Papers by Elizabeth Vargis
Raman spectroscopy (RS) has received increasing attention as a potential tool for clinical diagno... more Raman spectroscopy (RS) has received increasing attention as a potential tool for clinical diagnostics. However, the unknown comparability of multiple tissue RS systems remains a major issue for technique standardization and future multisystem trials. In this study, we evaluated potential factors affecting data collection and interpretation, utilizing the skin as an example tissue. The effects of contact pressure and probe angle were characterized as potential user-induced variability sources. Similarly , instrumentation-induced variability sources of system stability and system-dependent response were also analyzed on skin and a nonvolatile biological tissue analog. Physiologically induced variations were studied on multiple tissue locations and patients. The effect of variability sources on spectral line shape and dispersion was analyzed with analysis-of-variance methods, and a new metric for comparing spectral dispersion was defined. In this study, in vivo measurements were made on multiple sites of skin from five healthy volunteers, with four stand-alone fiber optic probe–based tissue RS systems. System stability and controlled user-induced variables had no effects on obtained spectra. By contrast, instrumentation and anatomical location of measurement were significant sources of variability. These findings establish the comparability of tissue Raman spectra obtained by unique systems. Furthermore, we suggest steps for further procedural and instrumentation standardization prior to broad clinical applications of the technique.
The molecular changes that occur with cervical remodelling during pregnancy are not completely un... more The molecular changes that occur with cervical remodelling during pregnancy are not completely understood. This study reviews Raman spectroscopy, an optical technique for detecting changes in the pregnant cervix, and reports preliminary studies on cervical remodelling in mice that suggest that the technique provides advantages over other methods. Conclusion: Raman spectroscopy is sensitive to biochemical changes in the pregnant cervix and has high potential as a tool for detecting premature cervical remodelling in pregnant women. Preterm birth (PTB), defined as delivery before 37 weeks of gestation, occurs in approximately 12% of pregnancies in the United States and is the leading cause of infant mortality (1–3). Temporary and long-term morbidity of the infant can result, costing an estimated $26 billion each year in the United States (1). Preterm birth is traditionally classified into two groups as follows: indicated preterm birth and spontaneous preterm birth. The former is induced labour or caesarean section, initiated by the patient's healthcare provider due to complications in the mother or foetus, and the latter occurs spontaneously with over half of the cases having an unknown cause, which is the syndrome focused on in this study (4). Poor understanding of cervical remodelling in preparation for labour and delivery is a contributory factor to the unacceptably high rates of spontaneous preterm birth over the past 30 years. However, the preterm birth rate has shown small and steady decreases since 2007 in the United States (5–8). Current clinical practice for assessing preterm birth risk begins by completing a detailed medical history to account for risk factors such as previous preterm birth. Transvaginal ultrasound measurements of the cervix have demonstrated that a short cervix (<25 mm prior to 24 weeks of gestation) is correlated with an increased risk of preterm birth (9). Patients with a history of preterm birth are recommended to have transvaginal ultrasound measurements every 2 weeks from 16 to 23 weeks of gestation (10). If a short cervix is identified, cervical cerclage (sewing the cervix closed), or prescribing progesterone may be recommended based on the patient's history and current situation (10,11). Such interventions based upon cervical length measures have significantly reduced preterm birth and perinatal death rates. These measurements have also demonstrated that cervical shortening occurs weeks to months before subsequent preterm birth, suggesting that the cervix might be a prime source of important early information about preterm birth risk. Clinicians need tools that can quantitatively and objectively obtain biochemical information from the cervix, so that they can assess the risk of premature remodelling to have adequate time to intervene. In vivo Raman spectros-copy has the potential to provide such a tool. Preliminary findings highlighted in this study demonstrate the ability of this technique to detect biochemical changes in the pregnant mouse cervix over the course of gestation and identify features that signify impending parturition. We still do not fully understand the cervical changes that take place during pregnancy. This review looks at how Raman spectroscopy can detect those changes and reports that preliminary studies in mouse models suggest that this optical technique is sensitive to biochemical changes in the cervix and provides advantages over other methods. We believe that Raman spectroscopy could provide a highly effective tool for detecting premature cervical changes during pregnancy.
The spatial organization of retinal pigment epithelial (RPE) cells grown in culture was controlle... more The spatial organization of retinal pigment epithelial (RPE) cells grown in culture was controlled using micropatterning techniques in order to examine the effect of patch size on cell health and differentiation. Understanding this effect is a critical step in the development of multiplexed high throughput fluidic assays and provides a model for replicating disease states associated with the deterioration of retinal tissue during age-related macular degeneration (AMD). Microcontact printing of fibronectin on polystyrene and glass substrates was used to promote cell attachment, forming RPE patches of controlled size and shape. These colonies mimic the effect of atrophy and loss-of-function that occurs in the retina during degenerative diseases such as AMD. After 72 h of cell growth, levels of vascular endothelial growth factor (VEGF), an important biomarker of AMD, were measured. Cells were counted and morphological indicators of cell viability and tight junction formation were assessed via fluorescence microscopy. Up to a twofold increase of VEGF expression per cell was measured as colony size decreased, suggesting that the local microenvironment of, and connections between, RPE cells influences growth factor expression leading to the initiation and progression of diseases such as AMD.
—Multiscale models that link sub-cellular, cellular and multicellular components offer powerful i... more —Multiscale models that link sub-cellular, cellular and multicellular components offer powerful insights in disease development. Such models need a realistic set of parameters to represent the physical and chemical mechanisms at the sub-cellular and cellular levels to produce high fidelity multicellular outcomes. However, determining correct values for some of the parameters is often difficult and expensive using high-throughput microfluidic approaches. This work presents an alternative approach that estimates cellular parameters from spatiotemporal data produced from bioengineered multicellular in vitro experiments. Specifically, we apply a search technique to an integrated cellular and multicellular model of retinal pigment epithelial (RPE) cells to estimate the binding rate and auto-regulation rate of vascular endothelial growth factor (VEGF). Understanding VEGF regulation is critical in treating age-related macular degeneration and many other diseases. The method successfully identifies realistic values for autoregulatory cellular parameters that reproduce the spatiotemporal in vitro experimental data.
Raman spectroscopy has enabled researchers to map the specific chemical makeup of surfaces, solut... more Raman spectroscopy has enabled researchers to map the specific chemical makeup of surfaces, solutions, and even cells. However, the inherent insensitivity of the technique makes it difficult to use and statistically complicated. When Raman active molecules are near gold or silver nanoparticles, the Raman intensity is significantly amplified. This phenomenon is referred to as surface-enhanced Raman spectroscopy (SERS). The extent of SERS enhancement is due to a variety of factors such as nanoparticle size, shape, material, and configuration. The choice of Raman reporters and protective coatings will also influence SERS enhancement. This review provides an introduction to how these factors influence signal enhancement and how to optimize them during synthesis of SERS nanoparticles.
The Breast Cancer Metastasis Suppressor 1 (BRMS1) is a nucleo-cytoplasmic protein that suppresses... more The Breast Cancer Metastasis Suppressor 1 (BRMS1) is a nucleo-cytoplasmic protein that suppresses cancer metastasis without affecting the growth of the primary tumor. Previous work has shown that it decreases the expression of protein mediators involved in chemoresistance. This study measured the biomechanical and biochemical changes in BRMS1 expression and the responses of BRMS1 to drug treatments on cancer cells in vitro. The results show that BRMS1 expression affects biomechanical properties by decreasing the Young's modulus and adhesion force of breast cancer cells after doxorubicin (DOX) exposure. Raman spectral bands corresponding to DNA/RNA, lipids and proteins were similar for all cells after DOX treatment. The expression of cytokines were similar for cancer cells after DOX exposure, although BRMS1 expression had different effects on the secretion of cytokines for breast cancer cells. The absence of significant changes on apoptosis, reactive oxygen species (ROS) expression and cell viability after BRMS1 expression shows that BRMS1 has little effect on cellular chemoresistance. Analyzing cancer protein expression is critical in evaluating therapeutics. Our study may provide evidence of the benefit of metastatic suppressor expression before chemotherapy.
Background: Surface-enhanced Raman scattering (SERS) is a powerful light scattering technique tha... more Background: Surface-enhanced Raman scattering (SERS) is a powerful light scattering technique that can be used for sensitive immunoassay development and cell labeling. A major obstacle to using SERS is the complexity of fabricating SERS probes since they require nanoscale characterization and optical uniformity. The light scattering response of SERS probes may also be modulated by the substrate used for SERS analysis. A typical SERS substrate such as quartz can be expensive. Polystyrene is a cheaper substrate option but can decrease the SERS response due to interfering Raman emission peaks and high background fluorescence. The goal of this research is to develop an optimized process for fabricating Raman-labeled nanoparticles for a SERS-based immunoassay on a polystyrene substrate. Results: We have developed a method for fabricating SERS nanoparticle probes for use in a light scattering immunoassay on a polystyrene substrate. The light scattering profile of both spherical gold nanoparticle and gold nanorod SERS probes were characterized using Raman spectroscopy and optical absorbance spectroscopy. The effects of substrate interference and autofluorescence were reduced by selecting a Raman reporter with a strong light scattering response in a spectral region where interfering substrate emission peaks are minimized. Both spherical gold nanoparticles and gold nanorods SERS probes used in the immunoassay were detected at labeling concentrations in the low pM range. This analytical sensitivity falls within the typical dynamic range for direct labeling of cell-surface biomarkers using SERS probes.
The objective of the study was to characterize the variations in normal cervical spectra because ... more The objective of the study was to characterize the variations in normal cervical spectra because of menopausal status and location within the menstrual cycle. Using the information obtained, the accuracy of Raman spectroscopy to diagnose low-grade squamous intraepithelial lesion (LGSIL) will be improved.
Raman spectroscopy (RS) has received increasing attention as a potential tool for clinical diagno... more Raman spectroscopy (RS) has received increasing attention as a potential tool for clinical diagnostics. However, the unknown comparability of multiple tissue RS systems remains a major issue for technique standardization and future multisystem trials. In this study, we evaluated potential factors affecting data collection and interpretation, utilizing the skin as an example tissue. The effects of contact pressure and probe angle were characterized as potential user-induced variability sources. Similarly, instrumentation-induced variability sources of system stability and system-dependent response were also analyzed on skin and a nonvolatile biological tissue analog. Physiologically induced variations were studied on multiple tissue locations and patients. The effect of variability sources on spectral line shape and dispersion was analyzed with analysis-ofvariance methods, and a new metric for comparing spectral dispersion was defined. In this study, in vivo measurements were made on multiple sites of skin from five healthy volunteers, with four stand-alone fiber optic probe-based tissue RS systems. System stability and controlled userinduced variables had no effects on obtained spectra. By contrast, instrumentation and anatomical location of measurement were significant sources of variability. These findings establish the comparability of tissue Raman spectra obtained by unique systems. Furthermore, we suggest steps for further procedural and instrumentation standardization prior to broad clinical applications of the technique.
Mammary development and tumorigenesis are profoundly influenced by signaling pathways under the c... more Mammary development and tumorigenesis are profoundly influenced by signaling pathways under the control of c-erbB2/ c-neu and estrogen receptor A (ERA). Signaling through ERA is essential for ductal growth during puberty. In mice overexpressing wild-type c-neu in mammary epithelial cells, Tg (c-neu), ductal growth is impaired. An impeded signaling through ERA is also observed in a subset of human mammary tumors that overexpress erbB2. However, ductal growth is also impaired in the absence of c-neu in mouse mammary epithelial cells. To resolve this apparent paradox, we examined the relationship between c-neu expression and estrogen/ERAdependent cell proliferation in pubertal Tg (c-neu). We report that proliferation in both terminal end buds and ducts is associated with ERA-positive cells, including those that coexpress c-neu, and is abolished in the absence of circulating estradiol. Tg (c-neu) contains hyperplastic mammary ducts with high proliferative index and coexpression of both ERA and c-neu in the dividing cells. These findings suggest that c-neu promotes ERA-dependent proliferation, and that this is responsible for the presence of hyperplastic ducts. Some of the hyperplastic ducts have acinar structures, indicative of morphologic differentiation. These ducts have low proliferative index and accompanied by a vast decrease in proliferation of ERA-positive cells, including those that express c-neu. As such, c-neu has dual but opposing effects on ERA-dependent proliferation in mammary epithelial cells. Therefore, depending on the physiologic setting, ductal morphogenesis will be compromised both in the absence and overexpression of c-neu, thus explaining the paradox. (Cancer Res 2006; 66(21): 10391-8)
BACKGROUND: Detecting human papillomaviruses (HPVs) infection in cervical cells is an exceedingly... more BACKGROUND: Detecting human papillomaviruses (HPVs) infection in cervical cells is an exceedingly important part of the clinical management of cervical dysplasia. Current guidelines in women's health outline the need for both the Papanicolaou test as well as high-risk HPV testing. Testing for HPV is expensive, is time-consuming, and requires experienced technicians. METHODS: Two sets of near-infrared Raman microspectroscopy experiments were conducted using a Raman confocal microscope system. First, Raman spectra were acquired from four different cell culture lines, two positive for HPV (HeLa, SiHa), one negative for HPV, but malignant (C33A), and one normal, HPV-negative line (NHEK). The three malignant lines were all derived from cervical cells. Second, Raman spectra were acquired from deidentified patient samples that were previously tested for the presence of high-risk HPV. RESULTS: The spectra from the cell culture lines and the patient samples contained many statistically significant differences. Using sparse multinomial logistic regression to classify the data led to classification accuracies of 89% to 97% for the cell culture samples and 98.5% for the patient samples. CONCLUSIONS: Raman microspectroscopy can be used to detect HPV and differentiate among specific HPV strains. This technique may provide health providers with a new method for quickly testing cell samples for the presence of HPV.
The goal of this research is to determine whether Raman spectroscopy (RS), an optical method that... more The goal of this research is to determine whether Raman spectroscopy (RS), an optical method that probes the vibrational modes of tissue components, can be used in vivo to study changes in the mouse cervix during pregnancy. If successful, such a tool could be used to detect cervical changes due to pregnancy, both normal and abnormal, in animal models and humans. For this study, Raman spectra were acquired before, during and after a 19-day mouse gestational period. In some cases, after Raman data was obtained, cervices were excised for structural testing and histological staining for collagen and smooth muscle. Various peaks of the Raman spectra, such as the areas corresponding to fatty acid content and collagen organization, changed as the cervix became softer in preparation for labor and delivery. These findings correspond to the increase in compliance of the tissue and the collagen disorganization visualized with the histological staining. The results of this study suggest that non-invasive RS can be used to study cervical changes during pregnancy, labor and delivery and can possibly predict preterm delivery before overt clinical manifestations, potentially lead to more effective preventive and therapeutic interventions.
Many groups have used Raman spectroscopy for diagnosing cervical dysplasia; however, there have b... more Many groups have used Raman spectroscopy for diagnosing cervical dysplasia; however, there have been few studies looking at the effect of normal physiological variations on Raman spectra. We assess four patient variables that may affect normal Raman spectra: Race/ethnicity, body mass index (BMI), parity, and socioeconomic status. Raman spectra were acquired from a diverse population of 75 patients undergoing routine screening for cervical dysplasia. Classification of Raman spectra from patients with a normal cervix is performed using sparse multinomial logistic regression (SMLR) to determine if any of these variables has a significant effect. Results suggest that BMI and parity have the greatest impact, whereas race/ethnicity and socioeconomic status have a limited effect. Incorporating BMI and obstetric history into classification algorithms may increase sensitivity and specificity rates of disease classification using Raman spectroscopy. Studies are underway to assess the effect of these variables on disease. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).
The risk of local recurrence for breast cancers is strongly correlated with the presence of a tum... more The risk of local recurrence for breast cancers is strongly correlated with the presence of a tumor within 1 to 2 mm of the surgical margin on the excised specimen. Previous experimental and theoretical results suggest that spatially offset Raman spectroscopy (SORS) holds much promise for intraoperative margin analysis. Based on simulation predictions for signal-to-noise ratio differences among varying spatial offsets, a SORS probe with multiple source-detector offsets was designed and tested. It was then employed to acquire spectra from 35 frozen-thawed breast tissue samples in vitro. Spectra from each detector ring were averaged to create a composite spectrum with biochemical information covering the entire range from the tissue surface to ∼2 mm below the surface, and a probabilistic classification scheme was used to classify these composite spectra as "negative" or "positive" margins. This discrimination was performed with 95% sensitivity and 100% specificity, or with 100% positive predictive value and 94% negative predictive value. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).
Raman spectroscopy has the potential to differentiate among the various stages leading to high-gr... more Raman spectroscopy has the potential to differentiate among the various stages leading to high-grade cervical cancer such as normal, squamous metaplasia, and low-grade cancer. For Raman spectroscopy to successfully differentiate among the stages, an applicable statistical method must be developed. Algorithms like linear discriminant analysis (LDA) are incapable of differentiating among three or more types of tissues. We developed a novel statistical method combining the method of maximum representation and discrimination feature (MRDF) to extract diagnostic information with sparse multinomial logistic regression (SMLR) to classify spectra based on nonlinear features for multiclass analysis of Raman spectra. We found that high-grade spectra classified correctly 95% of the time; low-grade data classified correctly 74% of the time, improving sensitivity from 92 to 98% and specificity from 81 to 96% suggesting that MRDF with SMLR is a more appropriate technique for categorizing Raman spectra. SMLR also outputs a posterior probability to evaluate the algorithm's accuracy. This combined method holds promise to diagnose subtle changes leading to cervical cancer.
In traditional immuno-polymerase chain reaction (immuno-PCR), a single antibody recognition event... more In traditional immuno-polymerase chain reaction (immuno-PCR), a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. Here we describe a nanoparticle-amplified immuno-PCR (NPA-IPCR) assay that combines antibody recognition of enzymelinked immunosorbent assay (ELISA) with a 50-fold nanoparticle valence amplification step prior to tag amplification by PCR. The assay detects a respiratory syncytial virus (RSV) surface protein using an antibody bound to a 15-nm gold nanoparticle cofunctionalized with thiolated DNA complementary to a hybridized 76-base tag DNA with a tag DNA/antibody ratio of 50:1. The presence of virus particles triggers the formation of a ''sandwich'' complex composed of the gold nanoparticle construct, virus, and an antibody-functionalized magnetic particle used for extraction. After extraction, DNA tags are released by heating to 95°C and detected via real-time PCR. The limit of detection of the assay was compared with ELISA and reversion transcription (RT) PCR using RSV-infected HEp-2 cell extracts. NPA-IPCR showed an approximately 4000-fold improvement in the limit of detection compared with ELISA and a 4-fold improvement compared with viral RNA extraction followed by traditional RT-PCR. NPA-IPCR offers a viable platform for the development of early-stage diagnostics requiring an exceptionally low limit of detection.
In this article, we present a method that provides prompt detection of the presence of cancer cel... more In this article, we present a method that provides prompt detection of the presence of cancer cells inside the 2mm margin of tissue surrounding the tumor after excision using spatially offset Raman spectroscopy (SORS). SORS was developed to detect subtle changes in soft tissue spectra in the 100-2000 μm range and tested on excised breast tissues. The results display a very high specificity and sensitivity (100% and 95%, respectively) of classification between positive and negative tumor margins. SORS is a clinically feasible method, suitable for the real-time, intraoperative assessment of tumor margins at the micrometer level.
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Papers by Elizabeth Vargis