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1. Optimized workflow for unknown screening using gas chromatography high‐resolution mass spectrometry expands identification of contaminants in silicone personal passive samplers

   https://doi.org/10.1002/rcm.9048  

   Travis, Steven C. ; Kordas, Katarzyna ; Aga, Diana S. ( February 2021 , Rapid Communications in Mass Spectrometry)

   Silicone wristbands have emerged as valuable passive samplers for monitoring of personal exposure to environmental contaminants in the rapidly developing field ofexposomics. Once deployed, silicone wristbands collect and hold a wealth of chemical information that can be interrogated using high‐resolution mass spectrometry (HRMS) to provide a broad coverage of chemical mixtures.

   Gas chromatography coupled to Orbitrap™ mass spectrometry (GC/Orbitrap™ MS) was used to simultaneously perform suspect screening (using in‐house database) and unknown screening (using vendor databases) of extracts from wristbands worn by volunteers. The goal of this study was to optimize a workflow that allows detection of low levels of priority pollutants, with high reliability. In this regard, a data processing workflow for GC/Orbitrap™ MS was developed using a mixture of 123 environmentally relevant standards consisting of pesticides, flame retardants, organophosphate esters, and polycyclic aromatic hydrocarbons as test compounds.

   The optimized unknown screening workflow using a search index threshold of 750 resulted in positive identification of 70 analytes in validation samples, and a reduction in the number of false positives by over 50%. An average of 26 compounds with high confidence identification, 7 level 1 compounds and 19 level 2 compounds, were observed in worn wristbands. The data were further analyzed via suspect screening and retrospective suspect screening to identify an additional 36 compounds.

   This study provides three important findings: (1) a clear evidence of the importance of sample cleanup in addressing complex sample matrices for unknown analysis, (2) a valuable workflow for the identification of unknown contaminants in silicone wristband samplers using electron ionization HRMS data, and (3) a novel application of GC/Orbitrap™ MS for the unknown analysis of organic contaminants that can be used in exposomics studies.

    

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2. Rapid Screening and Quantification of PFAS Enabled by SPME-DART-MS

   https://doi.org/10.1021/jasms.3c00088  

   Emmons, Ronald V. ; Fatigante, William ; Olomukoro, Aghogho A. ; Musselman, Brian ; Gionfriddo, Emanuela ( September 2023 , Journal of the American Society for Mass Spectrometry)

   Per- and polyfluoroalkyl substances (PFAS), an emerging class of toxic anthropogenic chemicals persistent in the environment, are currently regulated at the low part-per-trillion level worldwide in drinking water. Quantification and screening of these compounds currently rely primarily on liquid chromatography hyphenated to mass spectrometry (LC-MS). The growing need for quicker and more robust analysis in routine monitoring has been, in many ways, spearheaded by the advent of direct ambient mass spectrometry (AMS) technologies. Direct analysis in real time (DART), a plasma-based ambient ionization technique that permits rapid automated analysis, effectively ionizes a broad range of compounds, including PFAS. This work evaluates the performance of DART-MS for the screening and quantification of PFAS of different chemical classes, employing a central composite design (CCD) to better understand the interactions of DART parameters on their ionization. Furthermore, in-source fragmentation of the model PFAS was investigated based on the DART parameters evaluated. Preconcentration of PFAS from water samples was achieved by solid phase microextraction (SPME), and extracts were analyzed using the optimized DART-MS conditions, which allowed obtaining linear dynamic ranges (LDRs) within 10 and 5000 ng/L and LOQs of 10, 25, and 50 ng/L for all analytes. Instrumental analysis was achieved in less than 20 s per sample. 

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3. Investigation of the adsorption/desorption mechanism of perfluoroalkyl substances on HLB-WAX extraction phases for microextraction

   https://doi.org/10.1016/j.aca.2023.341206  

   Olomukoro, Aghogho A. ; DeRosa, Charlotte ; Gionfriddo, Emanuela ( June 2023 , Analytica Chimica Acta)

   The C–F alkyl structural backbone of per- and polyfluoroalkyl substances makes this class of molecules resistant to heat and degradation, leading to their high persistence and mobility in the environment and bioaccumulation in the tissues of living organisms. In this study, 15 PFAS with an alkyl chain length from C4 to C14, currently monitored by the U.S. Environmental Protection Agency (EPA), were preconcentrated by solid-phase microextraction (SPME) and analyzed by liquid chromatography-tandem mass spectrometry. The adsorption and desorption mechanisms of PFAS onto ion-exchange extraction phases was evaluated to understand the extraction process of PFAS from various environmental matrices under different conditions. This was achieved using two SPME geometries, namely fibers and thin films. The use of thin films resulted in a twofold improvement in extraction efficiency compared to fibers, especially for the short-chain PFAS. Methanol:water (80:20, v/v) was chosen as the optimized desorption solution, with ammonium formate added to minimize carryover. Extraction time profiles for both SPME geometries showed faster equilibration with thin films (30 min) compared to fibers (90–120 min). The linear dynamic range obtained with this method using fibers and thin films ranged from 10 to 5000 ng L􀀀 1 and 2.5–5000 ng L􀀀 1, respectively, with acceptable accuracy (70–130%) and precision (<15%). LOD ranged within 2.5–10 ng L􀀀 1 for fibers and 0.01–0.25 ng L􀀀 1 for thin films. Investigating the factors affecting PFAS recovery in complex samples enabled the quantitative assessment of PFAS contamination in various environmental water samples such as seawater, melted snow and biospecimens like human plasma. A 96-SPME holder was used for validation, which is compatible with sampling in 96-well plates and ensures high throughput in the analysis of real samples. The total concentration of PFAS detected in seawater and snow was 51.3 ng L􀀀 1 and 16.4 ng L􀀀 1, respectively. 

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4. The Temple University Digital Pathology Corpus: The Breast Tissue Subset

   https://doi.org/10.1109/SPMB52430.2021.9672275  

   Wevodau, Z. ; Doshna, B. ; Jhala, N. ; Akhtar, I. ; Obeid, I. ; Picone, J. ( December 2021 , Proceedings of the IEEE Signal Processing in Medicine and Biology Symposium (SPMB))

   Obeid, I. (Ed.)

   The Neural Engineering Data Consortium (NEDC) is developing the Temple University Digital Pathology Corpus (TUDP), an open source database of high-resolution images from scanned pathology samples [1], as part of its National Science Foundation-funded Major Research Instrumentation grant titled “MRI: High Performance Digital Pathology Using Big Data and Machine Learning” [2]. The long-term goal of this project is to release one million images. We have currently scanned over 100,000 images and are in the process of annotating breast tissue data for our first official corpus release, v1.0.0. This release contains 3,505 annotated images of breast tissue including 74 patients with cancerous diagnoses (out of a total of 296 patients). In this poster, we will present an analysis of this corpus and discuss the challenges we have faced in efficiently producing high quality annotations of breast tissue. It is well known that state of the art algorithms in machine learning require vast amounts of data. Fields such as speech recognition [3], image recognition [4] and text processing [5] are able to deliver impressive performance with complex deep learning models because they have developed large corpora to support training of extremely high-dimensional models (e.g., billions of parameters). Other fields that do not have access to such data resources must rely on techniques in which existing models can be adapted to new datasets [6]. A preliminary version of this breast corpus release was tested in a pilot study using a baseline machine learning system, ResNet18 [7], that leverages several open-source Python tools. The pilot corpus was divided into three sets: train, development, and evaluation. Portions of these slides were manually annotated [1] using the nine labels in Table 1 [8] to identify five to ten examples of pathological features on each slide. Not every pathological feature is annotated, meaning excluded areas can include focuses particular to these labels that are not used for training. A summary of the number of patches within each label is given in Table 2. To maintain a balanced training set, 1,000 patches of each label were used to train the machine learning model. Throughout all sets, only annotated patches were involved in model development. The performance of this model in identifying all the patches in the evaluation set can be seen in the confusion matrix of classification accuracy in Table 3. The highest performing labels were background, 97% correct identification, and artifact, 76% correct identification. A correlation exists between labels with more than 6,000 development patches and accurate performance on the evaluation set. Additionally, these results indicated a need to further refine the annotation of invasive ductal carcinoma (“indc”), inflammation (“infl”), nonneoplastic features (“nneo”), normal (“norm”) and suspicious (“susp”). This pilot experiment motivated changes to the corpus that will be discussed in detail in this poster presentation. To increase the accuracy of the machine learning model, we modified how we addressed underperforming labels. One common source of error arose with how non-background labels were converted into patches. Large areas of background within other labels were isolated within a patch resulting in connective tissue misrepresenting a non-background label. In response, the annotation overlay margins were revised to exclude benign connective tissue in non-background labels. Corresponding patient reports and supporting immunohistochemical stains further guided annotation reviews. The microscopic diagnoses given by the primary pathologist in these reports detail the pathological findings within each tissue site, but not within each specific slide. The microscopic diagnoses informed revisions specifically targeting annotated regions classified as cancerous, ensuring that the labels “indc” and “dcis” were used only in situations where a micropathologist diagnosed it as such. Further differentiation of cancerous and precancerous labels, as well as the location of their focus on a slide, could be accomplished with supplemental immunohistochemically (IHC) stained slides. When distinguishing whether a focus is a nonneoplastic feature versus a cancerous growth, pathologists employ antigen targeting stains to the tissue in question to confirm the diagnosis. For example, a nonneoplastic feature of usual ductal hyperplasia will display diffuse staining for cytokeratin 5 (CK5) and no diffuse staining for estrogen receptor (ER), while a cancerous growth of ductal carcinoma in situ will have negative or focally positive staining for CK5 and diffuse staining for ER [9]. Many tissue samples contain cancerous and non-cancerous features with morphological overlaps that cause variability between annotators. The informative fields IHC slides provide could play an integral role in machine model pathology diagnostics. Following the revisions made on all the annotations, a second experiment was run using ResNet18. Compared to the pilot study, an increase of model prediction accuracy was seen for the labels indc, infl, nneo, norm, and null. This increase is correlated with an increase in annotated area and annotation accuracy. Model performance in identifying the suspicious label decreased by 25% due to the decrease of 57% in the total annotated area described by this label. A summary of the model performance is given in Table 4, which shows the new prediction accuracy and the absolute change in error rate compared to Table 3. The breast tissue subset we are developing includes 3,505 annotated breast pathology slides from 296 patients. The average size of a scanned SVS file is 363 MB. The annotations are stored in an XML format. A CSV version of the annotation file is also available which provides a flat, or simple, annotation that is easy for machine learning researchers to access and interface to their systems. Each patient is identified by an anonymized medical reference number. Within each patient’s directory, one or more sessions are identified, also anonymized to the first of the month in which the sample was taken. These sessions are broken into groupings of tissue taken on that date (in this case, breast tissue). A deidentified patient report stored as a flat text file is also available. Within these slides there are a total of 16,971 total annotated regions with an average of 4.84 annotations per slide. Among those annotations, 8,035 are non-cancerous (normal, background, null, and artifact,) 6,222 are carcinogenic signs (inflammation, nonneoplastic and suspicious,) and 2,714 are cancerous labels (ductal carcinoma in situ and invasive ductal carcinoma in situ.) The individual patients are split up into three sets: train, development, and evaluation. Of the 74 cancerous patients, 20 were allotted for both the development and evaluation sets, while the remain 34 were allotted for train. The remaining 222 patients were split up to preserve the overall distribution of labels within the corpus. This was done in hope of creating control sets for comparable studies. Overall, the development and evaluation sets each have 80 patients, while the training set has 136 patients. In a related component of this project, slides from the Fox Chase Cancer Center (FCCC) Biosample Repository (https://www.foxchase.org/research/facilities/genetic-research-facilities/biosample-repository -facility) are being digitized in addition to slides provided by Temple University Hospital. This data includes 18 different types of tissue including approximately 38.5% urinary tissue and 16.5% gynecological tissue. These slides and the metadata provided with them are already anonymized and include diagnoses in a spreadsheet with sample and patient ID. We plan to release over 13,000 unannotated slides from the FCCC Corpus simultaneously with v1.0.0 of TUDP. Details of this release will also be discussed in this poster. Few digitally annotated databases of pathology samples like TUDP exist due to the extensive data collection and processing required. The breast corpus subset should be released by November 2021. By December 2021 we should also release the unannotated FCCC data. We are currently annotating urinary tract data as well. We expect to release about 5,600 processed TUH slides in this subset. We have an additional 53,000 unprocessed TUH slides digitized. Corpora of this size will stimulate the development of a new generation of deep learning technology. In clinical settings where resources are limited, an assistive diagnoses model could support pathologists’ workload and even help prioritize suspected cancerous cases. ACKNOWLEDGMENTS This material is supported by the National Science Foundation under grants nos. CNS-1726188 and 1925494. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. REFERENCES [1] N. Shawki et al., “The Temple University Digital Pathology Corpus,” in Signal Processing in Medicine and Biology: Emerging Trends in Research and Applications, 1st ed., I. Obeid, I. Selesnick, and J. Picone, Eds. New York City, New York, USA: Springer, 2020, pp. 67 104. https://www.springer.com/gp/book/9783030368432. [2] J. Picone, T. Farkas, I. Obeid, and Y. Persidsky, “MRI: High Performance Digital Pathology Using Big Data and Machine Learning.” Major Research Instrumentation (MRI), Division of Computer and Network Systems, Award No. 1726188, January 1, 2018 – December 31, 2021. https://www. isip.piconepress.com/projects/nsf_dpath/. [3] A. Gulati et al., “Conformer: Convolution-augmented Transformer for Speech Recognition,” in Proceedings of the Annual Conference of the International Speech Communication Association (INTERSPEECH), 2020, pp. 5036-5040. https://doi.org/10.21437/interspeech.2020-3015. [4] C.-J. Wu et al., “Machine Learning at Facebook: Understanding Inference at the Edge,” in Proceedings of the IEEE International Symposium on High Performance Computer Architecture (HPCA), 2019, pp. 331–344. https://ieeexplore.ieee.org/document/8675201. [5] I. Caswell and B. Liang, “Recent Advances in Google Translate,” Google AI Blog: The latest from Google Research, 2020. [Online]. Available: https://ai.googleblog.com/2020/06/recent-advances-in-google-translate.html. [Accessed: 01-Aug-2021]. [6] V. Khalkhali, N. Shawki, V. Shah, M. Golmohammadi, I. Obeid, and J. Picone, “Low Latency Real-Time Seizure Detection Using Transfer Deep Learning,” in Proceedings of the IEEE Signal Processing in Medicine and Biology Symposium (SPMB), 2021, pp. 1 7. https://www.isip. piconepress.com/publications/conference_proceedings/2021/ieee_spmb/eeg_transfer_learning/. [7] J. Picone, T. Farkas, I. Obeid, and Y. Persidsky, “MRI: High Performance Digital Pathology Using Big Data and Machine Learning,” Philadelphia, Pennsylvania, USA, 2020. https://www.isip.piconepress.com/publications/reports/2020/nsf/mri_dpath/. [8] I. Hunt, S. Husain, J. Simons, I. Obeid, and J. Picone, “Recent Advances in the Temple University Digital Pathology Corpus,” in Proceedings of the IEEE Signal Processing in Medicine and Biology Symposium (SPMB), 2019, pp. 1–4. https://ieeexplore.ieee.org/document/9037859. [9] A. P. Martinez, C. Cohen, K. Z. Hanley, and X. (Bill) Li, “Estrogen Receptor and Cytokeratin 5 Are Reliable Markers to Separate Usual Ductal Hyperplasia From Atypical Ductal Hyperplasia and Low-Grade Ductal Carcinoma In Situ,” Arch. Pathol. Lab. Med., vol. 140, no. 7, pp. 686–689, Apr. 2016. https://doi.org/10.5858/arpa.2015-0238-OA. 

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5. Improving the Quality of the TUSZ Corpus

   Rahman, Safwanur Hamid ( December 2020 , IEEE Signal Processing in Medicine and Biology Symposium (SPMB))

   Obeid, Iyad ; Selesnick, Ivan ; Picone, Joseph (Ed.)

   The Temple University Hospital Seizure Detection Corpus (TUSZ) [1] has been in distribution since April 2017. It is a subset of the TUH EEG Corpus (TUEG) [2] and the most frequently requested corpus from our 3,000+ subscribers. It was recently featured as the challenge task in the Neureka 2020 Epilepsy Challenge [3]. A summary of the development of the corpus is shown below in Table 1. The TUSZ Corpus is a fully annotated corpus, which means every seizure event that occurs within its files has been annotated. The data is selected from TUEG using a screening process that identifies files most likely to contain seizures [1]. Approximately 7% of the TUEG data contains a seizure event, so it is important we triage TUEG for high yield data. One hour of EEG data requires approximately one hour of human labor to complete annotation using the pipeline described below, so it is important from a financial standpoint that we accurately triage data. A summary of the labels being used to annotate the data is shown in Table 2. Certain standards are put into place to optimize the annotation process while not sacrificing consistency. Due to the nature of EEG recordings, some records start off with a segment of calibration. This portion of the EEG is instantly recognizable and transitions from what resembles lead artifact to a flat line on all the channels. For the sake of seizure annotation, the calibration is ignored, and no time is wasted on it. During the identification of seizure events, a hard “3 second rule” is used to determine whether two events should be combined into a single larger event. This greatly reduces the time that it takes to annotate a file with multiple events occurring in succession. In addition to the required minimum 3 second gap between seizures, part of our standard dictates that no seizure less than 3 seconds be annotated. Although there is no universally accepted definition for how long a seizure must be, we find that it is difficult to discern with confidence between burst suppression or other morphologically similar impressions when the event is only a couple seconds long. This is due to several reasons, the most notable being the lack of evolution which is oftentimes crucial for the determination of a seizure. After the EEG files have been triaged, a team of annotators at NEDC is provided with the files to begin data annotation. An example of an annotation is shown in Figure 1. A summary of the workflow for our annotation process is shown in Figure 2. Several passes are performed over the data to ensure the annotations are accurate. Each file undergoes three passes to ensure that no seizures were missed or misidentified. The first pass of TUSZ involves identifying which files contain seizures and annotating them using our annotation tool. The time it takes to fully annotate a file can vary drastically depending on the specific characteristics of each file; however, on average a file containing multiple seizures takes 7 minutes to fully annotate. This includes the time that it takes to read the patient report as well as traverse through the entire file. Once an event has been identified, the start and stop time for the seizure is stored in our annotation tool. This is done on a channel by channel basis resulting in an accurate representation of the seizure spreading across different parts of the brain. Files that do not contain any seizures take approximately 3 minutes to complete. Even though there is no annotation being made, the file is still carefully examined to make sure that nothing was overlooked. In addition to solely scrolling through a file from start to finish, a file is often examined through different lenses. Depending on the situation, low pass filters are used, as well as increasing the amplitude of certain channels. These techniques are never used in isolation and are meant to further increase our confidence that nothing was missed. Once each file in a given set has been looked at once, the annotators start the review process. The reviewer checks a file and comments any changes that they recommend. This takes about 3 minutes per seizure containing file, which is significantly less time than the first pass. After each file has been commented on, the third pass commences. This step takes about 5 minutes per seizure file and requires the reviewer to accept or reject the changes that the second reviewer suggested. Since tangible changes are made to the annotation using the annotation tool, this step takes a bit longer than the previous one. Assuming 18% of the files contain seizures, a set of 1,000 files takes roughly 127 work hours to annotate. Before an annotator contributes to the data interpretation pipeline, they are trained for several weeks on previous datasets. A new annotator is able to be trained using data that resembles what they would see under normal circumstances. An additional benefit of using released data to train is that it serves as a means of constantly checking our work. If a trainee stumbles across an event that was not previously annotated, it is promptly added, and the data release is updated. It takes about three months to train an annotator to a point where their annotations can be trusted. Even though we carefully screen potential annotators during the hiring process, only about 25% of the annotators we hire survive more than one year doing this work. To ensure that the annotators are consistent in their annotations, the team conducts an interrater agreement evaluation periodically to ensure that there is a consensus within the team. The annotation standards are discussed in Ochal et al. [4]. An extended discussion of interrater agreement can be found in Shah et al. [5]. The most recent release of TUSZ, v1.5.2, represents our efforts to review the quality of the annotations for two upcoming challenges we hosted: an internal deep learning challenge at IBM [6] and the Neureka 2020 Epilepsy Challenge [3]. One of the biggest changes that was made to the annotations was the imposition of a stricter standard for determining the start and stop time of a seizure. Although evolution is still included in the annotations, the start times were altered to start when the spike-wave pattern becomes distinct as opposed to merely when the signal starts to shift from background. This cuts down on background that was mislabeled as a seizure. For seizure end times, all post ictal slowing that was included was removed. The recent release of v1.5.2 did not include any additional data files. Two EEG files had been added because, originally, they were corrupted in v1.5.1 but were able to be retrieved and added for the latest release. The progression from v1.5.0 to v1.5.1 and later to v1.5.2, included the re-annotation of all of the EEG files in order to develop a confident dataset regarding seizure identification. Starting with v1.4.0, we have also developed a blind evaluation set that is withheld for use in competitions. The annotation team is currently working on the next release for TUSZ, v1.6.0, which is expected to occur in August 2020. It will include new data from 2016 to mid-2019. This release will contain 2,296 files from 2016 as well as several thousand files representing the remaining data through mid-2019. In addition to files that were obtained with our standard triaging process, a part of this release consists of EEG files that do not have associated patient reports. Since actual seizure events are in short supply, we are mining a large chunk of data for which we have EEG recordings but no reports. Some of this data contains interesting seizure events collected during long-term EEG sessions or data collected from patients with a history of frequent seizures. It is being mined to increase the number of files in the corpus that have at least one seizure event. We expect v1.6.0 to be released before IEEE SPMB 2020. The TUAR Corpus is an open-source database that is currently available for use by any registered member of our consortium. To register and receive access, please follow the instructions provided at this web page: https://www.isip.piconepress.com/projects/tuh_eeg/html/downloads.shtml. The data is located here: https://www.isip.piconepress.com/projects/tuh_eeg/downloads/tuh_eeg_artifact/v2.0.0/. 

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