Image Analysis Software Research Articles

Three-Dimensional Modeling and Quantitative Assessment of Mandibular Volume in Ectodermal Dysplasia: A Case Series.

Background and Objectives: Ectodermal dysplasia (ED)-a genetic disorder-is characterized by severe tooth deficiency. We compared the mandibular volume and the sagittal and horizontal mandibular widths between patients with ED (ED group) and individuals without tooth deficiency (control group) using three-dimensional modeling. We hypothesized that the mandibular volume differs in ED cases owing to congenital tooth deficiency. Materials and Methods: We used previously obtained cone-beam computed tomography (CBCT) images of 13 patients with ED. The control group data comprised retrospective CBCT images of patients of similar age and sex with a skeletal relationship of class 1. Further, using the three-dimensional image analysis software, the tooth crowns were separated from the mandible, the mandible was reconstructed and the gonion-to-gonion distance in the mandible was marked, the distance to the menton point was measured, and the distance between the two condyles was measured and compared with the control group. Results: Overall, 46.2% and 53.8% of the participants were men and women, respectively. In the ED group, the mean age of the participants was 15.46 (range, 6-24) years, and the mean number of mandibular teeth was 4.62. Notably, the edentulous mandible volume of the ED group (27.020 mm3) was statistically significantly smaller than that of the control group (49.213 mm3) (p < 0.001). There was no difference between the two groups in terms of the marked points. For data analysis, the Shapiro-Wilk test, independent samples t-test, and Mann-Whitney U test were used. Conclusions: It has been considered that mandible volume does not develop in ED cases because of missing teeth. Modern practices, such as the CBCT technique and three-dimensional software, may be effective in identifying the true morphologic features, especially in patients with genetic syndromes affecting the maxillofacial structure.

Abstract 1525: Integration of high-plex tumor-Immune phenotyping and checkpoint interactions for deeper spatial characterization of human cancer tissues

Abstract Background: Immune evasion is a key cancer hallmark, and one of the primary mechanisms is the PD-1/PD-L1 checkpoint axis. While immunotherapies that target these interactions have reshaped cancer treatment, the variable efficacy in patients is not fully understood. Due to the weak correlation between PD-1/PD-L1 expression measured by single-plex assays and observed clinical outcomes, there is a need to understand protein interactions at the cellular and subcellular level. Proximity Ligation Assays, detecting spatial interactions between ligand-receptor targets, provide insights into the activation of signaling pathways. Coupled with high-plex spatial phenotyping with tumor and immune profiling biomarkers, the integration of cellular and functional information will be beneficial for a deeper characterization of the tumor microenvironment (TME), understanding immune responses, and identifying spatial signatures for patient stratification and targeted treatment strategies. Methods: In this study, we phenotyped biopsies from head and neck cancer patients enrolled in immune checkpoint inhibitor therapies. We integrated Naveni® PD-1/PD-L1 proximity ligation assays and a customizable high-plex PhenoCode™ Signature Panel (CD3ε/CD8/CD20/CD68/PanCK) for high-throughput profiling of the TME utilizing the PhenoImager® HT 2.0 platform. Comprehensive bioinformatic analyses were conducted for whole-slide segmentation, identifying cellular phenotypes, spatial neighborhoods, functional interactions, and distinct spatial signatures via the open-source image analysis software QuPath. Results: Our analyses revealed key differences in the localization of the PD-1/PD-L1 interactions within the TME of head and neck cancers. Areas of PD-1/PD-L1 interactions are associated with immune cell types on the periphery of the tumor and some within the tumor-infiltrating lymphocytes. The PhenoCode Signature panels helped resolve the localization of PD-1/PD-L1 interactions to immune cell types within the TME and provided a dual workflow for spatial co-localization of interacting receptor/ligand interactions and cell phenotypes within the TME. Conclusions: By combining spatial immune profiling with Akoya’s PhenoCode Signature panels and protein-protein interaction data from Navinci’s Naveni® PD-1/PD-L1 assay, deep correlative insights of the TME can be applied to improve predictions of clinical outcomes. This innovative approach enriches our comprehension of underlying mechanisms and stands as a promising tool for refining patient selection and optimizing treatment outcomes in immunotherapy. Citation Format: Sara Bodbin, Ning Ma, Aditya Pratapa, Nadya Nikulina, James Monkman, Niyati Jhaveri, Hampus Elofsson, Subham Basu, Agata Zieba-Wicher, Arutha Kulasinghe. Integration of high-plex tumor-Immune phenotyping and checkpoint interactions for deeper spatial characterization of human cancer tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1525.

Abstract 6433: Anti-CD36 monoclonal antibody can sensitize the neoadjuvant chemotherapy response in PDAC by eliminating the senescent tertiary lymphatic structure

Abstract Objective: Pancreatic ductal adenocarcinoma (PDAC) is deadly and aggressive. Neoadjuvant chemotherapy (NAC) may reduce tumor volume and alter the resectability of the tumor. The efficacy of neoadjuvant chemotherapy in patients is still limited. Tertiary lymphoid structures (TLSs) develop in peripheral non-lymphoid tissues and are essential for the immune response and correlate with chemotherapy in various tumor types. However, more research is needed to determine how TLSs affect neoadjuvant chemotherapy in PDAC. We aim to determine how neoadjuvant chemotherapy affects tumor-infiltrating lymphocytes (TLSs) in PDAC patients. We also want to find a biomarker to boost PDAC neoadjuvant chemotherapy response. Methods: We retrospectively collected formalin-fixed paraffin embedded (FFPE) tissues from 115 PDAC patients without preoperative treatment and 72 patients pretreated with neoadjuvant chemotherapy. Multispectral images were processed using AI-powered image analysis software (Tissue Gnostics; Zeiss, Germany). The 10X Genomics platform was used to sequence single cells in tissue samples from five patients who received neoadjuvant chemotherapy (NAC) and five patients who had not received any treatment before analysis (pretreatment naïve, PN). TLS was separated using Laser Microdissection. The mouse model was created by orthotopically injecting tumor cells into the pancreas. Results: Patients who received NAC have a senescent microenvironment. High-throughput single-cell sequencing showed that the senescent gene signature score was enriched in the NAC group, especially in immune cells. The percentage of p16+ CD20+ B cells and P16+ CD3+ T cells in TLSs also increased in the NAC group. Spatial transcriptomics analysis exhibited that CD36 is highly expressed in senescent TLSs, which suggested that CD36 may be a marker for senescent TLSs. Moreover, CD36+ TLSs induced the infiltration of MDSCs and regulatory T cells. CD36+ TLSs were an independent prognostic factor for poor overall survival (OS) (HR=0.67, p=0.002) and progression-free survival (PFS) (HR=0.71, p=0.003). A mice orthotopic xenograft model showed that an anti-CD36 monoclonal antibody eliminated senescent TLSs in pancreatic tissue. MDSCs and Tregs in the surrounding tissue also decreased. Additionally, in vivo KPC mice model showed that the combination of an anti-CD36 and chemotherapy reduced tumor volume and increased survival compared to chemotherapy alone. Conclusion: NAC has the potential to induce the senescence of tumor-associated TLSs, a phenomenon that has been linked to unfavorable prognosis in PDAC. CD36 serves as a viable marker for senescent TLSs that can be targeted. The administration of an anti-CD36 monoclonal antibody can potentially enhance the efficacy of chemotherapy in PDAC by selectively eliminating CD36+ senescent TLSs in mouse models. Citation Format: Jingrui Yan, Tianxing Zhou, Chao Yang, Yongjie Xie, Jun Yu, Jihui Hao. Anti-CD36 monoclonal antibody can sensitize the neoadjuvant chemotherapy response in PDAC by eliminating the senescent tertiary lymphatic structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6433.

Abstract 5504: Integrating ultrahigh-plex spatial phenotyping: From discovery to clinical applications

Abstract Background The journey from discovering predictive spatial biomarkers (known as spatial signatures) for immunotherapy responses to their clinical application necessitates a cohesive approach bridging ultrahigh-plex discovery experiments with high-throughput translational studies. This study focuses on integrating Akoya Biosciences spatial multiplexed imaging technologies with advanced data analysis techniques for comprehensive spatial phenotyping across the discovery-to-clinical continuum. Methods Human formalin-fixed, paraffin-embedded (FFPE) cancer tissues were profiled using ultrahigh-plex PhenoCode™ Discovery panels to examine cell lineage, immune activation, and checkpoint markers via the PhenoCycler®-Fusion spatial biology platform. The subsequent use of PhenoCode™ Signature panels targeted specific biomarkers of immune profile, immune contexture, tumor-infiltrating lymphocytes (TIL), macrophage polarization, and T cell status via the PhenoImager® HT platform. Open-source whole slide image analysis software QuPath was employed for precise image analysis, including ROI segmentation, cell detection, classification, exploration of spatial interactions, and identification of distinct spatial signatures. Results Our analysis revealed distinct spatial relationships within various tumor types, quantifying immune cell distributions and their interactions. The ultrahigh-plex data correlated with high-throughput signature panel analyses, thus paving a new simplified way for targeted identification and development of predictive spatial signatures for immunotherapy outcomes. Conclusions The combined use of ultrahigh-plex discovery panels, high-throughput signature panels, and deep-learning image analysis provides a comprehensive understanding of cellular interactions in the tumor microenvironment. This integrated approach, utilizing Akoya's end-to-end workflows, accelerates the identification of predictive spatial biomarker signatures across various human tissue samples. Citation Format: Aditya Pratapa, Ning Ma, Niyati Jhaveri. Integrating ultrahigh-plex spatial phenotyping: From discovery to clinical applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5504.

Abstract 1027: Immunohistochemistry (IHC) to assess tumor cell-associated OX40 expression in lymphoma PDXs

Abstract Background. Tumor necrosis factor receptor superfamily member 4, or OX40, is an immunostimulatory receptor usually expressed in activated T-cells. The OX40-OX40L (ligand of OX40) interaction plays a crucial role in the enhancement of T-cell proliferation, survival, and cytokine production, including those in tumor microenvironment (TME) leading to increased anti-tumor immunity1. OX40 agonist antibodies have been widely tested for immunotherapy for solid tumors2,3. Interestingly, in the treatment of lymphoma, the situation may become more complicate, where OX40 could also be found to be expressed on lymphoma cells, in addition to TME. This phenomenon might or might not impact OX40 agonist therapy. This report aims at developing an immunohistochemistry (IHC) assay to assess tumor-associated OX40 by utilizing morphology approach to understand OX40 expression in lymphoma cells, as opposed on tumor-infiltrate T-lymphocytes (TILs) present in TME. FFPE samples from patient-derived xenografts (PDXs), recapitulating the main pathological features of the original tumors, but exclusive of human TILs, were used as the first stage of the assay development, avoiding TIL interference prior to human sample testing. Methods. Ninety leukemia and lymphoma PDXs are comprehensively annotated, including RNAseq and histopathology (HuBaseTM: https://hubase.crownbio.com). IHC staining of PDX FFPE slides or TMA (tissue microarray) of various cancers was performed using a commercial OX40 antibody (CST, #61637) and Bond RX Automated IHC/ISH Staining System (Leica Biosystems), followed by whole slide imaging by NanoZoomer NDP2.0-HT Digital Slide System (Hamamatsu) and quantitative analysis by HALOTM image analysis software (Indica labs). OX40 IHC scores were also compared to the RNA expression determined by RNAseq. Results. A total of 90 leukemia and lymphoma PDXs were comprehensively evaluated. RNASeq data (human mRNA) indeed revealed OX40 gene expression (cutoff as Log2 (FPKM)&amp;gt;3) in 8.9% of models (8/90 models); on the other hand, IHC data also confirmed these OX40 protein expression; and the IHC scores largely correlated with the RNA data. Together, our data firmly confirmed the expression of OX40 in certain hematological cancers, beyond just in TME. The ongoing studies is investigating the characteristics of the OX40 positive lymphoma/leukemia, and the preliminary data will be presented in the annual conference. Furthermore, we are also using the same assay to explore other cancer types beyond hematological cancers. Conclusion. A tumor associated OX40 expression IHC workflow was preliminarily established and validated to facility the OX40 immunotherapy development. Citation Format: Jie Lin, Xiaolong Tu, Likun Zhang, Hongjiang Yu, Cheng JIang, Tao Yang, Henry Qixiang Li. Immunohistochemistry (IHC) to assess tumor cell-associated OX40 expression in lymphoma PDXs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1027.

Abstract 65: Differential impacts of PD-L1 expression in tumor vs. stroma on survival in esophageal squamous cell carcinoma

Abstract Background: Esophageal squamous cell carcinoma (ESCC) has high incidence and recurrence rates, particularly in advanced stages. While the combined positive score (CPS) and tumor proportional score (TPS) are standard benchmarks for PD-L1 assessment in ESCCs, they face challenges such as reliance on subjective human interpretation and the uncertain clinical significance of strong staining intensities. Our study utilizes H-scores for PD-L1 expression and separates analysis of tumor and stroma, providing additional layers of information that complement current methods and contribute to a more comprehensive understanding of the role of PD-L1 in ESCC. Methods: This retrospective single-center cohort analysis involved 194 cases of ESCC who underwent surgical resection. We performed a comprehensive evaluation of PD-L1 expression within the tumor and stroma using whole slide images (WSI). QuPath image analysis software was used to classify regions as tumor or stroma and quantify the PD-L1 expression H-score. Results: Using optimal H-score cutoffs determined from the minimum P-value method, the Kaplan-Meier survival analysis showed that higher PD-L1 expression was significantly linked with better post-operative survival across all compartments. This correlation was most pronounced in the stroma (P&amp;lt;0.001). However, in multivariate Cox regression analysis, only high tumoral PD-L1 expression emerged as an independent predictor of favorable prognosis (HR=0.47, 95% CI: 0.27-0.81, P=0.007). The non-significant contribution of stromal PD-L1 in this model could be attributed to its association with advanced pT and pN stages, and the presence of lymphatic and venous invasion (P&amp;lt;0.001, respectively). Interestingly, stromal PD-L1 expression was associated with a longer duration of cytotoxic chemotherapy (cutoff 8 months, Median H-score 98.28 vs 55.24, P=0.026), but no significant correlation was observed with the duration of immune checkpoint inhibitor treatment. Conclusions: The study underscores the complex role of PD-L1 in ESCC, with tumoral PD-L1 expression emerging as an independent prognostic factor, and stromal PD-L1 expression found to be related with tumor progression and the duration of cytotoxic chemotherapy response. Citation Format: Tomohiro Murakami, Satoru Furuhashi, Yuuki Sakai, Kenichi Sekimori, Ryoma Haneda, Eisuke Booka, Tomohiro Matsumoto, Hirotoshi Kikuchi, Yoshihiro Hiramatsu, Hiroya Takeuchi. Differential impacts of PD-L1 expression in tumor vs. stroma on survival in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 65.

Abstract 5525: Tissue-resident cytotoxic T cell infiltration predicts metachronous colorectal cancer metastasis

Abstract Background: Identifying patients at high risk for metachronous colorectal cancer metastasis is essential for improving prognosis. The balance between host immune regulation and tumor immune evasion ultimately permits or prevents cancer progression. Lymphocytic infiltration in colorectal cancer can predict long-term clinical outcomes. However, these lymphocytes' origin, subtype and function have yet to be adequately explored. We investigated the association between the development of metastasis and CD8+T cells expressing the integrin protein CD103, a marker of tissue-resident memory T cells. Methods: In this retrospective, case-control matched study, we conducted multiplex immunofluorescence staining of tumor samples from 124 patients with colorectal cancer. Tissue microarrays containing representative cores of the central tumor, invasive margin, and adjacent normal tissue were immunostained for CD3, CD8, CD103, pSMAD3, and cytokeratin, using tyramide signal amplification. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kruskal-Wallis one-way analysis of variance with subsequent Dunn’s test for pair-wise comparisons was performed using the Bonferroni method of multiple-comparison correction. Results: Central tumor and invasive margin CD3+ densities were associated with synchronous metastasis. Compared to non-metastatic patients, those who went on to develop metachronous metastasis (n=42) had a low proportion of intra-epithelial CD8+CD103+ T cells at the central tumor (non-metastatic: 32.8%, IQR 15.0-42.7%; metachronous metastasis: 12.9%, IQR 2.08-28.4%; P = 0.013). Similarly, patients with metachronous metastasis had a low proportion of stromal CD8+CD103+ T cells at the invasive margin (non-metastatic: 3.51%, IQR 1.83-6.55%; metachronous metastasis: 1.75%, IQR 0.62-3.72%; P = 0.019). On multivariate Cox regression of histological features, amongst patients with metastasis, significant overall survival prognostic markers included a low proportional CD8+CD103+ T cell infiltration (HR 2.41, 95%CI 1.16-5.01, P = 0.019), mucinous features (HR 4.28, 95%CI 1.98-9.24, P &amp;lt; 0.001), and the presence of perineural invasion (HR 2.75, 95%CI 1.28-5.89, P &amp;lt; 0.001). Conclusion: CD8+CD103+ lymphocytes may protect from the development of colorectal metastasis. Proportional CD8+CD103+ T cell infiltration is a promising prognostic marker in colorectal adenocarcinoma for the development of metachronous metastasis, and, amongst patients with metastasis, it is a prognostic marker of overall survival. Citation Format: Ryan Cohen, Tracey Lee-Pullen, Timothy Miller, Shadi Pirasteh, Cameron Platell, Katie Meehan, Kathy Fuller, Melanie McCoy. Tissue-resident cytotoxic T cell infiltration predicts metachronous colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5525.

Foaming and Physico-Mechanical Properties of Geopolymer Pastes Manufactured from Post-Metallurgical Recycled Slag.

This paper presents a research program aimed towards developing a method of producing lightweight, porous geopolymer composites for the construction industry based on industrial wastes. A direct method involving the addition of chemicals is currently most commonly used to produce the porous mineral structure of a geopolymer matrix. This relies on a reaction in a highly alkaline environment of the geopolymer to produce a gas (usually hydrogen or oxygen) that forms vesicles and creates a network of pores. This paper demonstrates the feasibility of producing a slag-based geopolymer paste foamed with aluminum powder, taking into account different parameters of fresh paste production: the mixing duration, its speed and the timing of foaming agent addition. The foaming process of the fresh paste in terms of the volumetric changes and temperature development of the fresh paste during the curing of the material are observed. After hardening, the physical properties (density and porosity) as well as the mechanical parameters (compressive strength and work of damage) are determined for the nine manufactured foamed pastes. Image analysis software was used to assess the porosity distribution of the material across the cross-section of the samples. The results enabled the design of the mixing procedure to be adopted during the manufacture of such composites.

Exploring water-absorbing capacity: a digital image analysis of seeds from 120 wheat varieties

Wheat is a staple food crop that provides a significant portion of the world's daily caloric intake, serving as a vital source of carbohydrates and dietary fiber for billions of people. Seed shape studies of wheat typically involve the use of digital image analysis software to quantify various seed shape parameters such as length, width, area, aspect ratio, roundness, and symmetry. This study presents a comprehensive investigation into the water-absorbing capacity of seeds from 120 distinct wheat lines, leveraging digital image analysis techniques facilitated by SmartGrain software. Water absorption is a pivotal process in the early stages of seed germination, directly influencing plant growth and crop yield. SmartGrain, a powerful image analysis tool, was employed to extract precise quantitative data from digital images of wheat seeds, enabling the assessment of various seed traits in relation to their water-absorbing capacity. The analysis revealed significant transformations in seed characteristics as they absorbed water, including changes in size, weight, shape, and more. Through statistical analysis and correlation assessments, we identified robust relationships between these seed traits, both before and after water treatment. Principal Component Analysis (PCA) and Agglomerative Hierarchical Clustering (AHC) were employed to categorize genotypes with similar trait patterns, providing insights valuable for crop breeding and genetic research. Multiple linear regression analysis further elucidated the influence of specific seed traits, such as weight, width, and distance, on water-absorbing capacity. Our study contributes to a deeper understanding of seed development, imbibition, and the crucial role of water absorption in wheat. These insights have practical implications in agriculture, offering opportunities to optimize breeding programs for improved water absorption in wheat genotypes. The integration of SmartGrain software with advanced statistical methods enhances the reliability and significance of our findings, paving the way for more efficient and resilient wheat crop production. Significant changes in wheat seed shape parameters were observed after imbibition, with notable increases in area, perimeter, length, width, and weight. The length-to-width ratio (LWR) and circularity displayed opposite trends, with higher values before imbibition and lower values after imbibition.

Noninvasive and Reliable Quantification of Anteromedial Rotatory Knee Laxity: A Pilot Study on Healthy Individuals

Background: Anteromedial rotatory instability (AMRI) of the knee is a complex and severe condition caused by injury to the anterior cruciate ligament and/or the medial collateral ligament. Clinical studies dealing with AMRI are rare, and objective measurements are nonexistent. Purpose/Hypothesis: The objectives of this study were, first, to quantify anteromedial rotatory knee laxity in healthy individuals using a noninvasive image analysis software and, second, to assess intra- and interrater reliability and equivalence in measuring anteromedial knee translation (AMT). It was hypothesized that AMT could be reliably quantified using a noninvasive image analysis software. Study design: Cohort study; Level of evidence, 3. Methods: This prospective proof-of-concept study included healthy individuals aged 16 to 40 years with no history of knee injury or surgery. Three adhesive surface markers were placed on predefined landmarks on the medial side of the knee. Three independent investigators examined anteromedial rotatory knee laxity with an anterior drawer test in different tibial rotations (neutral tibial rotation, 15° of external tibial rotation, and 15° of internal tibial rotation). The entire examination of each knee was recorded, and AMT including the side-to-side difference (SSD) was assessed using a freely available and validated image analysis software (PIVOT iPad application). Group comparisons were performed using a 1-way analysis of variance with Bonferroni-adjusted post hoc analysis. Intraclass correlation coefficients (ICCs) were calculated to assess inter- and intrarater reliability of AMT measurements. Equivalence of measurements was evaluated using the 2 one-sided t-test procedure. Results: Anteromedial rotatory knee laxity was assessed in 30 knees of 15 participants (53% male) with a mean age of 26.2 ± 3.5 years. In all 3 raters, the highest AMT was observed in neutral tibial rotation (range of means, 2.2-3.0 mm), followed by external tibial rotation (range of means, 2.0-2.4 mm) and internal tibial rotation (range of means, 1.8-2.2 mm; P < .05). Intrarater reliability of AMT (ICC, 0.88-0.96) and SSD (ICC, 0.61-0.96) measurements was good to excellent and moderate to excellent, respectively. However, interrater reliability was poor to moderate for AMT (ICC, 0.44-0.73) and SSD (ICC, 0.12-0.69) measurements. Statistically significant equivalence of AMT and SSD measurements was observed between and within raters for almost all testing conditions. Conclusion: Anteromedial rotatory knee laxity could be quantified using a noninvasive image analysis software, with the highest AMT observed during neutral tibial rotation in uninjured individuals. Reliability and equivalence of measurements were good to excellent within raters and moderate between raters.

The Global Track Concept for Assessment of Engaging Hill-Sachs Defects in Anterior Shoulder Instability

Background: The glenoid track concept is used to determine preoperatively whether a Hill-Sachs defect is engaging or not. Currently, the glenoid track concept relies on measurements of bony structures as well as on the confines and elasticity of the rotator cuff as a reference point, which varies extensively among individuals and therefore limits the reliability and accuracy of this concept. Purpose: To evaluate the reliability of the global track concept, which determines the angular distance of the Hill-Sachs defect from the center of the articular surface of the humeral head as a new reference point with the help of an automated image analysis software and 3-dimensional analysis of the humeral head. Study Design: Controlled laboratory study. Methods: Computed tomography scans of 100 patients treated for anterior shoulder instability with different sizes of Hill-Sachs defects were evaluated manually by 2 orthopaedic surgeons independently using the software OsiriX as well as automatically by using a dedicated prototype software (ImFusion). Obtained manual and automated measurements included the Hill-Sachs length, Hill-Sachs width, and Hill-Sachs depth of the defect; the Hill-Sachs interval (HSI); and the glenoid width for the glenoid track concept, as well as the angular distance of the Hill-Sachs defect from the center of the articular surface of the humeral head (global track concept). The reliability of the different measurement techniques was compared by calculating intraclass correlation coefficients (ICCs). Results: There was a significant difference for all obtained parameters comparing manual and automatic measurements. For manually obtained parameters, measurements referring to bony boundaries (glenoid width, Hill-Sachs length, and Hill-Sachs width) showed good to excellent agreement (ICC, 0.86, 0.82, and 0.62, respectively), while measurements referring to soft tissue boundaries (HSI and glenoid track; ICC, 0.56 and 0.53, respectively) or not directly identifiable reference points (center of articular surface and global track) only showed fair reliability (ICC middle excursion, 0.42). When the same parameters were measured with the help of an automated software, good reliability for the glenoid track concept and excellent reliability for the global track concept in the middle excursion were achieved. Conclusion: The present study showed that the more complex global track measurements of humeral defects are more reliable than the current standard HSI and glenoid track measurements. However, this is only true when automated software is used to perform the measurements. Clinical Relevance: Future studies using the new proposed method in combination with an automated software need to be conducted to determine critical threshold values for defects prone to engagement.

Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman’s correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70–23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05–39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, −49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

Characterization of real-time cine MR imaging distortion on 0.35 T MRgRT with concentric cine imaging QA phantom

Objective. Real-time MRgRT uses 2D-cine imaging for target tracking and motion evaluation. Rotation of gantry induced B 0 off-resonance, resulting in image artifacts and imaging isocenter-shift precluding MR-guided arc therapy. Standard MRI phantoms designed for higher resolution images face challenges when low-resolution cine imaging is needed to achieve high frame rates. This work aimed to examine the spatial accuracy including geometric distortion and isocenter shift in real-time during gantry rotation on a 0.35 T MR-Linac using the concentric Cine imaging quality assurance (QA) phantom and its associated image analysis software. Approach. The Cine imaging QA phantom consists of two concentric shells of low-T1 mineral oil and a central alignment structure. The phantom was scanned on three different MRI systems; 0.55 T Siemens Free.Max, 1.5 T Philips Ingenia, and 0.35 T ViewRay MRIdian MR-Linac using 2D balanced steady-state free precession (bSSFP) imaging sequence. In addition, bSSFP cine MRI with the banding artifact correction was tested on 0.35 T ViewRay MR-Linac. Images from the MR-Linac were acquired with the Linac gantry stationary and rotating from gantry 300°→ 0° and vice versa. Three orthogonal image planes were scanned excluding the 1.5 T Philips Ingenia, where only the axial plane was scanned. The image analysis software calculated the distortion values as well as the isocenter position for each cine frame. Main results. The geometric distortion of cine imaging on MRIs and MR-Linac at gantry stationary are within 1 mm while the substantial geometric distortion of 2 and 2.2 mm were observed on 0.35 T MR-Linac while rotating the gantry clockwise (300°→ 0°) and counterclockwise 0°→ 300° respectively. The average imaging isocenter shift was 0.1 mm for both MRIs and the static gantry and imaging isocenter shift of ≤1.5 mm was observed during the gantry rotation. The imaging isocenter shift decreased by 1 ± 0.2 mm clockwise and counterclockwise with B 0 compensation. Significance. The concentric Cine imaging QA phantom and its associated software effectively demonstrate the image distortion on real-time cine imaging on regular MRIs and 0.35 T MR-Linac. The results of significant geometric distortion with a rotating gantry in the MR-Linac system require further investigation to alleviate the extent of the image distortion.

OLEAtool: An open-source software for morphopalynological research in Olea europaea L. pollen

In this paper we present OLEAtool, a new software tool for palynological research to facilitate morphological analysis and measurements of Olea pollen. OLEAtool is a macro extension for use with ImageJ, an open-access and freely available image analysis software, and was developed as a component of the OLEA-project. This larger project examines olive tree expansion and mosaic landscape formation on the Balearic Islands. Pollen analysis of both fossil and modern grains has been proven useful for characterizing cultivars and therefore an important method for studying olive tree cultivation in the Mediterranean. However, these methods still struggle with distinguishing between wild and cultivated varieties. Traditional morphological analysis of pollen grains can be a difficult and time-consuming task. However, OLEAtool dramatically increases the speed of collecting data on pollen grains, expands the number of variables an analyst can measure, and greatly enhances the replicability of morphological analysis.

A simplified version of rapid susceptibility testing of bacteria and yeasts using optical nanomotion detection.

We present a novel optical nanomotion-based rapid antibiotic and antifungal susceptibility test. The technique consisted of studying the effects of antibiotics or antifungals on the nanometric scale displacements of bacteria or yeasts to assess their sensitivity or resistance to drugs. The technique relies on a traditional optical microscope, a video camera, and custom-made image analysis software. It provides reliable results in a time frame of 2-4 h and can be applied to motile, non-motile, fast, and slowly growing microorganisms. Due to its extreme simplicity and low cost, the technique can be easily implemented in laboratories and medical centers in developing countries.

Using the traditional microscope for mineral grain orientation determination: A prototype image analysis pipeline for optic-axis mapping (POAM).

This paper reports on the development of an open-source image analysis software 'pipeline' dedicated to petrographic microscopy. Using conventional rock thin sections and images from a standard polarising microscope, the pipeline can classify minerals and subgrains into objects and obtain information about optic-axis orientation. Five metamorphic rocks were chosen to test and illustrate the method. Thin sections were imaged using reflected and cross- and plane-polarised transmitted light. Images were taken at different angles of the polariser and analyser (360° with 10° steps), both with and without the full-lambda plate. The resulting image stacks were analysed with a modular pipeline for optic-axis mapping (POAM). POAM consists of external and internal software packages that register, segment, classify, and interpret the visible light spectra using object-based image analysis (OBIAS). The mapped fields-of-view and grain orientation stereonets of interest are presented in the context of whole-slide images. Two innovations are reported. First, we used hierarchical tree region merging on blended multimodal images to classify individual grains of rock-forming minerals into objects. Second, we assembled a new optical mineralogy algorithm chain that identifies the mineral slow axis orientation. The c-axis orientation results were verified with scanning electron microscopy electron backscattered diffraction (SEM-EBSD) data. For quartz (uniaxial) in a granite mylonite the test yielded excellent correspondence of c-axis azimuth and good agreement for inclination. For orthorhombic orthopyroxene in a deformed garnet harzburgite, POAM produced acceptable results for slow axis azimuth. In addition, the method identified slight anisotropy in garnet that would not be appreciated by traditional microscopy. We propose that our method is ideally suited for two commonly performed tasks in mineralogy. First, for mineral grain classification of entire thin sections scans on blended images to provide automated modal abundance estimates and grain size distribution. Second, for prospective fields of view of interest, POAM can rapidly generate slow axis crystal orientation maps from multiangle image stacks on conventionally prepared thin sections for targeting detailed SEM-EBSD studies.

Analysis of Osteosarcoma Cell Lines and Patient Tissue Using a 3D In Vivo Tumor Model—Possible Effects of Punicalagin

Osteosarcomas are the most common primary malignant bone tumors and mostly affect children, adolescents, and young adults. Despite current treatment options such as surgery and polychemotherapy, the survival of patients with metastatic disease remains poor. In recent studies, punicalagin has reduced the cell viability, angiogenesis, and invasion in cell culture trials. The aim of this study was to examine the effects of punicalagin on osteosarcomas in a 3D in vivo tumor model. Human osteosarcoma biopsies and SaOs-2 and MG-63 cells, were grown in a 3D in vivo chorioallantoic membrane (CAM) model. After a cultivation period of up to 72 h, the tumors received daily treatment with punicalagin for 4 days. Weight measurements of the CAM tumors were performed, and laser speckle contrast imaging (LSCI) and a deep learning-based image analysis software (CAM Assay Application v.3.1.0) were used to measure angiogenesis. HE, Ki-67, and Caspase-3 staining was performed after explantation. The osteosarcoma cell lines SaOs-2 and MG-63 and osteosarcoma patient tissue displayed satisfactory growth patterns on the CAM. Treatment with punicalagin decreased tumor weight, proliferation, and tumor-induced angiogenesis, and the tumor tissue showed pro-apoptotic characteristics. These results provide a robust foundation for the implementation of further studies and show that punicalagin offers a promising supplementary treatment option for osteosarcoma patients. The 3D in vivo tumor model represents a beneficial model for the testing of anti-cancer therapies.

Using infrared thermography to determine changes in teat skin surface temperature after machine milking in dairy cows

Mastitis is among the costliest diseases affecting dairy cows, partly due to the resulting permanent reduction in the quantity and quality of milk produced. Most mastitis cases involve pathogenic organisms entering the cow's mammary gland through the teat canal. The teat has natural defenses against these pathogens that can be disrupted during milk harvesting. These disruptions of the teat's circulatory system and tissue integrity can predispose them to mastitis. Traditionally, machine milking–induced changes in teat blood circulation and tissue integrity have been assessed by means of manual evaluation and ultrasonography. Infrared thermography has previously been shown to produce precise and consistent measurements of skin surface temperatures (SST) on cows' hind teats. Our objective was to describe the variability in the teat SST following machine milking. Describing the variability in teat SST before and after milking could be useful to guide further studies to elucidate the physiology of the effects of milking on teat defense mechanisms. In this observational study, thermographic images of both hind teats from 140 cows immediately pre- and postmachine milking were analyzed. The average SST were subsequently determined at the proximal, middle, and distal aspects of each hind teat using image analysis software. The LSM (95% CI) from general linear mixed models of the pre- and postmilking SST, respectively, were 33.6 (33.5–33.8)°C and 35.4 (35.3–35.5)°C at the proximal aspect, 33.2 (33.1–33.4)°C and 35.2 (35.1–35.3)°C at the middle aspect, and 32.3 (32.1–32.5)°C and 34.0 (33.9–34.1)°C at the distal aspect. The observed increase in SST from pre- to postmilking SST at all 3 aspects of the teat suggest that some of the variability in the SST can be attributed to the milking event. Future research is warranted to investigate the biological relevance of SST changes during machine milking and any potential change in teat defense mechanisms, risk of mastitis, or other pathologies.

Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342.

Adeno-associated viral transduction allows the introduction of nucleic fragments into cells and is widely used to modulate gene expressions in vitro and in vivo. It enables the study of genetic functions and disease mechanisms and, more recently, serves as a tool for gene repair. To achieve optimal transduction performance for a given cell type, selecting an appropriate serotype and the number of virus particles per cell, also known as the multiplicity of infection, is critical. Fluorescent proteins are one of the common reporter genes to visualize successfully transduced cells and assess transduction efficiencies. Traditional methods of measuring fluorescence-positive cells are endpoint analysis by flow cytometry or manual counting with a fluorescence microscope. However, the flow cytometry analysis does not allow further measurement in a test run, and manual counting by microscopy is time-consuming. Here, we present a method that repeatedly evaluates transduction efficiencies by adding the DNA-stain Hoechst 33342 during the transduction process combined with a microscope or live-cell imager and microplate image analysis software. The method achieves fast, high-throughput, reproducible, and real-time post-transduction analysis and allows for optimizing transduction parameters and screening for a proper approach.

Accelerated corneal crosslinking with 20'-soaking hydroxypropyl methyl cellulose/riboflavin vs conventional crosslinking with 30'-soaking dextran/riboflavin.

To evaluate and compare functional and structural outcomes of accelerated corneal crosslinking (A-CXL) using riboflavin with hydroxypropyl methyl cellulose (HPMC) vs conventional corneal crosslinking (C-CXL) using riboflavin with dextran. American University of Beirut Medical Center, Beirut, Lebanon. Retrospective analysis. Retrospective analysis of 83 eyes of 73 patients with mild to moderate keratoconus. First group (n = 44 eyes) underwent C-CXL using a 30-minute riboflavin/dextran soaking between June 2014 and March 2016. Second group (n = 39 eyes) underwent A-CXL using a 20-minute riboflavin/HPMC soaking between April 2016 and December 2017. Patients were evaluated preoperatively and at 1, 3, and 12 months postoperatively. Main outcome measures were simulated keratometry (simK), maximum axial keratometry (Kmax), demarcation line depth, and haze intensity measured using optical coherence tomography-based image analysis software. Demarcation line (DL) was 298.30 ± 64.60 μm and 335.61 ± 99.76 μm for C-CXL and A-CXL groups, respectively ( P = .04). Haze profile was similar for both groups. The mean simK values were reduced from 46.93 ± 3.50 and 46.44 ± 2.93 preoperatively to 46.18 ± 3.65 and 45.54 ± 2.78 at 12 months postoperatively, for C-CXL and A-CXL, respectively ( P = .003 for both groups). The mean Kmax decreased from 52.46 ± 4.82 and 51.50 ± 3.87 preoperatively to 51.30 ± 4.42 and 50.30 ± 3.52 postoperatively, for the C-CXL and A-CXL, respectively ( P < .001 for both groups). There was no difference in the simK and Kmax changes between the C-CXL and A-CXL groups ( P = .814 and P = .913), visual acuity, and refraction between the 2 groups ( P > .05). A-CXL with a 20-minute riboflavin/HPMC soaking produced deeper DL and similar corneal haze, topographic, refractive, and visual results to C-CXL with a 30-minute riboflavin/dextran soaking.