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1.  Identification of Inhibitors of Triacylglyceride Accumulation in Muscle Cells: Comparing HTS Results from 1536-well Plate-Based and High-Content Platforms 
Excess caloric consumption leads to triacylglyceride (TAG) accumulation in tissues that do not typically store fat, such as skeletal muscle. This ectopic accumulation alters cells, contributing to the pathogenesis of metabolic syndrome, a major health problem worldwide. We developed a 1536-well assay to measure intracellular TAG accumulation in differentiating H9c2 myoblasts. For this assay, cells were incubated with oleic acid to stimulate TAG accumulation prior to adding compounds. We used Nile red as a fluorescent dye to quantify TAG content with a microplate-reader. The cell nuclei were counterstained with DAPI nuclear stain to assess cell count and filter cytotoxic compounds. In parallel, we developed an image-based assay in H9c2 cells to measure lipid accumulation levels via high-content analysis, exploiting the dual emission spectra characteristic of Nile red staining of neutral and phospholipids. Using both approaches, we successfully screened ~227,000 compounds from the NIH Library. The screening data from the plate-reader and IC50 values correlated with that from the Opera QEHS cell imager. The 1536-well plate-reader assay is a powerful HTS platform to identify potent inhibitors of TAG accumulation to better understand the molecular pathways involved in lipid metabolism that lead to lipotoxicity.
PMCID: PMC3901053  PMID: 23989452
H9c2 cardiomyocytes; human primary myocytes; lipid accumulation; Nile red fluorescence; 1536-well High throughput screening; High content analysis; phenotypic screening
2.  Quantitative Automated Image Analysis System with Automated Debris Filtering for the Detection of Breast Carcinoma Cells 
Acta Cytologica  2011;55(3):271-280.
To develop an intraoperative method for margin status evaluation during breast conservation therapy (BCT) using an automated analysis of imprint cytology specimens.
Study Design
Imprint cytology samples were prospectively taken from 47 patients undergoing either BCT or breast reduction surgery. Touch preparations from BCT patients were taken on cut sections through the tumor to generate positive margin controls. For breast reduction patients, slide imprints were taken at cuts through the center of excised tissue. Analysis results from the presented technique were compared against standard pathologic diagnosis. Slides were stained with cytokeratin and Hoechst, imaged with an automated fluorescent microscope, and analyzed with a fast algorithm to automate discrimination between epithelial cells and noncellular debris.
The accuracy of the automated analysis was 95% for identifying invasive cancers compared against final pathologic diagnosis. The overall sensitivity was 87% while specificity was 100% (no false positives). This is comparable to the best reported results from manual examination of intraoperative imprint cytology slides while reducing the need for direct input from a cytopathologist.
This work demonstrates a proof of concept for developing a highly accurate and automated system for the intraoperative evaluation of margin status to guide surgical decisions and lower positive margin rates.
PMCID: PMC3101495  PMID: 21525740
Imprint cytology; Breast conservation therapy; Computer-aided diagnosis; Fluorescent microscopy
3.  Automated Microscopy to Evaluate Surgical Specimens Via Touch Prep in Breast Cancer 
Annals of surgical oncology  2009;16(3):709-720.
Breast conservation therapy is the standard treatment for breast cancer; however, 20–50% of operations have a positive margin leading to secondary procedures. The standard of care to evaluate surgical margins is based on permanent section. Imprint cytology (touch prep) has been used to evaluate surgical samples, but conventional techniques require an experienced cytopathologist for correct interpretation. An automated image screening process has been developed to discern cancer cells from normal epithelial cells. This technique is based on cellularity of the imprint specimen and does not require expertise in cytopathology.
A rapid immunofluorescent staining technique coupled with automated microscopy was used to classify specimens as cancer vs. noncancer based on the density of epithelial cells captured on touch prep of tumor cross-sections. The results of the automated analysis vs. a manual screen of ten 20× fields were compared to the pathology interpretation on permanent section.
A total of 34 consecutive cases were analyzed: 10 normal cases, and 24 cancer cases. The cross-section specimens for invasive cancer were correctly classified in at least 65% of the cases by using manual microscopy and at least 83% by using automated microscopy. The manual and automated microscopy correlated well for measurements of epithelial cell density (R2 = 0.64); however, the automated microscopy was more accurate.
This preliminary study using an automated system for intraoperative interpretation does not require a cytopathologist and shows that rapid, low-resolution imaging can correctly identify cancer cells for invasive carcinoma in surgical specimens. Therefore, automated determination of cellularity in touch prep is a promising technique for future margin interpretation of breast conservation therapy.
PMCID: PMC3154836  PMID: 19137376

Results 1-3 (3)