skin is investigated in vivo using polarized videoreflectometry. An incident beam (linearly polarized, wavelength 650 nm) is focused at the sample surface. Two types of tissuelike media are used as controls to verify the technique: isotropic delrin and highly anisotropic demineralized bone with a priori knowledge of preferential orientation of collagen fibers. Equi-intensity profiles of light, backscattered from the sample, are fitted with ellipses that appear to follow the orientation of the collagen fibers. The ratio of the ellipse semiaxes is well correlated with the ratio of reduced scattering coefficients obtained from radial intensity distributions. Variation of equi-intensity profiles with distance from the incident beam is analyzed for different initial polarization states of the light and the relative orientation of polarization filters for incident and backscattered light. For the anisotropic media (demineralized bone and human and mouse skin), a qualitative difference between intensity distributions for cross- and co-polarized orientations of the polarization analyzer is observed up to a distance of 1.5 to 2.5 mm from the entry point. The polarized videoreflectometry of the skin may be a useful tool to assess skin fibrosis resulting from radiation treatment.
Increasing evidence suggests that inflammation may contribute to the process of carcinogenesis. This is the basis of several clinical trials evaluating potential chemopreventive drugs. These trials require quantitative assessments of inflammation, which, for the oral epithelium, are traditionally provided by histopathological evaluation. To reduce patient discomfort and morbidity of tissue biopsy procedures, we develop a noninvasive alternative using diffuse reflectance spectroscopy to measure epithelial thickness as an index of tissue inflammation. Although any optical system has the potential for probing near-surface structures, traditional methods of accounting for scattering of photons are generally invalid for typical epithelial thicknesses. We develop a single-scattering theory that is valid for typical epithelial thicknesses. The theory accurately predicts a distinctive feature that can be used to quantify epithelial thickness given intensity measurements with sources at two different angles relative to the tissue surface. This differential measure approach has acute sensitivity to small, layer-related changes in scattering coefficients. To assess the capability of our method to quantify epithelial thickness, detailed Monte Carlo simulations and measurements on phantom models of a two-layered structure are performed. The results show that the intensity ratio maximum feature can be used to quantify epithelial thickness with an error less than 30% despite fourfold changes in scattering coefficients and 10-fold changes in absorption coefficients. An initial study using a simple two-source, four-detector probe on patients shows that the technique has promise. We believe that this new method will perform well on patients with diverse tissue optical characteristics and therefore be of practical clinical value for quantifying epithelial thickness in vivo.
Recent studies suggest that inflammatory cell products may contribute to the evolution of particular cancers leading to new chemoprevention trials exploring the benefit of anti-inflammatory drugs such as aspirin and related products. As part of a prospective trial evaluating this anti-inflammatory strategy for oral cancer, we evaluated a non-invasive optical system to determine if we could use an indirect measure of oral inflammation, mucosal thickness, as a monitoring parameter to evaluate the effectiveness of anti-inflammatory drug therapy. Diffuse reflectance spectroscopy has the potential for probing near-surface structures, however, traditional methods for accounting for scattering of photons are generally invalid for typical epithelial thicknesses. Monte Carlo simulations have shown that, with proper scaling, a simple photon model may be used to predict photon behavior under these conditions. A differential measure, which is very sensitive to small changes, has been shown to have the potential to quantify epithelial thickness. A simple prototype device has been brought from desk, to bench and bedside in a rapid manner to fill a need for a non-invasive measure of oral inflammation. From the theory, a simple feature has been identified that corresponds to patient oral inflammation. Preliminary results from this work are presented and indicate that further development of the approach to enable quantification of epithelial thickness in vivo is warranted.
Photodynamic therapy (PDT) is a surface oriented, locally cytotoxic intervention being investigated for oncologic therapy. Surfaces such as the pleura or the peritoneum are frequency involved with primary or metastatic cancer, and the chance for cure in such situations is low due to the inability to eradicate all the disease. A series of investigations has been performed at the National Cancer Institute since 1985 studying the possible use of PDT for large cavity treatment. This report details the original methodology, immediate results, and overall feasibility of the delivery of intrapleural PDT to patients after debulking of primary and malignant neoplasms in the chest which were considered to be 'non-curative' by standard surgical techniques alone. From this original feasibility study, an ongoing Phase I trial has developed to determine the maximum amount of this therapy which can be delivered safely to the thorax.
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