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Kato-Katz slide preservation technique: Extension of viability and the benefits for schistosomiasis control programs

The World Health Organization recommends the use of the Kato-Katz method in the procedures of schistosomiasis control programs. Studies show the importance of a fast reading of the slides due to the decline of their viability, with the appearance of fungi or desiccation of the sample, which hinders diagnosis. It is necessary to establish a procedure to improve the long-term preservation of these Kato-Katz slides in order to accomplish the following: (1) preserve the slides for future quality control procedures and readings; (2) allow for the production of durable materials for training; and (3) train health professionals involved in diagnosing schistosomiasis. Therefore, this study aims to test a slide preservation methodology for these purposes. The results showed that the modifications made to the experimental slides demonstrated that egg loss was within the expected range and the limit accepted by quality control standards, as well as improved the diagnostic durability of the slides during the preservation times tested. We concluded that the application of the preservation technique to the slides promoted stabilization and permanence for long-term storage.

Deep Learning and Transfer Learning for Malaria Detection.

Infectious disease malaria is a devastating infectious disease that claims the lives of more than 500,000 people worldwide every year. Most of these deaths occur as a result of a delayed or incorrect diagnosis. At the moment, the manual microscope is considered to be the most effective equipment for diagnosing malaria. It is, on the other hand, time-consuming and prone to human error. Because it is such a serious global health issue, it is important that the evaluation process be automated. The objective of this article is to advocate for the automation of the diagnosis process in order to eliminate the need for human intervention in the process. Convolutional neural networks (CNNs) and other deep-learning technologies, such as image processing, are being utilized to evaluate parasitemia in microscopic blood slides in order to enhance diagnostic accuracy. The approach is based on the intensity characteristics of Plasmodium parasites and erythrocytes, which are both known to be variable. Images of infected and noninfected erythrocytes are gathered and fed into the CNN models ResNet50, ResNet34, VGG-16, and VGG-19, which are all trained on the same dataset. The techniques of transfer learning and fine-tuning are employed, and the outcomes are contrasted. The VGG-19 model obtained the best overall performance given the parameters and dataset that were evaluated.

MO253: Interstitial Fibrosis in Anca-Associated Vasculitis: MPO Versus PR3-A Single-Center Experience

Abstract BACKGROUND AND AIMS Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a necrotizing vasculitis characterized by inflammation of small blood vessels, being the kidney one of the most frequently affected organs. AAV has a high morbidity and mortality, developing rapidly progressive renal failure that could lead to end-stage renal disease (ESRD). The pathogenesis is complex and multifactorial; macrophages have turned out to be a key element in inflammation and also in fibrosis. As already described in previous publications, PR3-AAV and MPO-AAV show clinical-demographic differences and different renal phenotypes on kidney biopsy. Histopathological subgrouping into four classes (focal, crescentic, mixed and sclerotic, as defined by Berden et al.) was proposed to predict long-term renal survival rates, poorest in the sclerotic class. This classification, validated by our group and multiple others, is still under review. The aim of this study is to demonstrate whether interstitial fibrosis in AAV is at least as important as glomerular sclerosis in renal prognosis, being a differentiating element between MPO and PR3 vasculitis. METHOD We performed a retrospective single-center study of 95 AAV-patients (78 MPO-AAV and 17 PR3-AAV) diagnosed by renal biopsy from 1991 to 2021 and at least 1-year follow-up. Clinical and laboratory variables, presence and type of ANCA, immunosuppressant therapy and renal/patient survival were evaluated. Sixty-two kidney biopsy samples have been analyzed: histomorphometric quantification has been carried out using MetaMorph® software on trichrome-stained slides in order to measure the degree of fibrosis. Continuous variables were described using measures of central tendency and dispersion (mean and standard deviation), and categorical variables using frequency tables and percentages. Student's t-tests or Mann–Whitney U tests, as appropriate, were performed to evaluate differences in mean values, and chi-squared tests were used to evaluate differences in qualitative variables. Statistical significance was defined as P-values <.05. All P-values were two-sided. RESULTS PR3-AAV population was predominantly male (70%) with a mean age at diagnosis of 62 years and a mean follow-up of 54 months, while MPO-AAV population was mainly female (65.8%), mean age 66 years and follow-up 65 months. There were no statistically significant differences in renal function between MPO and PR3-AAV at diagnosis. However, our data showed that MPO-AAV had worse renal function than PR3-AAV at the end of follow-up (P = .01). Furthermore, it should be highlighted that renal function improved throughout the follow-up (P < .01 and P = .05 in MPO and PR3-AAV, respectively). From a histological point of view: MPO-AAV manifests more interstitial fibrosis at diagnosis than PR3-AAV (P = .01). However, there were no statistically differences in the amount of glomerular sclerosis. It is noteworthy that PR3-AAV showed more crescentic proliferation at diagnosis (P = .03) but less fibrotic crescents than MPO-AAV (not reaching statistical significance). CONCLUSION The method we used allows a quantitative assessment of renal fibrosis. Our data confirm that renal prognosis is better in PR3-AAV than in MPO-AAV, which could be explained by a greater interstitial fibrosis, as well as more fibrotic crescentic in MPO-AAV at diagnosis.

Raman Microscopic Identification of Microorganisms on Metal Surfaces via Support Vector Machines.

An easy, inexpensive, and rapid method to identify microorganisms is in great demand in various areas such as medical diagnostics or in the food industry. In our study, we show the development of several predictive models based on Raman spectroscopy combined with support vector machines (SVM) for 21 species of microorganisms. The microorganisms, grown under standardized conditions, were placed on a silver mirror slide to record the data for model development. Additional data was obtained from microorganisms on a polished stainless-steel slide in order to validate the models in general and to assess possible negative influences of the material change on the predictions. The theoretical prediction accuracies for the most accurate models, based on a five-fold cross-validation, are 98.4%. For practical validation, new spectra (from stainless-steel surfaces) have been used, which were not included in the calibration data set. The overall prediction accuracy in practice was about 80% and the inaccurate predictions were only due to a few species. The development of a database provides the basis for further investigations such as the application and extension to single-cell analytics and for the characterization of biofilms.

Using deep learning to identify bladder cancers with FGFR-activating mutations from histology images.

BackgroundIn recent years, the fibroblast growth factor receptor (FGFR) pathway has been proven to be an important therapeutic target in bladder cancer. FGFR‐targeted therapies are effective for patients with FGFR mutation, which can be discovered through genetic sequencing. However, genetic sequencing is not commonly performed at diagnosis, whereas a histologic assessment of the tumor is. We aim to computationally extract imaging biomarkers from existing tumor diagnostic slides in order to predict FGFR alterations in bladder cancer.MethodsThis study analyzed genomic profiles and H&E‐stained tumor diagnostic slides of bladder cancer cases from The Cancer Genome Atlas (n = 418 cases). A convolutional neural network (CNN) identified tumor‐infiltrating lymphocytes (TIL). The percentage of the tissue containing TIL (“TIL percentage”) was then used to predict FGFR activation status with a logistic regression model.ResultsThis predictive model could proficiently identify patients with any type of FGFR gene aberration using the CNN‐based TIL percentage (sensitivity = 0.89, specificity = 0.42, AUROC = 0.76). A similar model which focused on predicting patients with only FGFR2/FGFR3 mutation was also found to be highly sensitive, but also specific (sensitivity = 0.82, specificity = 0.85, AUROC = 0.86).ConclusionTIL percentage is a computationally derived image biomarker from routine tumor histology that can predict whether a tumor has FGFR mutations. CNNs and other digital pathology methods may complement genome sequencing and provide earlier screening options for candidates of targeted therapies.

POS1315 USEFULNESS OF SYNOVIAL BIOPSY IN THE DIFFERENTIAL DIAGNOSIS AND AS POSSIBLE PREDICTOR OF RESPONSE TO TREATMENT IN JUVENILE IDIOPATHIC ARTHRITIS

Background:While synovial biopsy is an invasive procedure and is not required for the diagnosis of juvenile idiopathic arthritis (JIA), it may be useful in doubtful cases.Objectives:Aims of the study were i.to verify the role of synovial biopsy in the differential diagnosis of JIA and ii. to review the pathology slides in order to evaluate possible associations of a histologic pattern with response to treatment.Methods:We reviewed data from medical records of patients under the age of 18 years who underwent a synovial biopsy requested by our Pediatric Rheumatology Unit over the last 10 years. We collected information on demographic, clinical, laboratory, radiological, histopathological characteristics, as well as treatment response (in particular, remission at the last visit and number of examination, number of biologic drugs used). Among variables in the histologic score, number of layers in the synovial lining and inflammatory infiltrate (0-5) were compared to clinical status at last visit. Potential differences in variables between responders and non responders were assessed by unpaired t-test or non-parametric Mann-Whitney test, as appropriate.Results:We identified 64 patients (40F, 24M) with a median age at disease onset of 9 years (range 1-15) and a median follow-up time of 161 months (range 8-1160). We recognized two groups of interest: patients with a known JIA diagnosis (28/64) and patients with unknown diagnosis (36/64) at the moment of synovial biopsy. In the group with known JIA, most underwent the procedure during orthopedic surgery, and in all cases the histology was consistent with JIA. Among the unknown diagnosis group, in 19 cases results were consistent with a chronic synovitis, while among the other 17 histology could lead to a diagnosis of other conditions in 6 cases (foreign body and villonodular synovitis n=2 each, sarcoidosis and osteochondromatosis n=1 each). In the remaining 11 the final diagnoses were varied (mostly genetic forms eg skeletal dysplasia, CACP, Thiemann disease).Between the two groups we identified 46 patients with a definite JIA diagnosis. At the last follow-up visit 29 of them were in clinical remission, albeit on medication. The remaining 17 had a severe course of disease, with persistent activity and use of at least two biologic drugs. In 26 cases we could evaluate the correlation between status at last visit and number of layers/inflammatory infiltrate, but no statistical significant correlation was found.Conclusion:Despite its limited use nowadays, synovial biopsy may still be a useful tool in patients whose diagnosis is unclear. In our study, while it confirmed the suspicion in most cases, in other instances it allowed the diagnosis of rare conditions that would have been otherwise missed. No association between disease course and histological features in a small JIA cohort was found. We are currently expanding the study with a larger series.Disclosure of Interests:None declared

Airborne fungal spore monitoring: between analyst proficiency testing

This study presents the results of a Europe-wide training and Quality Control (QC) exercise carried out within the framework of the European Aerobiology Society’s QC Working Group. The main aim of this exercise was to examine the feasibility of carrying out a QC exercise for fungal spore monitoring in Europe, using a similar methodology to the one previously used for pollen. The QC survey was conducted in two parts: (1) Coordinators of national and regional aerobiological networks in Europe involved in the monitoring of atmospheric fungal spores were invited to complete a questionnaire survey related to their network and asked whether they were interested in taking part in an external inter-laboratory QC exercise; (2) Participating networks performed an inter-laboratory ring test with the same sample slide in order to determine the reproducibility of identifying and counting two fungal spore taxa (Alternaria and Epicoccum) in air samples collected by a Hirst-type volumetric spore trap. Participants were instructed to read five separate longitudinal transects in the “effective collecting area” of the slide. Reproducibility of analysis was determined following the method previously used in the European Aerobiology Society’s QC exercises for pollen. Thirty-two counters from 16 national or regional networks in Europe participated in the QC exercise. Coefficients of Variation (CV%) ranged from 23.0 to 22.5 when reading one transect and from 14.0 to 16.0 when reading five transects for Alternaria and Epicoccum, respectively. Considering a CV% of 30 as the limit for fungal spores, no significant differences were observed between the absolute errors from two, three, four and five transects. The only significant difference was between one and five transects. We recommend that fungal spore analysis should be carried out on about 5% of the slide (two transects in this study) because results were not significantly different to five transects.

Petrographical and microfacies study of Sinjar formation in Bazyan anticline, Sulaimaniyah region (Northern Iraq)

Purpose. The work studies Sinjar formation (upper Paleocene lower Eocene) within the Bazyan anticline in the Sulaimaniyah region (northeastern Iraq). The facies analysis is based on the petrographic study of limestone deposits and is essential to identify their nature. The sediments that formed Sinjar formation have a high content of skeletal granules which include a small amount of red algae and residues of unicellular organisms-foraminifera. Methodology. The method of work included two main aspects: field study and laboratory work. The first part included the field description of the rocky excavation and modeling of 10 rocks samples from it within the Bazyan section. As for the second main aspect, it included the preparation of 10 slides for petrographic study of Sinjar Formation rocks followed by a facial analysis of these deposits. Findings. Micro facial analysis showed that the sequences of Sinjar formation consist of three main facies. The wacky limestone facies bears benthic foraminifera. The facies of compact limestone bears the benthic foraminifera and red algae while the compact wacky limestone facies bears the fossils. Originality. Wacky limestone facies bearing benthic foraminifera, compacted limestone facies bearing benthic foraminifera and red algae, and wacky limestone facies bearing benthic foraminifera were selected and studied. Thus, according to the facial analysis, Sinjar formation was deposited within the open shelf before the formation of the main deposit. Practical value. Samples of rocks were obtained from the Bazyan anticline for further production of slides in order to study the facies of this anticline, which allowed showing its lithostratigraphic column.

Laser microdissection applications in histology: an open way to molecular studies

One of the most fascinating aspects of the use of a laser beam in the field of biology has emerged with the development of devices able to perform fine dissections of biological tissues. Laser microdissection can collect phenotypically identical cells from tissue regions laid on a microscope slide in order to make differential molecular analyses on these microdissected cells. Laser microdissection can be used many areas including oncology to specify molecular mechanisms that enable to adapt a treatment related to diagnosis and research in biology, but also forensic science for tissue selection, neurology for post-mortem studies on patients with Alzheimer's disease, for clonality studies from cell cultures and cytogenetics to decipher chromosomal rearrangements. This technology represents the missing link between clinical observations and the intrinsic physiological mechanisms of biological tissues and its major applications will be addressed here.

Comparison of Fractional Order PID Controller and Sliding Mode Controller with Computational Tuning Algorithm

The industry processes involving punching, lifting, and digging usually require high precision, high force and long operating hours that increase the prestige in the usage of the electrohydraulic actuator (EHA) system. These processes with the companion of the EHA system usually possess high dynamic complexities that are hard to be controlled and require well-designed and powerful control system. Therefore, this paper will involve the examination of the designed controllers which is applied to the EHA system. Firstly, the conventional proportional-integral-derivative (PID) controller which is the famous controller in the industry is designed. Then, the improved PID controller, which is known as the fractional order PID (FO-PID) controller is designed. After that, the design of the gradually famous robust controller in the education field, which is the sliding mode controller (SMC) is performed. Since the controller's parameters are essentially influencing the performance of the controller, the meta-heuristic optimization method, which is the particle swarm optimization (PSO) tuning method is applied. The variation in the system's parameter is applied to evaluate the performance of the designed controllers. Referring to the outcome analysis, the increment of 59.3% is obtained in the comparison between PID and FOPID, while the increment of 67.13% is obtained in the comparison of the PID with the SMC controller. As a conclusion, all of the controllers perform differently associated with their own advantages and disadvantages.

Comparison of Technical Terms and Consciousness of Blended Classes in ‘AI Technology’ and ‘Artificial Intelligence'

<p style="text-align:justify">Target subject is a module called ‘AI Technology’, which applied the ideas of blended learning. Firstly, lecture-style teaching was conducted with presentation slides in order to explain the contents of a textbook. Secondly, students were required to do exercises and quizzes. By using the last eight weeks, they were asked to create presentation slides outside a class to introduce the up-to-date topics on artificial intelligence. These slides were mutually evaluated among them so that they developed their own slides based on the feedback before the tenth week of the course for the second round of mutual evaluations. Questionnaires concerning students’ understanding technical terms of the field and consciousness-raising towards competence were also conducted before and after the programs. The learning effects of a module in ‘AI Technology’ are compared with my previous research outcome of the module, ‘Artificial Intelligence’. The reasons of difference between both modules are discussed. This paper reports their results.</p>

Evaluation of Proliferative Marker Ki-67 in Adenoid Cystic Carcinoma: A Retrospective Study

Adenoid cystic carcinoma (ACC) is a malignant tumor of salivary gland origin. Although the histologic appearance of ACC is low grade, management of this malignancy is a distinct therapeutic challenge because of its tendency for perineural involvement and potential for distant metastasis. Ki-67 antigen is expressed during the G1, S, G2 and M phases in the cell cycle but is absent in the quiescent G0 phase in tissue sections. Aim of the study was to review hematoxylin and eosin stained slides in order to confirm the previous histopathological diagnosis as per the criteria given by World Health Organisation (WHO) and to evaluate the expression of cell proliferation marker, Ki-67 antigen in Adenoid cystic carcinoma and correlate the expression of Ki-67 antigen histopathologically with different grades in Adenoid cystic carcinoma. Tissue samples of 32 cases (12 males and 20 females) were selected from minor salivary glands with age range from 21 to 70 years. Two paraffin-embedded sections of these total 32 cases each of 4 µm thick were cut on a rotary microtome. One section was stained using hematoxylin and Eosin (H&E) and the other was used for Immunohistochemical staining with Ki-67 antigen. Among these 32 cases of Adenoid cystic carcinoma, Histologically 14 (43.75%) tumors were classified in grade I, 8 (25%) were in grade II, and 10 (31.25%) were in grade III. The average percentage of Ki-67 expression was 27.12% in grade I, 34.43% in grade II and 38.45% in grade III. Ki-67 immunoreactivity increased with increase in histopathological grades of ACC. Since Ki-67 is a useful marker for assessing the proliferative potential of tumors, the prognosis of patients can definitely be predicted.

An insight into the cytotoxicity, genotoxicity, and mutagenicity of smoked cigarette butt leachate by using Allium cepa as test system

Smoked cigarette butt (SCB) discharged in the environment became an issue of unknown consequences for plants. Thus, we aim at assessing the impact of water containing SBC leachate on the meristem cells of Allium cepa roots. We defined the following experimental groups: negative control (water), positive control (cyclophosphamide); water with SCB leachate at environmental concentration (1.9μg/L of nicotine) (EC1× group) and water with SCB leachate concentration 1000 times higher than EC1 (EC1000× group). Mitotic index, total number of abnormal cells, index of abnormal cells per mitotic/phase, relative growth index, and inhibition index were calculated after 48 exposure hours. Root meristems were used to prepare slides in order to investigate chromosomal and nuclear abnormalities. According to our data, plants exposed to SCB leachate presented low relative growth index, high inhibition index, large number of abnormal cells, and high abnormality frequency at metaphase/anaphase. The exposed A. cepa recorded a wide variety of abnormalities such as diagonal metaphase/anaphase, metaphase/anaphase presenting chromosome fragments, binucleated cells, displaced nucleus, chromosome bridges, micronuclei, necrotic cells, stick metaphase, chromosome adherence, notched nucleus, among other cell disturbances. The chemicals in the SBC leachate had aneugenic and clastogenic effect on the genetic material of the tested plants, either when they acted individually, synergistically, or additively. Thus, our study is a pioneer in reporting that the mere disposal of cigarette butts in the environment can have cytotoxic, genotoxic, and mutagenic effects on plants.

Abstract B095: An automated tissue staining and quantitative digital image analysis pipeline for quantification of DNA damage repair at the single-cell level

Abstract Background: The repair of double-stranded breaks in DNA occurs through homologous recombination (HR) or nonhomologous enjoining (NHEJ) pathways. Germline mutations in DNA damage repair enzymes reduce efficiency of DNA damage repair (DDR) and increase cancer risk, but also the sensitivity to DNA damaging agents, such as cisplatinum and PARP1 inhibitors. Because the regulation of DDR occurs through protein-protein interactions and post-translational modifications, genomic methods are insufficient to identify cancers with defective DDR pathways. The formation of DDR protein complexes underlies strict spatial regulation. Therefore, the measurement of DDR efficiency requires in situ detection of DDR foci by visualizing the location of multiple proteins within the nucleus. This can be accomplished by multiantibody immunofluorescent staining combined with digital image analysis. The main goal of the project is to develop tissue staining assays for protein complexes involved in DDR using 5 antibodies per slide and quantitative digital image analysis pipelines to integrate data from consecutive slides in order to establish a DDR index of a patient’s prostate cancer. Methods: We developed 2 fully automated 5-plex fluorescent (RPA32, RAD51, Ku80, XRCC4, pH2AX) (53BP1, pDNA-PK, PARP1, PAR, AR) antibody protocols on the Ventana Discovery autostainer. The fluorescent assays were combined with an automated cytokeratin (CK), Ki-67, and Geminin chromogenic staining protocol on the exact same tissue. The panels were applied to a TMA of high-grade prostate cancer and for comparison to a bladder and a rectal cancer TMA slide. Slides were imaged after the fluorescent portion of the assay was completed and subsequently processed for staining with CK (yellow) and Ki-67 (teal) and Geminin (brown). Chromogenic and fluorescent images were coregistered. To integrate images from adjacent slides, a transformation matrix was applied to distort the images in order to maximize the overlap of nuclear DAPI stains. The nuclear staining of each antibody was quantified through intensity, area, and texture measurements and compared in nuclei positive and negative for pH2AX. After histogram normalization and subtraction of background and autofluorescence, the amount of colocalized pixels between antibody pairs was determined. Results: We have demonstrated feasibility of automated multiplex staining protocols that increase the reproducibility and future clinical utility of DDR assays. Specific staining of individual antibodies was identified as a punctate nuclear staining pattern in a fraction of cancer cells, and multiple parameters of the staining pattern (intensity, stained area, subnuclear distribution) were quantified using digital image analysis. DDR occurred in high-grade and low-grade prostate cancer as determined by the percentage of cancer cells that stained positive for pH2AX. pH2AX staining was also noticed in normal epithelium (weakly proliferative), stroma (nonproliferative), and inflammatory cells (proliferative). At least 5 nuclei from each compartment (tumor, normal, stroma, and inflammation) per TMA core were selected for analyses based on pH2AX positivity. Using a consistent image analysis pipeline to set staining intensity thresholds, overlapping pixels between RPA32-RAD51, Ku80-XRCC4, 53BP1-pDNAPK, and the overlap of pixels of each protein complex with AR were determined. The intracase and intercase variance of DDR complexes was compared for each cell type (cancer, normal epithelium, stroma, and inflammation) and correlated with the amount of cell proliferation. Conclusion: We demonstrate feasibility of measuring DDR complexes in single nuclei. The assay has the potential to be used in a CLIA-certified laboratory and the quantitative measurements from the multiplex DDR assays can be compared to those from other OMICS platforms, such as mutation burden and copy number alterations. Funding: Precision Medicine initiative and Translational Research Core at Cedars-Sinai Medical Center. Citation Format: Joshua Saylor, Eric Weterings, Nathan Ellis, James Hinton, Esteban Roberts, Anne Cress, Beatrice Knudsen. An automated tissue staining and quantitative digital image analysis pipeline for quantification of DNA damage repair at the single-cell level [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B095.

Abstract 2104: Near-infrared fluorescent tumor histology for multiplexed phenotypic identification of immunosuppressive cells

Abstract The purpose of our study was to develop near-infrared fluorophore-labeled antibodies to overcome autofluorescence interference in the visible spectrum, thereby enabling enhanced multiplexed identification of immune cells in human tumor tissue sections. Towards multiplexing, we conjugated each monoclonal primary antibody to a unique hapten tag. Detection of hapten-tagged primary antibodies was accomplished using high-affinity anti-hapten rabbit monoclonal secondary antibodies, which were each conjugated to a near-infrared fluorophore with Emax = 594, 650, 731, or 800 nm. For identification of immunosuppressive cells in tumor tissues, we used formalin-fixed, paraffin-embedded human tissue microarrays comprising malignant breast or prostate tumor core sections. Microarrays contained 12-60 patient biopsy cores with duplicate sections per case mounted on standard glass slides. Two antibody panels to detect immunosuppressive cell phenotypes were used in our study. The T regulatory panel targeted the antigens CD4, CD8, FOXP3, and LAG-3, allowing for phenotypic identification of CD4+ T helper cells, CD8+ cytotoxic T cells, CD4+ FOXP3+ Tregs, and highly immunosuppressive CD4+ FOXP3+ LAG-3+ Tregs. The macrophage panel targeted the antigens CD3, CD20, CD68, and CD163 for identification of CD3+ T cells, CD20+ B cells, CD68+ M1 macrophages, and highly immunosuppressive CD163+ M2 macrophages. Widefield fluorescence microscopy was used to image the stained tumor microarrays, utilizing a high-radiance mercury arc lamp, high-sensitivity digital CMOS camera, and single-band optical filter sets to detect near-infrared fluorescence. Triplicate images were taken per tissue core. Prior to image analysis, a spectral unmixing algorithm was created using a reference set of single-stained tissue slides in order to deconvolute minor spectral overlap of near-infrared fluorophores. The algorithm was then applied to the multiplexed digital raw images, and immunophenotyping analysis was conducted to enumerate cells of interest. Image analysis results indicated unique patterns of distribution of immune cells in tumor tissues correlated with tumor grade and stage. In breast cancer tissues, higher levels of LAG-3+ immunosuppressive cells were found in tumors of the aggressive, treatment-resistant "triple-negative" tumor subtype. In prostate cancer tissues, an increase in CD68+ macrophages and loss of immunostimulatory CD3+ T cells and CD20+ B cells were found to be associated with advanced tumor grade and Gleason score. The use of near-infrared fluorophores and hapten-tagged detection antibodies facilitated enhanced, rapid multiplexed phenotyping of immunosuppressive cells in tumor tissue. These findings demonstrate our approach to be a potentially paradigm-shifting technology for detection of immune infiltrates in patient samples. Citation Format: Amy Flor, Annalise Vaccarello, Matt Levin, Helen Snyder, David Schwartz, Stephen Kron. Near-infrared fluorescent tumor histology for multiplexed phenotypic identification of immunosuppressive cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2104.

Terahertz transmission vs reflection imaging and model-based characterization for excised breast carcinomas.

This work presents experimental and analytical comparison of terahertz transmission and reflection imaging modes for assessing breast carcinoma in excised paraffin-embedded human breast tissue. Modeling for both transmission and reflection imaging is developed. The refractive index and absorption coefficient of the tissue samples are obtained. The reflection measurements taken at the system's fixed oblique angle of 30° are shown to be a hybridization of TE and TM modes. The models are validated with transmission spectroscopy at fixed points on fresh bovine muscle and fat tissues. Images based on the calculated absorption coefficient and index of refraction of bovine tissue are successfully compared with the terahertz magnitude and phase measured in the reflection mode. The validated techniques are extended to 20 and 30 μm slices of fixed human lobular carcinoma and infiltrating ductal carcinoma mounted on polystyrene microscope slides in order to investigate the terahertz differentiation of the carcinoma with non-cancerous tissue. Both transmission and reflection imaging show clear differentiation in carcinoma versus healthy tissue. However, when using the reflection mode, in the calculation of the thin tissue properties, the absorption is shown to be sensitive to small phase variations that arise due to deviations in slide and tissue thickness and non-ideal tissue adhesion. On the other hand, the results show that the transmission mode is much less sensitive to these phase variations. The results also demonstrate that reflection imaging provides higher resolution and more clear margins between cancerous and fibroglandular regions, cancerous and fatty regions, and fibroglandular and fatty tissue regions. In addition, more features consistent with high power pathology images are exhibited in the reflection mode images.

Ultrasensitive and visual detection of squamous cell carcinoma antigen based on a silver-enhanced sandwich immunoassay using AuNPs@g-C3N4 nanocomposites

Based on gold label silver stain (GLSS) method, a sandwich-type immunoassay for visual, ultrasensitive and specific detection of squamous cell carcinoma antigen (SCCA) was constructed using gold nanoparticles@graphite- like carbon nitride (AuNPs@g-C3N4). In this assay, the capture SCCA antibody (Ab1) was immobilized on the silanized slides via the Schiff’s base links, and the AuNPs@g-C3N4 nanocomposite labeled by the detection SCCA antibody (Ab2) acted as the signal probe. The detection mechanism was as follows: Ab1 was immobilized onto the silanized slides in order to catch the target antigen SCCA, and then allowed the immobilization of the signal probe (Ab2) via the interaction between antigen and antibody. Compared with conventional GLSS method in which AuNPs are directly immobilized on Ab2, much more AuNPs corresponded to the antigen SCCA through AuNPs@g-C3N4 because g-C3N4 possessed larger surface area and loaded much more AuNPs than Ab2. Therefore, more AuNPs were exposed to silver ions during staining, more silver ions were reduced, and the sensitivity improved 1000-fold. The average grey values were linearly related to the logarithm of SCCA concentration from 0.001ng/mL to 100ng/mL, with high specificity, good reproducibility and long-term stability. Results suggested that the proposed sandwich immunoassay based on GLSS method is a simple, sensitive, selective, and visual tool for the detection of SCCA.

Integrating Clinical Medicine with the Basic Sciences; Pulmonology Learning Modules for Medical Students

Many medical students find memorizing the detailed of their basic science courses difficult and daunting, especially if they do not know what the application of that subject is during their medical practice. The overall objective of this study was to encourage students to adopt a more anatomical approach to clinical skills and also to show explicitly the relevance of pulmonary anatomy and embryology, thus moving away from rote memorization of physical exam skills and anatomical knowledge.In order to accomplish the goal of interactive case‐based modules we used the Articulate Engage software suite to create two modules that progress slide by slide in order to encourage a logical flow from patient presentation through to treatment. Using Articulate we were also able to add interactive labeled diagrams to review pulmonary anatomy, pictures to study clinical skills, and integrated quizzes to test student knowledge. The pulmonary modules were evaluated objectively from the survey questionnaire results and subjectively from the comments from the results. Before the modules 20% of students felt they knew the pulmonary materials well or very well. After the modules 58% of students felt they knew the pulmonary materials well or very well. Over 90% of students surveyed strongly or very strongly wanted to see more modules like these modules in their program.

Ion beam analysis and co-sputtering simulation (CO-SS) of bi-metal films produced by magnetron co-sputtering

Magnetron sputtering is widely used to deposit thin films on different types of substrates. An important application of this method is to make multicomponent thin films using co-sputtering, where two or more elements are included in the target. The thickness and elemental composition of the films depend on the experimental parameters used, the system geometry and the spatial distribution of the elements in the target. If the target is made of two spatially separate pieces of the materials, then the composition of the deposit depends on a combination of the relative areas, the sputtering yield and the angular distribution of the emission of the sputtered flux of each material. In this work, a co-sputtering simulation program, known as CO-SS, was developed to simulate the thickness and composition of metal films produced by DC magnetron sputtering (Al) and co-sputtering (Al+Ti). The CO-SS code models the angular distribution of particles ejected by sputtering from the target, where this is assumed to vary as cosnβ, where n is a free parameter and β is the angle of ejection relative to the normal to the surface of the target, and the sputtering yield of each material. The program also takes into account other geometry factors such as the distance between the target and the substrate, and the size of the substrate.Rutherford backscattering (RBS) using 4He was employed to measure the thickness and the composition of the films deposited on glass cover slides in order to assess the CO-SS program. The film thickness was also measured by profilometry. The CO-SS code was found to accurately model the experimental results for both the Al and Ti/Al films. The CO-SS code is freely available for use from http://demonstrations.wolfram.com/CoSputteringSimulationCOSS/.

Whole slide imaging in pathology: advantages, limitations, and emerging perspectives

Significant technologic gains have led to the adoption of innovative digital imaging solutions in pathology. Whole slide imaging (WSI), which refers to scanning of conventional glass slides in order to produce digital slides, is the most recent imaging modality being employed by pathology departments worldwide. WSI continues to gain traction among pathologists for diagnostic, educational, and research purposes. This article provides a technologic review of WSI platforms and covers clinical and nonclinical pathology applications of these imaging systems. Barriers to adoption of WSI include limiting technology, image quality, problems with scanning all materials (eg, cytology slides), cost, digital slide storage, inability to handle high-throughput routine work, regulatory barriers, ergonomics, and pathologists' reluctance. Emerging issues related to clinical validation, standardization, and forthcoming advances in the field are also addressed.