Diagnostic Performance of Ex Vivo Fluorescence Confocal Microscopy in the Assessment of Diagnostic Biopsies of the Prostate

Background: Fluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. The method has been well established for the diagnosis of tumors in dermatological specimens. Methods: We compare intraoperative diagnoses of the real-time application of FCM in pre-therapeutic prostate biopsies (35 patients, total number of biopsy specimens: n = 438) with the findings of conventional histology. Results: Prostate carcinoma was reliably diagnosed in all patients. Depending on scan quality and experience of the examiner, smaller lesions of well differentiated carcinoma (ISUP1) could not be consistently differentiated from reactive changes. Furthermore, in some cases there was difficulty to distinguish ISUP grade 2 from ISUP grade 1 tumors. ISUP grades 3–5 were reliably detected in FCM. Conclusion: Despite some limitations, FCM seems to be an effective tool for the timely assessment of prostate biopsies enabling reliable diagnosis of prostate cancer in patients requiring therapy.

Myofibril orientation as a metric for characterizing heart disease

Myocyte disarray is a hallmark of cardiomyopathy. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been due to a paucity of methods for objective and quantitative analysis. Here we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority as compared to conventional histological assessments. Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle sigma). At a sarcomere length (SL) of 1.9 microns, the angle sigma was 0.23 +/- 0.01 rad, decreased to 0.19 +/- 0.01 rad at a SL of 2.1 microns, and further decreased to 0.15 +/- 0.01 rad at a SL of 2.3 microns (p<0.0001). Angle sigma was significantly larger in R403Q (a MYH7 HCM model) porcine myocardium (0.24 +/- 0.01 rad) compared to WT myocardium (0.14 +/- 0.005 rad, p<0.0001) as well as in human heart failure tissue (0.19 +/- 0.006 rad) when compared to non-failing samples (0.17 +/- 0.007 rad, p=0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared to healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological methods that directly assess myocyte orientation and only indirectly assess myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen human myocardium provides a new tool for investigating the structural bases of cardiomyopathies.

Chronic Active Antibody-Mediated Rejection Is Associated With the Upregulation of Interstitial But Not Glomerular Transcripts

Molecular assessment of renal allografts has already been suggested in antibody-mediated rejection (ABMR), but little is known about the gene transcript patterns in particular renal compartments. We used laser capture microdissection coupled with quantitative RT-PCR to distinguish the transcript patterns in the glomeruli and tubulointerstitium of kidney allografts in sensitized retransplant recipients at high risk of ABMR. The expressions of 13 genes were quantified in biopsies with acute active ABMR, chronic active ABMR, acute tubular necrosis (ATN), and normal findings. The transcripts were either compartment specific (TGFB1 in the glomeruli and HAVCR1 and IGHG1 in the tubulointerstitium), ABMR specific (GNLY), or follow-up specific (CXCL10 and CX3CR1). The transcriptional profiles of early acute ABMR shared similarities with ATN. The transcripts of CXCL10 and TGFB1 increased in the glomeruli in both acute ABMR and chronic active ABMR. Chronic active ABMR was associated with the upregulation of most genes (SH2D1B, CX3CR1, IGHG1, MS4A1, C5, CD46, and TGFB1) in the tubulointerstitium. In this study, we show distinct gene expression patterns in specific renal compartments reflecting cellular infiltration observed by conventional histology. In comparison with active ABMR, chronic active ABMR is associated with increased transcripts of tubulointerstitial origin.

Identification of molecular biomarkers associated with disease progression in the testis of bulls infected with Besnoitia besnoiti

Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, five well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fibrosis was investigated and complemented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense inflammatory infiltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+ cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of differentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant findings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely affected and sterile bulls, and all these genes could be prognostic markers of sterility.

3D cyclorama for digital unrolling and visualisation of deformed tubes

Colonic crypts are tubular glands that multiply through a symmetric branching process called crypt fission. During the early stages of colorectal cancer, the normal fission process is disturbed, leading to asymmetrical branching or budding. The challenging shapes of the budding crypts make it difficult to prepare paraffin sections for conventional histology, resulting in colonic cross sections with crypts that are only partially visible. To study crypt budding in situ and in three dimensions (3D), we employ X-ray micro-computed tomography to image intact colons, and a new method we developed (3D cyclorama) to digitally unroll them. Here, we present, verify and validate our ‘3D cyclorama’ method that digitally unrolls deformed tubes of non-uniform thickness. It employs principles from electrostatics to reform the tube into a series of onion-like surfaces, which are mapped onto planar panoramic views. This enables the study of features extending over several layers of the tube’s depth, demonstrated here by two case studies: (i) microvilli in the human placenta and (ii) 3D-printed adhesive films for drug delivery. Our 3D cyclorama method can provide novel insights into a wide spectrum of applications where digital unrolling or flattening is necessary, including long bones, teeth roots and ancient scrolls.

Ultrastaging Using Ex Vivo Sentinel Lymph Node Mapping and One-Step Nucleic Acid Amplification (OSNA) in Gastric Cancer: Experiences of a European Center

Background: In this study, the effectiveness of One-step nucleic acid amplification (OSNA) in combination with ex vivo SLN mapping is compared with conventional histology including immunohistochemistry. Methods: LNs were retrieved from gastrectomy specimens in an unfixed state. After ex vivo SLN mapping using methylene-blue, LNs were sliced to provide samples for histology and OSNA. Results: In total, 334 LNs were retrieved in the fresh state from 41 patients. SLN detection was intended in 40 cases but was successful in only 29, with a correct LN status prediction in 23 cases (79%). Excluding one case out of 41 with a failure likely caused by a processing error, OSNA showed a high effectiveness with sensitivity, specificity, and accuracy rates of 85.4%, 93.5%, and 92.4%, respectively. The LN status could be predicted in all but one case, in which the single positive LN was not eligible for OSNA testing. Moreover, OSNA evaluation led to upstaging from N0 to N+ in three cases (14%). Conclusion: The ex vivo SLN protocol used resulted in a relatively poor detection rate. However, the OSNA method was not hampered by this detection rate and proved its potential to increase the sensitivity of metastases detection.

Brain virtual histology with X-ray phase-contrast tomography Part I: whole-brain myelin mapping in white-matter injury models

White-matter injury leads to severe functional loss in many neurological diseases. Myelin staining on histological samples is the most common technique to investigate white-matter fibers. However, tissue processing and sectioning may affect the reliability of 3D volumetric assessments. The purpose of this study was to propose an approach that enables myelin fibers to be mapped in the whole rodent brain with microscopic resolution and without the need for strenuous staining. With this aim, we coupled in-line (propagation-based) X-ray phase-contrast tomography (XPCT) to ethanol-induced brain sample dehydration. We here provide the proof-of-concept that this approach enhances myelinated axons in rodent and human brain tissue. In addition, we demonstrated that white-matter injuries could be detected and quantified with this approach, using three animal models: ischemic stroke, premature birth and multiple sclerosis. Furthermore, in analogy to diffusion tensor imaging (DTI), we retrieved fiber directions and DTI-like diffusion metrics from our XPCT data to quantitatively characterize white-matter microstructure. Finally, we showed that this non-destructive approach was compatible with subsequent complementary brain sample analysis by conventional histology. In-line XPCT might thus become a novel gold-standard for investigating white-matter injury in the intact brain. This is Part I of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part II shows how in-line XPCT enables the whole-brain 3D morphometric analysis of amyloid-β (Aβ) plaques.HighlightsX-ray phase-contrast tomography (XPCT) enables myelin mapping of the whole brainXPCT detects and quantifies white-matter injuries in a range of diseasesFiber directions and anisotropy metrics can be retrieved from XPCT dataXPCT is compatible with subsequent conventional histology of brain samplesXPCT is a powerful virtual histology tool that requires minimal sample preparationGraphical Abstract

The Immunopathology of Giant Cell Arteritis Across Disease Spectra

Giant cell arteritis (GCA) is a granulomatous systemic vasculitis of large- and medium-sized arteries that affects the elderly. In recent years, advances in diagnostic imaging have revealed a greater degree of large vessel involvement than previously recognized, distinguishing classical cranial- from large vessel (LV)- GCA. GCA often co-occurs with the poorly understood inflammatory arthritis/bursitis condition polymyalgia rheumatica (PMR) and has overlapping features with other non-infectious granulomatous vasculitides that affect the aorta, namely Takayasu Arteritis (TAK) and the more recently described clinically isolated aortitis (CIA). Here, we review the literature focused on the immunopathology of GCA on the background of the three settings in which comparisons are informative: LV and cranial variants of GCA; PMR and GCA; the three granulomatous vasculitides (GCA, TAK, and CIA). We discuss overlapping and unique features between these conditions across clinical presentation, epidemiology, imaging, and conventional histology. We propose a model of GCA where abnormally activated circulating cells, especially monocytes and CD4+ T cells, enter arteries after an unknown stimulus and cooperate to destroy it and review the evidence for how this mechanistically occurs in active disease and improves with treatment.

Automated analysis of rabbit knee calcified cartilage morphology using micro-computed tomography and deep learning segmentation

PurposeOnly little is known how calcified cartilage (CC) structure changes during exercise, aging and disease. CC thickness (CC.Th) can be analyzed using conventional histological sections. Micro-computed tomography (μCT) allows for three-dimensional (3D) imaging of mineralized tissues, however, the segmentation between bone and CC is challenging. Here, we present state-of-the-art deep learning segmentation for μCT images to enable assessment of CC morphology.MethodsSixteen knees from twelve New Zealand White rabbits were dissected into osteochondral samples from six anatomical regions: lateral and medial femoral condyles, lateral and medial tibial plateaus, femoral groove and patella (n = 96). Samples were imaged with μCT and processed for conventional histology. Manually segmented CC from the histology and reconstructed μCT images was used as the gold standard to train segmentation models with different encoder-decoder architectures. The models with the greatest out-of-fold evaluation Dice score were used for automated CC.Th analysis. Subsequently, the automated CC.Th analysis was compared across a total of 24 regions, co-registered between the imaging modalities, using Pearson correlation and Bland-Altman analyses. Finally, the anatomical variation in CC.Th was assessed via a Linear Mixed Model analysis.ResultsThe best segmentation models yielded average Dice scores of 0.891 and 0.807 for histology and μCT segmentation, respectively. The correlation between the co-registered regions across the modalities was strong (r = 0.897). The Bland-Altman analysis yielded a bias of 21.9 μm and a standard deviation of 21.5 μm between the methods. Finally, both methods could separate the CC morphology between the patella, femoral, and tibial regions (p < 0.001).ConclusionThe presented method allows for ex vivo 3D assessment of CC.Th in an automated and non-destructive manner. We demonstrated its utility by quantifying CC.Th in different anatomical regions. CC.Th was the thickest in the patella and the thinnest in the tibial plateau.Graphical abstractWe present a μCT-based method with deep learning segmentation for analyzing calcified cartilage thickness (CC.Th). The method is compared throughout the study against conventional histology. The comparison against co-registered regions yielded a strong Pearson correlation (r = 0.90). Both methods were able to separate the CC.Th properties between tibia, femur, and patella.