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Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high-resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with thionin, hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.

Absorption dyes are widely used in traditional cytology and pathology clinical practice, while fluorophores and nanoparticles are more often used in biologic research. Optical projection tomographic microscopy (OPTM) is a platform technology that can image the same specimen in multiple modes in 3D, providing morphologic and molecular information concurrently and in exact co-registration. The depth-of-field of a high numerical aperture objective is extended by scanning the focal plane through the sample to generate an optical projection image. Samples of cells or tissue are brought into the OPTM instrument through a microcapillary tube filled with optical index-matching gel. Multiple optical projection images are taken from different perspectives by rotating the tube. Computed tomography (CT) algorithms are applied to these optical projection images to reconstruct 3D structure of the sample. Image segmentation and analysis based on these 3D images provide quantitative biosignatures for cancer diagnosis that represents a clear improvement over conventional 2D image analysis. In this article, we introduce the OPTM platform, optical Cell-CT, and Tissue-CT instruments, and some applications using these OPTM instruments.

Background: The objective of this study was to assess the frequency of premalignant and malignant cells in sputum from patients with lung cancer and to measure the dependence of these cells on cancer stage, histologic type, tumor size, and tumor location., Methods: This analysis included 444 patients with lung cancer. First, all patients were asked to produce sputum spontaneously; then, they underwent sputum induction. Slide preparations of the sputa were screened for the presence of abnormal cells., Results: Of all patients with lung cancer who had produced adequate specimens, 74.6% had sputum that was positive for premalignant or worse cells, whereas 48.7% had sputum that was positive for malignant cells alone. Surprisingly, the presence of premalignant or worse cells in sputum depended only moderately on disease stage (82.9% of stage IV cancers vs 65.9% of stage I cancers), tumor size (78.6% of tumors >2 cm vs 64.7% of tumors

Objective: To study the feasibility of AutoPap System location-guided screening and to evaluate the accuracy of the AutoPap System in selecting locations of potentially abnormal cellular material, the accuracy of triaging slides using the selected locations and the improvement in laboratory accuracy and workload reduction resulting from a combination of location-guided rescreening and AutoPap System primary screening., Study Design: For the study, 683 conventionally prepared cervical cytologic smears (263 WNL, 184 atypical squamous cells of undetermined significance/atypical glandular cells of undetermined significance, 173 low grade squamous intraepithelial lesions, 55 high grade squamous intraepithelial lesions and 8 cancer slides) were acquired from nine laboratories. The study slides were independently screened to establish study reference diagnoses. The slides were processed on the AutoPap System and screened by cytotechnologists who were guided by the field of view (FOV) locations provided by the AutoPap System. The location-guided information was used either for initial manual screening (location-guided screening or after initial manual screening of the entire slide (location-guided rescreening). The results were compared to the study reference diagnoses. Sensitivity and workload reduction figures were estimated for different slide populations., Results: The accuracy of location-guided slide triage was high and did not vary significantly over different slide populations. Also, for > 80% of the abnormal slides, the AutoPap System-selected FOV slide locations did contain the most diagnostic cells, which were used to assign an accurate diagnosis to the slide. By combining location-guided rescreening with AutoPap primary screening, the estimated overall location-guided screening false negative fraction (FNF) for LSIL+ slides was approximately 40 times lower than the cytotechnologist-only FNFs, and this improvement was achieved-even at a no review rate of 70%., Conclusion: The feasibility study results showed that the AutoPap System location-guided screening process can triage slides accurately and enhance the overall accuracy of slide diagnosis. The combination of location-guided rescreening and AutoPap primary screening could improve the accuracy and workload of the laboratory.

Recent clinical trials indicated that the ThinPrep method of sample preparation has greater diagnostic sensitivity than the conventional direct Papanicolaou smear. The authors hypothesized that nonhomogeneous cell sampling during transfer from the sampling device to the microscope slide was a contributing factor to the reduced accuracy of the conventional direct Pap smears in these trials. To test this hypothesis, four direct smear methods were compared with the newly developed, fluid-based, filter-transfer method. Counts of epithelial cells on conventional smears showed that only a fraction of the available epithelial cells on the sampling devices (medians, 6.5% to 62.5%) was actually deposited on the slides. In all 27 cases studied with the ThinPrep method, equivalent diagnostic material was obtained on each of the replicate slides prepared per specimen. This identifies a new source of error, preparation error, in conventional smears.

A retrospective study was conducted to assess and confirm the significance of normal-appearing endometrial cells detected in cervical cytologic smears in the second half of the menstrual cycle or in the postmenopausal period. Of 440 women with normal endometrial cells identified on routine Papanicolaou smears, 179 underwent further endometrial evaluation. Endometrial disease was identified in 64 (35.7%) of those patients having endometrial sampling and/or hysterectomy within 12 months of the cytologic evaluations. These lesions included 21 cases (11.7%) of endometrial polyps, 23 cases (12.9%) of endometrial hyperplasia, and 20 cases (11.2%) of adenocarcinoma. The frequency of endometrial cancer was positively associated with age (P less than .01). Five of 20 women with endometrial cancer were asymptomatic.