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Understanding the correlation between tissue architecture, health status, and mechanical properties is essential for improving material models and developing tissue engineering scaffolds. Since structural-based material models are state of the art, there is an urgent need for experimentally obtained structural parameters. For this purpose, the medial layer of nine human abdominal aortas was simultaneously subjected to equibiaxial loading and multi-photon microscopy. At each loading interval of 0.02, collagen and elastin fibers were imaged based on their second-harmonic generation signal and two-photon excited autofluorescence, respectively. The structural alterations in the fibers were quantified using the parameters of orientation, diameter, and waviness. The results of the mechanical tests divided the sample cohort into the ruptured and non-ruptured, and stiff and non-stiff groups, which were covered by the findings from histological investigations. The alterations in structural parameters provided an explanation for the observed mechanical behavior. In addition, the waviness parameters of both collagen and elastin fibers showed the potential to serve as indicators of tissue strength. The data provided address deficiencies in current material models and bridge multiscale mechanisms in the aortic media. STATEMENT OF SIGNIFICANCE: Available material models can reproduce, but cannot predict, the mechanical behavior of human aortas. This deficiency could be overcome with the help of experimentally validated structural parameters as provided in this study. Simultaneous multi-photon microscopy and biaxial extension testing revealed the microstructure of human aortic media at different stretch levels. Changes in the arrangement of collagen and elastin fibers were quantified using structural parameters such as orientation, diameter and waviness. For the first time, structural parameters of human aortic tissue under continuous loading conditions have been obtained. In particular, the waviness parameters at the reference configuration have been associated with tissue stiffness, brittleness, and the onset of atherosclerosis.

Current clinical practice for aneurysmatic interventions is often based on the maximum diameter of the vessel and/or on the growth rate, although rupture can occur at any diameter and growth rate, leading to fatality. For 27 medial samples obtained from 12 non-aneurysmatic (control) and 9 aneurysmatic human descending thoracic aortas we examined: the mechanical responses up to rupture using uniaxial extension tests of circumferential and longitudinal specimens; the structure of these tissues using second-harmonic imaging and histology, in particular, the content proportions of collagen, elastic fibers and smooth muscle cells in the media. It was found that the mean failure stresses were higher in the circumferential directions (Control-C 1474 kPa; Aneurysmatic-C 1446 kPa), than in the longitudinal directions (Aneurysmatic-L 735 kPa; Control-L 579 kPa). This trend was the opposite to that observed for the mean collagen fiber directions measured from the loading axis (Control-L > Aneurysmatic-L > Aneurysmatic-C > Control-C), thus suggesting that the trend in the failure stress can in part be attributed to the collagen architecture. The difference in the mean values of the out-of-plane dispersion in the radial/longitudinal plane between the control and aneurysmatic groups was significant. The difference in the mean values of the mean fiber angle from the circumferential direction was also significantly different between the two groups. Most specimens showed delamination zones near the ruptured region in addition to ruptured collagen and elastic fibers. This study provides a basis for further studies on the microstructure and the uniaxial failure properties of (aneurysmatic) arterial walls towards realistic modeling and prediction of tissue failure. Statement of Significance A data set relating uniaxial failure properties to the microstructure of non-aneurysmatic and aneurysmatic human thoracic aortic medias under uniaxial extension tests is presented for the first time. It was found that the mean failure stresses were higher in the circumferential directions, than in the longitudinal directions. The general trend for the failure stresses was Control-C > Aneurysmatic-C > Aneurysmatic-L > Control-L, which was the opposite of that observed for the mean collagen fiber direction relative to the loading axis (Control-L > Aneurysmatic-L > Aneurysmatic-C > Control-C) suggesting that the trend in the failure stress can in part be attributed to the collagen architecture. This study provides a first step towards more realistic modeling and prediction of tissue failure.

This case report is about myopericytomatosis, a recently described rare tumor entity with only a dozen cases found in PubMed. Histologically, this tumor is characterized by diffuse infiltration of innumerable discrete myopericytoma-like nodules composed of myoid cells in a perivascular distribution. PDGFRB mutation has been linked to myopericytomatosis as well as other myopericytic tumors. The aim of this paper is to share the clinical presentation and expand the spectrum of genetic findings. We report a case of myopericytomatosis arising around the left ankle of a 73-year-old man. MRI, biopsy, and histopathology were the main diagnostic steps. Targeted next-generation sequencing was carried out to analyze the tumor sample. We performed local excision for tumor mass reduction, as wide resection was impossible a priori based on the delicate anatomical region with important structures in close proximity. Genetic analysis revealed a missense mutation of PDGFRB. PDGFRB alteration seems to play a pathogenic role in myopericytomatosis as highlighted in previously reported cases. Currently, myopericytomatosis is best treated surgically.

Peripheral vascular trauma due to injuries of the upper and lower limbs are life-threatening, and their treatment require rapid diagnosis and highly-qualified surgical procedures. Experienced surgeons have recognized that subclavian arteries, affected by injuries of the upper limbs, require a more careful handling due to fragility than common iliac arteries, which are may be affected by injures of the lower limbs. We investigated these two artery types with comparable diameter to evaluate the differences in the biomechanical properties between subclavian and iliac arteries. Human subclavian and common iliac arteries of 14 donors either from the right or the left side (age: 63 yrs, SD: 19 , 9 female and 5 male) were investigated. Extension-inflation-torsion experiments at different axial strains (0–20%), transmural pressures (0–200 mmHg) and torsion ( ± 25 ° ) on preconditioned arterial tubes were performed. Residual stresses in both circumferential and axial direction were determined. Additionally, the microstructure of the tissues was determined via second-harmonic generation imaging and by histological investigations. At physiological conditions ( p i = 13.3 kPa, λ z = 1.1 ) common iliac arteries revealed higher Cauchy stresses in circumferential and axial directions but a more compliant response in the circumferential direction than subclavian arteries. Both arteries showed distinct stiffer behavior in circumferential than in axial direction. Circumferential stiffness of common iliac arteries at physiological conditions increased significantly with aging ( r = - 0 . 67 , p = 0 . 02 ). The median inversion stretches, where the axial force is basically independent of the transmural pressure, were determined to be 1.05 for subclavian arteries and 1.11 for common iliac arteries. Both arteries exhibited increased torsional stiffness, when either axial prestretch or inflation pressure was increased. Residual stresses in the circumferential direction were significantly lower for subclavian arteries than for common iliac arteries at measurements after 30 min ( p = 0.05 ) and 16 hrs ( p = 0.01 ). Investigations of the collagen microstructure revealed different collagen fiber orientations and dispersions in subclavian and iliac arteries. The difference in the collagen microstructure revealed further that the adventitia seems to contribute significantly to the passive mechanical response of the tested arteries at physiological loadings. Histological investigations indicated pronounced thickened intimal layers in subclavian and common iliac arteries, with a thickness comparable to the adventitial layer. In conclusion, we obtained biomechanical differences between subclavian and common iliac arteries, which possibly resulted from their different mechanical loadings/environments and respective in vivo movements caused by their anatomical locations. The biomechanical differences explored in this study are well reflected by the microstructure of the collagen and the histology of the investigated arteries, and the results can improve trauma patient care and endovascular implant design. Statement of Significance During surgical interventions surgeons experienced that subclavian arteries (SAs) supplying the upper extremities, appear more fragile and prone to damage during surgical repair than common iliac arteries (CIAs), supplying the lower extremities. To investigate this difference in a systematic way the aim of this study was to compare the biomechanical properties of these two arteries from the same donors in terms of geometry, extension-inflation-torsion behavior, residual stresses, microstructure, and histology. In regard to cardiovascular medicine the material behavior of aged human arteries is of crucial interest. Moreover, the investigation of SA is important as it can help to improve surgical procedures at this challenging location. Over the long-term it might well be of value in the construction of artificial arteries for substituting native arteries. In addition, the analysis of mechanical stresses can improve design and material choice for endovascular implants to optimize long-term implant function.

Summary Molecular diagnostics in personalized medicine increasingly relies on the combination of a variety of analytical technologies to characterize individual diseases and to select patients for targeted therapies. The gold standard for tissue-based diagnostics is fixation in formalin and embedding in paraffin, which results in excellent preservation of morphology but negatively impacts on a variety of molecular assays. The formalin-free, non-cross-linking PAXgene tissue system preserves morphology in a similar way to formalin, but also preserves biomolecules essentially in a similar way to cryopreservation, which markedly widens the spectrum, sensitivity and accuracy of molecular analytics. In this study, we have developed and tested a protocol for PAXgene-fixed and paraffin-embedded tissues for fluorescent in situ hybridization (FISH). The implementation of a 24-h formalin postfixation step of slides from PAXgene-fixed and paraffin-embedded tissues allowed us to use the assays approved for formalin-fixed and paraffin-embedded tissues. The equivalence of the methodologies was demonstrated by FISH analysis of HER2 amplification in breast cancer cases. The 24-h postfixation step of the slides used for FISH can be well integrated in the routine diagnostic workflow and allows the remaining PAXgene-fixed and paraffin-embedded tissue to be used for further molecular testing.

Osteochondromas rarely induce vascular complications by mechanical compression. We present the case of a subclavian artery pseudoaneursym caused by an osteochondroma of the scapula in a 67-year-old male. The diagnosis was based on a previous history of multiple exostoses, computed tomography and magnetic resonance imaging, as well as the local vascular clinical status of the lesion. Surgical treatment consisted of vascular and orthopaedic intervention. First, the vascular surgeon implanted a bypass of the subclavian artery from the ventral aspect, enabling the orthopaedic surgeon to resect the osteochondroma from the dorsal aspect. The patient recovered with full function. Vascular pseundoaneurysms should be taken into consideration in patients with osteochondromas, especially with a known history of multiple hereditary exostoses.

Background In Ewing sarcomas (ES), histological response to polychemotherapy is the main prognostic factor. We aimed at evaluating the histological response separately for the extraosseous and intraosseous tumor compartment as well as its prognostic influence. Methods Thirty-one patients with ES and marked soft tissue expansion, treated at our department between January 2006 and December 2015, were retrospectively included. Data was taken from medical records. Original histologic specimens of the resected tumors were re-evaluated separately for intra- and extraosseous tumor regression according to Salzer-Kuntschik regression grading. Multivariate survival analysis with stepwise backward variable selection was calculated to determine the impact of extraosseous and intraosseous regression on prognosis. Results All patients had received chemotherapy, 15 (48.4%) had been administered preoperative radiotherapy. Extraosseous tumor regression was significantly worse than intraosseous regression (Wilcoxon signed-rank test, p = 0.018). While neither intraosseous nor extraosseous tumor regression had an impact on overall survival, extraosseous complete remission had a beneficial impact on event-free-survival in the multivariate analysis (Cox-regression; hazard ratio: 0.148, 95% confidence interval 0.031-0.707, p = 0.017). Conclusions On average, regression of ES seems to be worse in the extraosseous tumor compartment following preoperative chemotherapy. Moreover, extraosseous tumor regression may have a stronger prognostic influence on event-free survival than intraosseous regression.

Arterial walls can be regarded as composite materials consisting of collagen fibers embedded in an elastic matrix and smooth muscle cells. Remodeling of the structural proteins has been shown to play a significant role in the mechanical behavior of walls during pathogenesis of abdominal aortic aneurysms (AAA). In this study, we systematically studied the change in the microstructure, histology and mechanics to link them to AAA disease progression. We performed biaxial extension tests, second-harmonic generation imaging and histology on 15 samples from the anterior part of AAA walls harvested during open aneurysm surgery. Structural data were gained by fitting to a bivariate von Mises distribution and yielded the mean fiber direction and in- and out-of-plane fiber dispersions of collagen. Mechanical and structural data were fitted to a recently proposed material model. Additionally, the mechanical data were used to derive collagen recruitment points in the obtained stress-stretch curves. We derived 14 parameters from histology such as smooth muscle cell-, elastin-, and abluminal adipocyte content. In total, 22 parameters were obtained and statistically evaluated. Based on the collagen recruitment points we were able to define three different stages of disease progression. Significant differences in elastin content, collagen orientation and adipocyte contents were discovered. Nerves entrapped inside AAA walls pointed towards a significant deposition of newly formed collagen abluminally, which we propose as neo-adventitia formation. We were able to discriminate two types of remodeled walls with a high collagen content - potentially safe and possibly vulnerable walls with a high adipocyte content inside the wall and significant amounts of inflammation. The study yielded a hypothesis for disease progression, derived from the systematic comparison of mechanical, microstructural and histological changes in AAAs. STATEMENT OF SIGNIFICANCE: Remodeling of the structural proteins plays an important role in the mechanical behavior of walls during pathogenesis of abdominal aortic aneurysms (AAA). We analyzed changes in the microstructure, histology and biomechanics of 15 samples from the anterior part of AAA walls and, for the first time, linked the results to three different stages of disease progression. We identified significant differences in elastin content, collagen orientation, adipocyte contents, and also a deposition of newly formed collagen forming a neoadventitia. We could discriminate two types of remodeled walls: (i) potentially safe and (ii) possibly vulnerable associated with inflammation and a high amount of adipocytes.

RNA from matched FFPE, PFPE and cryo preserved human tissues, stored for up to seven years at 22°C and 4°C, examined for integrity and usability in quantitative RT-PCR

Usual ductal hyperplasia (UDH) of the breast is generally regarded as a nonneoplastic proliferation, albeit loss of heterozygosity has long been reported in a part of these lesions. To gain deeper insights into the molecular drivers of these lesions, an extended mutation profiling was performed. The coding regions of 409 cancer-related genes were investigated by next-generation sequencing in 16 cases of UDH, nine unassociated with neoplasia (classic) and seven arising within papillomas. Phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) activation was investigated by phosphorylated AKT, mTOR, and S6 immunohistochemistry. Of 16 lesions, 10 (63%) were mutated; 56% of classic lesions were unassociated with neoplasia, and 71% of lesions arose in papillomas. Fourteen missense mutations were detected: PIK3CA [6 (43%) of 14], AKT1 [2 (14%) of 14], as well as GNAS, MTOR, PIK3R1, LPHN3, LRP1B, and IGF2R [each 1 (7%) of 14]. Phosphorylated mTOR was seen in 83% and phosphorylated S6 in 86% of evaluable lesions (phospho-AKT staining was technically uninterpretable). In conclusion, UDH displays mutations of the phosphatidylinositol 3-kinase/AKT/mTOR axis at different levels, with PIK3R1, MTOR, and GNAS mutations not previously described. Specifically, oncogenic G-protein activation represents a yet unrecognized route to proliferation in UDH. On the basis of evidence of activating mutations, loss of heterozygosity, and a mass forming proliferation, we propose that UDH is most appropriately viewed as an early neoplastic intraductal proliferation.

In the multidisciplinary field of heart research it is of utmost importance to identify accurate myocardium material properties for the description of phenomena such as mechano-electric feedback or heart wall thickening. A rationally-based material model is required to understand the highly nonlinear mechanics of complex structures such as the passive myocardium under different loading conditions. Unfortunately, to date there are no experimental data of human heart tissues available to estimate material parameters and to develop adequate material models. This study aimed to determine biaxial extension and triaxial shear properties and the underlying microstructure of the passive human ventricular myocardium. Using new state-of-the-art equipment, planar biaxial extension tests were performed to determine the biaxial extension properties of the passive ventricular human myocardium. Shear properties of the myocardium were examined by triaxial simple shear tests performed on small cubic specimens excised from an adjacent region of the biaxial extension specimens. The three-dimensional microstructure was investigated through second-harmonic generation (SHG) microscopy on optically cleared tissues, which emphasized the 3D orientation and dispersion of the myofibers and adjacent collagen fabrics. The results suggest that the passive human LV myocardium under quasi-static and dynamic multiaxial loadings is a nonlinear, anisotropic (orthotropic), viscoelastic and history-dependent soft biological material undergoing large deformations. Material properties of the tissue components along local microstructural axes drive the nonlinear and orthotropic features of the myocardium. SHG microscopy investigation revealed detailed information about the myocardial microstructure due to its high resolution. It enabled the identification of structural parameters such as the fiber and the sheet orientations and corresponding dispersions. With this complete set of material data, a sophisticated material model and associated material parameters can be defined for a better description of the biomechanical response of the ventricular myocardium in humans. Such a model will lead to more accurate computational simulations to better understand the fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases. Statement of Significance Unfortunately, to date there are no experimental data of human heart tissues available for material parameter estimation and the development of adequate material models. In this manuscript novel biaxial tensile and shear test data at different specimen orientations are presented, which allowed to adequately capture the direction-dependent material response. With these complete sets of mechanical data, combined with their underlying microstructural data (also presented herein), sophisticated material models and associated material parameters can be defined for the description of the mechanical behavior of the ventricular myocardium in humans. Such models will lead to accurate computational simulations to better understand the fundamental underlying ventricular mechanics, a step needed in the improvement of medical treatment of heart diseases.

Development of aortic aneurysms includes significant morphological changes within the tissue: collagen content increases, elastin content reduces and smooth muscle cells degenerate. We seek to quantify the impact of these changes on the passive mechanical response of aneurysms in the supra-physiological loading range via mechanical testing and constitutive modeling. We perform uniaxial extension tests on circumferentially and axially oriented strips from five thoracic (65.6 years ± 13.4, mean ± SD) and eight abdominal (63.9 years ± 11.4) aortic fusiform aneurysms to investigate both continuous and discontinuous softening during supra-physiological loading. We determine the significance of the differences between the fitted model parameters: diseased thoracic versus abdominal tissues, and healthy (Weisbecker et al., J. Mech. Behav. Biomed. Mater. 12, 93-106, 2012) versus diseased tissues. We also test correlations among these parameters and age, Body Mass Index (BMI) and preoperative aneurysm diameter, and investigate histological cuts. Tissue response is anisotropic for all tests and the anisotropic pseudo-elastic damage model fits the data well for both primary loading and discontinuous softening which we interpret as damage. We found statistically relevant differences between model parameters fitted to diseased thoracic versus abdominal tissues, as well as between those fitted to healthy versus diseased tissues. Only BMI correlated with fitted model parameters in abdominal aortic aneurysmal tissues. publisher: Elsevier articletitle: Human thoracic and abdominal aortic aneurysmal tissues: Damage experiments, statistical analysis and constitutive modeling journaltitle: Journal of the Mechanical Behavior of Biomedical Materials articlelink: http://dx.doi.org/10.1016/j.jmbbm.2014.10.003 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved. ispartof: Journal of the Mechanical Behavior of Biomedical Materials vol:41 pages:92-107 ispartof: location:Netherlands status: published

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin intermediate filaments are obligatory heteropolymers, which may result in dissociation between RNA and protein expression levels in the event that the homeostasis of the expression of the proper keratin partners is disturbed. Furthermore, variable accessibility of epitopes on keratin polypeptides due to conformational changes may lead to false negative results. Preanalytical effects, such as warm/cold ischemia, fixation, tissue processing, and embedding may result in false negative or inappropriate reactions. An experimental design for how to systematically test preanalytical effects and to validate immunohistochemistry protocols is presented. This kind of evaluation should be performed for each antigen and antibody since the various epitopes recognized by antibodies may behave differently. In this context, one has to be aware that different cell structures may be affected or modified differently by various preanalytical procedures and may thus require different preanalytical and staining protocols.

Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin intermediate filaments are obligatory heteropolymers, which may result in dissociation between RNA and protein expression levels in the event that the homeostasis of the expression of the proper keratin partners is disturbed. Furthermore, variable accessibility of epitopes on keratin polypeptides due to conformational changes may lead to false negative results. Preanalytical effects, such as warm/cold ischemia, fixation, tissue processing, and embedding may result in false negative or inappropriate reactions. An experimental design for how to systematically test preanalytical effects and to validate immunohistochemistry protocols is presented. This kind of evaluation should be performed for each antigen and antibody since the various epitopes recognized by antibodies may behave differently. In this context, one has to be aware that different cell structures may be affected or modified differently by various preanalytical procedures and may thus require different preanalytical and staining protocols.

Background Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. Methods Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. Results All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. Conclusion PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (k w) = 0.73 (95 % confidence interval (CI), 0.41–0.94)), with a higher agreement between the reference evaluation and the PFPE samples (k w = 0.86 (95 % CI, 0.67–1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis.

One goal of cardiac research is to perform numerical simulations to describe/reproduce the mechanoelectrical function of the human myocardium in health and disease. Such simulations are based on a complex combination of mathematical models describing the passive mechanical behavior of the myocardium and its electrophysiology, i.e., the activation of cardiac muscle cells. The problem in developing adequate constitutive models is the shortage of experimental data suitable for detailed parameter estimation in specific functional forms. A combination of shear and biaxial extension tests with different loading protocols on different specimen orientations is necessary to capture adequately the direction-dependent (orthotropic) response of the myocardium. In most experimental animal studies, where planar biaxial extension tests on the myocardium have been conducted, the generated shear stresses were neither considered nor discussed. Hence, in this study a method is presented which allows the quantification of shear deformations and related stresses. It demonstrates an approach for experimenters as to how the generation of these shear stresses can be minimized during mechanical testing. Experimental results on 14 passive human myocardial specimens, obtained from nine human hearts, show the efficiency of this newly developed method. Moreover, the influence of the clamping technique of the specimen, i.e., the load transmission between the testing device and the tissue, on the stress response is determined by testing an isotropic material (Latex). We identified that the force transmission between the testing device and the specimen by means of hooks and cords does not influence the performed experiments. We further showed that in-plane shear stresses definitely exist in biaxially tested human ventricular myocardium, but can be reduced to a minimum by preparing the specimens in an appropriate manner. Moreover, we showed whether shear stresses can be neglected when performing planar biaxial extension tests on fiber-reinforced materials. The used method appears to be robust to quantify normal and shear deformations and corresponding stresses in biaxially tested human myocardium. This method can be applied for the mechanical characterization of any fiber-reinforced material using planar biaxial extension tests.

Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.

In a previous study we were able to accurately fit experimental data on arterial tissues at supra-physiological loads using a material model that accounts for softening/damage only in the portion of the model associated with the collagen fibers (Weisbecker et al., 2012). Naturally, this result leads to the hypothesis that the softening behavior is related only to the collagen fibers, and not to the matrix material. In this study we test this hypothesis by conducting uniaxial extension tests on elastase and collagenase treated tissues and on untreated control specimens from the media of human thoracic aortas. We relate structural changes in the tissue after enzyme treatment to changes in the corresponding mechanical behavior. Collagenase treated tissue does not exhibit any softening behavior under quasi-static cyclic loading, a result supporting our hypothesis. Conversely, elastase treated tissue exhibits continuous softening under the same loading conditions, indicating that the integrity of the tissue is destroyed upon removal of the elastin. Finally, we fit isotropic and anisotropic constitutive models to the mechanical response of the collagenase treated arterial tissue, while our anisotropic model better approximates the response of collagenase treated arterial tissues, we show that an isotropic matrix model is sufficient to accurately reproduce the mechanical response of untreated control specimens, consistent with current practice in the literature. QC 20131001

Metabolomic profiles of tissues could greatly contribute to advancements in personalized medicine but are influenced by differences in adopted preanalytical procedures; nonhomogeneous pre- and post-excision ischemia times are potential sources of variability. In this study, we monitored the impact of ischemia on the metabolic profiles, acquired with high-resolution magic-angle-spinning H-1 NMR, of 162 human liver samples collected during and up to 6 h after routine surgery. The profiles changed significantly as a function of intraoperative warm ischemia (WI) and postresection cold ischemia (CI) time, with significant variations in the concentration of the same 16 metabolites. Therefore, a tight control of the preanalytical phase is essential for reliable metabolomic analyses of liver diseases. The NMR profiles provide a reliable "fingerprint" of ischemia and have predictive value: the best-performing predictive models are found to discriminate extreme time points of CI (0' vs 360') in the training set with cross-validation accuracy of similar to 90%; samples in the validation cohort can discriminate short (= 180') CI with an accuracy of similar to 80%. For WI, the corresponding figures are 95.6 and 92%, respectively. Therefore, ischemia NMR profiles might become a tool for tissue quality control in biobanks.

Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples.

Soft biological tissues such as aortic walls can be viewed as fibrous composites assembled by a ground matrix and embedded families of collagen fibres. Changes in the structural components of aortic walls such as the ground matrix and the embedded families of collagen fibres have been shown to play a significant role in the pathogenesis of aortic degeneration. Hence, there is a need to develop a deeper understanding of the microstructure and the related mechanics of aortic walls. In this study, tissue samples from 17 human abdominal aortas (AA) and from 11 abdominal aortic aneurysms (AAA) are systematically analysed and compared with respect to their structural and mechanical differences. The collagen microstructure is examined by analysing data from second-harmonic generation imaging after optical clearing. Samples from the intact AA wall, their individual layers and the AAA wall are mechanically investigated using biaxial stretching tests. A bivariate von Mises distribution was used to represent the continuous fibre dispersion throughout the entire thickness, and to provide two independent dispersion parameters to be used in a recently proposed material model. Remarkable differences were found between healthy and diseased tissues. The out-of-plane dispersion was significantly higher in AAA when compared with AA tissues, and with the exception of one AAA sample, the characteristic wall structure, as visible in healthy AAs with three distinct layers, could not be identified in AAA samples. The collagen fibres in the abluminal layer of AAAs lost their waviness and exhibited rather straight and thick struts of collagen. A novel set of three structural and three material parameters is provided. With the structural parameters fixed, the material model was fitted to the mechanical experimental data, giving a very satisfying fit although there are only three material parameters involved. The results highlight the need to incorporate the structural differences into finite-element simulations as otherwise simulations of AAA tissues might not be good predictors for the actual in vivo stress state.

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding Integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules. (J Mol Diagn 2012, 14:458-466. http://dx.doi.org/10.1016/j.jmoldx.2012.05.002)

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System ( PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for ( immuno) histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.

Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.

U. Oelmueller 1, K.F. Becker 2, M. Heinrich 3, L. Krieger 3, M. Kubista 4, M. Pazzagli 5, P. Riegman6, P. Turano7, C. Viertler 8 , K. Zatloukal 8. 1MDx Development, QIAGEN GmbH, Hilden, Germany; 2Department of Pathology, Technical University of Munich, Munchen, Germany; 3DIN Institute, DIN Deutsches Institut fur Normung e. V., Berlin, Germany; 4TATAA Biocenter AB, TATAA Biocenter AB, Goeteborg, Sweden; 5Dept. of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy; 6Department of Pathology, Erasmus Medcial Center, Rotterdam, Netherlands Antilles; 7Magnetic Resonance Center (CERM), University of Florence, Florence, Italy; 8Institute of Pathology, Medical University of Graz, Graz, Austria

The quality of human tissue specimens can have a significant impact on analytical data sets for biomarker research. The aim of this study was to characterize fluctuations of protein and phosphoprotein levels in human tissue samples during the preanalytical phase. Eleven intestine and 17 liver specimens were surgically resected, aliquoted, and either snap-frozen or fixed in formalin immediately or exposed to different ischemic conditions before preservation. Protein levels in the resultant samples were investigated by reverse phase protein array, Western blot analysis, and liquid chromatography tandem mass spectrometry. Our data revealed that the degree of sensitivity of proteins and phosphoproteins to delayed preservation varied between different patients and tissue types. For example, up-regulation of phospho-p42/44 MAPK in intestine samples was seen in some patients but not in others. General trends toward up- or down regulation of most proteins were not evident due to pronounced interpatient variability but signal intensities of only a few proteins, such as cytokeratin 18, were altered from baseline in postresection samples. In contrast, glyceraldehyde 3:phosphate dehydrogenase was found to be stable during periods of cold ischemia. Our study represents a proper approach for studying potential protein fluctuations in tissue specimens for future biomarker development programs.

Pre-analytical factors can greatly influence the outcome of molecular analyses in medical diagnostics and research. This also applies to in situ staining techniques such as immunohistochemistry (IHC), where different types of tissue fixation methods lead to different modifications of proteins and thus can affect differently the detection by antibodies. For formalin-fixed paraffin-embedded (FFPE) tissue, antigen retrieval is applied in order to reverse the negative effects of formalin and re-establish immunoreactivity. Most antibodies and protocols used in IHC are optimized for FFPE tissue, but not for paraffin-embedded tissue treated with other fixatives such as non-crosslinking fixatives. We report results from systematic studies on distinct pre-analytical conditions in IHC, immunofluorescence and electron microscopy. Parameters investigated are the impact of crosslinking and non-crosslinking fixatives (comparing formalin and PAXgene Tissue fixation) on whole tissue, subcellular structures and organelles, as well as on ultrastructure. The results generated show that minor changes in antigen retrieval conditions may have a major impact on IHC results and that protocols optimized for crosslinking fixatives may not be used for other fixatives without re-validation. Key antigen retrieval parameters such as buffers with different pH and duration of microwave treatment must be tested systematically for each antibody and fixation protocol.

Tumor heterogeneity is considered a major cause for therapy resistance in colorectal cancer. Sub-populations of cells with different genetic alterations may exist in spatially distinct areas. Upon therapy, resistant sub-clones may enrich and ultimately lead to disease progression. Although ample data are available on tumors which are heterogeneous on a morphological level, only little is known about morphologically homogeneous tumors. We aimed to investigate if morphologically homogeneous colorectal cancer can harbor a heterogeneous genetic landscape. We chose to microdissect six morphologically homogeneous colorectal carcinomas into several areas and performed next-generation sequencing (NGS) to identify tumors with genetic heterogeneity. We then applied an mRNA-based in situ mutation detection technology based on padlock probes to localize and visualize mutations directly in the tumor tissue. In three out of six tumors, NGS revealed a high rate of variability of mutations between different tumor areas. We selected two cases for in situ mutation detection to visualize genetic heterogeneity. In situ mutation detection confirmed differences in mutant allele frequencies between different tumor areas of morphological homogeneous tumors. We conclude that genetic heterogeneity in morphologically homogeneous colorectal cancer is an observable, but underreported event. Our results illustrate the power of in situ mutation analysis to visualize genetic heterogeneity directly in tumor tissue. Electronic supplementary material The online version of this article (doi:10.1007/s00418-017-1557-5) contains supplementary material, which is available to authorized users.