Peer creation and sharing of mnemonics in collaborative documents for pathology education: a pilot study.

BACKGROUND: Mnemonic techniques are memory aids that could help improve memory encoding, storage, and retrieval. Using the brain's natural propensity for pattern recognition and association, new information is associated with something familiar, such as an image, a structure, or a pattern. This should be particularly useful for learning complex medical information. Collaborative documents have the potential to revolutionize online learning because they could increase the creativity, productivity, and efficiency of learning. The purpose of this study was to investigate the feasibility of combining peer creation and sharing of mnemonics with collaborative online documents to improve pathology education. METHODS: We carried out a prospective, quasi-experimental, pretest-posttest pilot study. The intervention group was trained to create and share mnemonics in collaborative documents for pathological cases, based on histopathological slides. The control group compared analog and digital microscopy. RESULTS: Both groups consisted of 41 students and did not reveal demographic differences. Performance evaluations did not reveal significant differences between the groups' pretest and posttest scores. Our pilot study revealed several pitfalls, especially in instructional design, time on task, and digital literacy, that could have masked possible learning benefits. CONCLUSIONS: There is a gap in evidence-based research, both on mnemonics and on CD in pathology didactics. Even though, the combination of peer creation and sharing of mnemonics is very promising from a cognitive neurobiological standpoint, and collaborative documents have great potential to promote the digital transformation of medical education and increase cooperation, creativity, productivity, and efficiency of learning. However, the incorporation of such innovative techniques requires meticulous instructional design by teachers and additional time for students to become familiar with new learning methods and the application of new digital tools to promote also digital literacy. Future studies should also take into account validated high-stakes testing for more reliable pre-posttest results, a larger cohort of students, and anticipate technical difficulties regarding new digital tools.

A family with limb girdle muscular dystrophy type 1B and multiple exostoses.

INTRODUCTION: Next-generation sequencing in cases of hereditary neuromuscular disorders often yields multiple candidate gene variants. Here, we describe a case with mutations in two genes, lamin A/C (LMNA) and exostosin glycosyltransferase 2 (EXT2), which led to hereditary myopathy combined with multiple exostoses. CASE HISTORY: A 51-year-old German woman with a history of removal of multiple exostoses during childhood presented with proximal limb-girdle muscular dystrophy and a newly diagnosed cardiomyopathy with atrioventricular conduction block. Because her younger son had exostoses and her younger brother had died at age 44 after heart transplantation due to dilated cardiomyopathy, an autosomal dominant inheritance was suspected. RESULTS: Muscle biopsy revealed features of chronic myopathy associated with focal myofibrillar disintegration. Electron microscopy showed myonuclear, myofibrillar, and Z-disc alterations, accumulations of granulofilamentous material, and a large sporadic osmiophilic inclusion body reminiscent of a nemaline body. Mendeliome and Sanger sequencing detected both a c.1129>T LMNA mutation of known pathogenicity and a c.1101_1102delAG (E368Kfs*18) truncating EXT2 mutation in the patient and her affected son. DISCUSSION: The clinical, genetic, and muscle biopsy findings suggest that both mutations are pathogenic. The EXT2 mutation was most likely responsible for the multiple exostoses phenotype in mother and son, whereas the myopathy was probably caused by a combined effect of the LMNA and EXT2 mutations.

Buzz groups facilitate collaborative learning and improve histopathological competencies of students.

INTRODUCTION: Traditional teacher-centered histopathology training is based on theoretical lectures and practical tutorials. We hypothesize that learning outcomes improve if students are activated by demonstrating cardinal features of slides to each other and discussing their pathogenesis. Buzz groups (BGs) could facilitate this. The aim of this study was to investigate the effectiveness of student-centered BGs, i.e., peer-teaching, versus traditional teacher-centered histopathology teaching. Furthermore, we compared digital with analog microscopy. MATERIALS AND METHODS: In addition to traditional guided instruction and explanation of slides, neighboring students demonstrated to each other histopathological features and discussed associated pathogenesis. RESULTS: After only 4 course lessons, the BG students did much better than the control group (ANCOVA p = 0.002; F = 9.7). Then the control group also applied the BG technique. After another 4 lessons, the control group was able to catch up almost completely (ANCOVA p = 0.36; F = 0.9). Overall, there was no difference in time on task. DISCUSSION: Collaborative BGs improve the learning of histopathological competencies. They motivate and activate students to learn. The course also increased the appreciation of students for histopathology. For BGs, digital microscopy was better suited than traditional analog microscopy. The application of BGs in the context of analyzing microscopic images should be disseminated and studied on larger cohorts.

Development and characterization of an ex vivo arterial long-term proliferation model for restenosis research.

One of the main limitations of percutaneous coronary interventions is the restenosis, occurring in small-diameter arteries, and efforts are high to find improved intracoronary devices to prevent in-stent-restenosis. Aim of this study was to produce a new in vitro test platform for restenosis research, suitable for long-term cell proliferation and migration studies in stented vessels. Fresh segments of porcine coronary arteries were obtained for decellularization and were then reseeded with human coronary artery endothelial (HCAEC) and human coronary artery smooth muscle cells (HCASMC). Subsequently, bare metal stents (BMS) and drug eluting stents (DES), respectively, were implanted and the segments were reseeded with HCAEC and HCASMC for up to three months. The stented segments were examined at time zero and after 2, 4, 6, 8 and 12 weeks by histochemical and immunohistochemical characterization and the reseeded areas before and after stent implantation were measured. We have found that cells formed multiple layers after three months, and the detection with both CD31 and a-smooth muscle actin specific antibodies showed that HCAEC and HCASMC are adherent and growing in several layers. Furthermore, we could show a significantly smaller proliferation area in DES (70% ± 3.5%), compared to BMS (17% ± 2.3%). These data are similar to animal and human studies. Therefore, this vessel model might appear as an initial benchmark for testing new anti-proliferative endovascular therapies and consequently helps to reduce animal experiments in this research area.

Coherent anti-Stokes Raman scattering and two photon excited fluorescence for neurosurgery.

OBJECTIVE: There is no established method for in vivo imaging during biopsy and surgery of the brain, which is capable to generate competitive images in terms of resolution and contrast comparable with histopathological staining. METHODS: Coherent anti-Stokes Raman scattering (CARS) and two photon excited fluorescence (TPEF) microscopy are non-invasive all optical imaging techniques that are capable of high resolution, label-free, real-time, nondestructive examination of living cells and tissues. They provide image contrast based on the molecular composition of the specimen which allows the study of large tissue areas of frozen tissue sections ex vivo. RESULTS: Here, preliminary data on 55 lesions of the central nervous system are presented. The generated images very nicely demonstrate cytological and architectural features required for pathological tumor typing and grading. Furthermore, information on the molecular content of a probe is provided. The tool will be implemented into a biopsy needle or endoscope in the near future for in vivo studies. CONCLUSION: With this promising multimodal imaging approach the neurosurgeon might directly see blood vessels to minimize the risk for biopsy associated hemorrhages. The attending neuropathologist might directly identify the tumor and guide the selection of representative specimens for further studies. Thus, collection of non-representative material could be avoided and the risk to injure eloquent brain tissue minimized.

Expanding multimodal microscopy by high spectral resolution coherent anti-Stokes Raman scattering imaging for clinical disease diagnostics.

Over the past years fast label-free nonlinear imaging modalities providing molecular contrast of endogenous disease markers with subcellular spatial resolution have been emerged. However, applications of these imaging modalities in clinical settings are still at the very beginning. This is because single nonlinear imaging modalities such as second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) have only limited value for diagnosing diseases due to the small number of endogenous markers. Coherent anti-Stokes Raman scattering (CARS) microscopy on the other hand can potentially be added to SHG and TPEF to visualize a much broader range of marker molecules. However, CARS requires a second synchronized laser source and the detection of a certain wavenumber range of the vibrational spectrum to differentiate multiple molecules, which results in increased experimental complexity and often inefficient excitation of SHG and TPEF signals. Here we report the application of a novel near-infrared (NIR) fiber laser of 1 MHz repetition rate, 65 ps pulse duration, and 1 cm(-1) spectral resolution to realize an efficient but experimentally simple SGH/TPEF/multiplex CARS multimodal imaging approach for a label-free characterization of composition of complex tissue samples. This is demonstrated for arterial tissue specimens demonstrating differentiation of elastic fibers, triglycerides, collagen, myelin, cellular cytoplasm, and lipid droplets by analyzing the CARS spectra within the C-H stretching region only. A novel image analysis approach for multispectral CARS data based on colocalization allows correlating spectrally distinct pixels to morphologic structures. Transfer of this highly precise but compact and simple to use imaging approach into clinical settings is expected in the near future.

A compact microscope setup for multimodal nonlinear imaging in clinics and its application to disease diagnostics.

The past years have seen increasing interest in nonlinear optical microscopic imaging approaches for the investigation of diseases due to the method's unique capabilities of deep tissue penetration, 3D sectioning and molecular contrast. Its application in clinical routine diagnostics, however, is hampered by large and costly equipment requiring trained staff and regular maintenance, hence it has not yet matured to a reliable tool for application in clinics. In this contribution implementing a novel compact fiber laser system into a tailored designed laser scanning microscope results in a small footprint easy to use multimodal imaging platform enabling simultaneously highly efficient generation and acquisition of second harmonic generation (SHG), two-photon excited fluorescence (TPEF) as well as coherent anti-Stokes Raman scattering (CARS) signals with optimized CARS contrast for lipid imaging for label-free investigation of tissue samples. The instrument combining a laser source and a microscope features a unique combination of the highest NIR transmission and a fourfold enlarged field of view suited for investigating large tissue specimens. Despite its small size and turnkey operation rendering daily alignment dispensable the system provides the highest flexibility, an imaging speed of 1 megapixel per second and diffraction limited spatial resolution. This is illustrated by imaging samples of squamous cell carcinoma of the head and neck (HNSCC) and an animal model of atherosclerosis allowing for a complete characterization of the tissue composition and morphology, i.e. the tissue's morphochemistry. Highly valuable information for clinical diagnostics, e.g. monitoring the disease progression at the cellular level with molecular specificity, can be retrieved. Future combination with microscopic probes for in vivo imaging or even implementation in endoscopes will allow for in vivo grading of HNSCC and characterization of plaque deposits towards the detection of high risk plaques.

Tumor margin identification and prediction of the primary tumor from brain metastases using FTIR imaging and support vector machines.

Infrared spectroscopy enables the identification of tissue types based on their inherent vibrational fingerprint without staining in a nondestructive way. Here, Fourier transform infrared microscopic images were collected from 22 brain metastasis tissue sections of bladder carcinoma, lung carcinoma, mamma carcinoma, colon carcinoma, prostate carcinoma and renal cell carcinoma. The scope of this study was to distinguish the infrared spectra of carcinoma from normal tissue and necrosis and to use the infrared spectra of carcinoma to determine the primary tumor of brain metastasis. Data processing follows procedures that have previously been developed for the analysis of Raman images of these samples and includes the unmixing algorithm N-FINDR, segmentation by k-means clustering, and classification by support vector machines (SVMs). Upon comparison with the subsequent hematoxylin and eosin stained tissue sections of training specimens, correct classification rates of the first level SVM were 98.8% for brain tissue, 98.4% for necrosis and 94.4% for carcinoma. The primary tumors were correctly predicted with an overall rate of 98.7% for FTIR images of the training dataset by a second level SVM. Finally, the two level discrimination models were applied to four independent specimens for validation. Although the classification rates are slightly reduced compared to the training specimens, the majority of the infrared spectra of the independent specimens were assigned to the correct primary tumor. The results demonstrate the capability of FTIR imaging to complement histopathological tools for brain tissue diagnosis.

Advances in optical biopsy--correlation of malignancy and cell density of primary brain tumors using Raman microspectroscopic imaging.

Raman spectroscopy is a promising tool towards biopsy under vision as it provides label-free image contrast based on intrinsic vibrational spectroscopic fingerprints of the specimen. The current study applied the spectral unmixing algorithm vertex component analysis (VCA) to probe cell density and cell nuclei in Raman images of primary brain tumor tissue sections. Six Raman images were collected at 785 nm excitation that consisted of 61 by 61 spectra at a step size of 2 micrometers. After data acquisition the samples were stained with hematoxylin and eosin for comparison. VCA abundance plots coincided well with histopathological findings. Raman spectra of high grade tumor cells were found to contain more intense spectral contributions of nucleic acids than those of low grade tumor cells. Similarly, VCA endmember signatures of Raman images from high grade gliomas showed increased nucleic acid bands. Further abundance plots and endmember spectra were assigned to tissue containing proteins and lipids, and cholesterol microcrystals. Since no sample preparation is required, an important advantage of the Raman imaging methodology is that all tissue components can be observed - even those that may be lost in sample staining steps. The results demonstrate how morphology and chemical composition obtained by Raman imaging correlate with histopathology and provide complementary, diagnostically relevant information at the cellular level.

In vivo characterization of atherosclerotic plaque depositions by Raman-probe spectroscopy and in vitro coherent anti-stokes Raman scattering microscopic imaging on a rabbit model.

Visualization as well as characterization of inner arterial plaque depositions is of vital diagnostic interest, especially for the early recognition of vulnerable plaques. Established clinical techniques provide valuable visual information but cannot deliver information about the chemical composition of individual plaques. Here, we employ Raman-probe spectroscopy to characterize the plaque compositions of arterial walls on a rabbit model in vivo, using a miniaturized filtered probe with one excitation and 12 collection fibers integrated in a 1 mm sleeve. Rabbits were treated with a cholesterol-enriched diet. The methodology can improve the efficiency of animal experiments and shows great potential for applications in cardiovascular research. In order to further characterize the plaque depositions visually, coherent anti-Stokes Raman scattering (CARS) microscopy images have been acquired and are compared with the Raman-probe results.

Interpreting CARS images of tissue within the C-H-stretching region.

Single band coherent anti-Stokes Raman scattering (CARS) microscopy is one of the fastest implementation of nonlinear vibrational imaging allowing for video-rate image acquisition of tissue. This is due to the large Raman signal in the C-H-stretching region. However, the chemical specificity of such images is conventionally assumed to be low. Nonetheless, CARS imaging within the C-H-stretching region enables detection of single cells and nuclei, which allows for histopathologic grading of tissue. Relevant information such as nucleus to cytoplasm ratio, cell density, nucleus size and shape is extracted from CARS images by innovative image processing procedures. In this contribution CARS image contrast within the C-H-stretching region is interpreted by direct comparison with Raman imaging and correlated to the tissue composition justifying the use of CARS imaging in this wavenumber region for biomedical applications.

Towards automated segmentation of cells and cell nuclei in nonlinear optical microscopy.

Nonlinear optical (NLO) imaging techniques based e.g. on coherent anti-Stokes Raman scattering (CARS) or two photon excited fluorescence (TPEF) show great potential for biomedical imaging. In order to facilitate the diagnostic process based on NLO imaging, there is need for an automated calculation of quantitative values such as cell density, nucleus-to-cytoplasm ratio, average nuclear size. Extraction of these parameters is helpful for the histological assessment in general and specifically e.g. for the determination of tumor grades. This requires an accurate image segmentation and detection of locations and boundaries of cells and nuclei. Here we present an image processing approach for the detection of nuclei and cells in co-registered TPEF and CARS images. The algorithm developed utilizes the gray-scale information for the detection of the nuclei locations and the gradient information for the delineation of the nuclear and cellular boundaries. The approach reported is capable for an automated segmentation of cells and nuclei in multimodal TPEF-CARS images of human brain tumor samples. The results are important for the development of NLO microscopy into a clinically relevant diagnostic tool.

Nonlinear microscopy, infrared, and Raman microspectroscopy for brain tumor analysis.

Contemporary brain tumor research focuses on two challenges: First, tumor typing and grading by analyzing excised tissue is of utmost importance for choosing a therapy. Second, for prognostication the tumor has to be removed as completely as possible. Nowadays, histopathology of excised tissue using haematoxylin-eosine staining is the gold standard for the definitive diagnosis of surgical pathology specimens. However, it is neither applicable in vivo, nor does it allow for precise tumor typing in those cases when only nonrepresentative specimens are procured. Infrared and Raman spectroscopy allow for very precise cancer analysis due to their molecular specificity, while nonlinear microscopy is a suitable tool for rapid imaging of large tissue sections. Here, unstained samples from the brain of a domestic pig have been investigated by a multimodal nonlinear imaging approach combining coherent anti-Stokes Raman scattering, second harmonic generation, and two photon excited fluorescence microscopy. Furthermore, a brain tumor specimen was additionally analyzed by linear Raman and Fourier transform infrared imaging for a detailed assessment of the tissue types that is required for classification and to validate the multimodal imaging approach. Hence label-free vibrational microspectroscopic imaging is a promising tool for fast and precise in vivo diagnostics of brain tumors.

Prospective study of p-[123I]iodo-L-phenylalanine and SPECT for the evaluation of newly diagnosed cerebral lesions: specific confirmation of glioma.

PURPOSE: The differentiation between gliomas, metastases and gliotic or inflammatory lesions by imaging techniques remains a challenge. Gliomas frequently exhibit increased uptake of radiolabelled amino acids and are thus amenable to PET or SPECT imaging. Recently, p-[123I]iodo-L-phenylalanine (IPA) was validated for the visualization of glioma by SPECT and received orphan drug status. Here we investigated its diagnostic performance for differentiating indeterminate brain lesions. METHODS: This prospective open study included 67 patients with newly diagnosed brain lesions suspicious for glioma (34 without and 33 with contrast enhancement in the MRI scan). Patients received 250 MBq IPA intravenously after overnight fasting. SPECT images at 30 min and 3 h post-injection were iteratively reconstructed and visually interpreted after image fusion with an MRI brain scan (fluid-attenuated inversion recovery sequence or T1-weighted contrast-enhanced image). Findings were correlated with results of stereotactic or open biopsies or serial imaging. RESULTS: Twenty-seven low-grade (2 WHO I, 25 WHO II) and 24 high-grade gliomas (1 WHO III, 23 WHO IV), 3 metastases originating from lung cancer as well as 13 non-neoplastic lesions were proven. All non-neoplastic lesions and all metastases were negative with IPA SPECT. Forty gliomas were true-positive (TP) and 11 false-negative (FN) findings (8 WHO II, 1 WHO III, 2 WHO IV) occurred. There were no false-positive (FP) findings. For the differentiation of primary brain tumours and non-neoplastic lesions, sensitivity and specificity were 78 and 100%. In 34 lesions without contrast enhancement in MRI, IPA SPECT resulted in 17 TP, 8 true-negative, 9 FN and no FP findings (sensitivity 65%, specificity 100%). CONCLUSION: In patients with suspected glioma, IPA SPECT shows a high specificity, but especially in low-grade gliomas FN findings may occur. Due to the high positive predictive value a positive finding allows a suspected glioma to be confirmed.

A case with coincidental diagnosis of primary central nervous system lymphoma and lymph node sarcoidosis.

Primary central nervous system lymphoma (PCNSL) is rare. Clinical and histological differential diagnosis of systemic lymphoma and sarcoidosis continues to be a challenge. The first case report in the German and English literature of PCNSL and synchronous sarcoidosis is presented. Synchronous mediastinal lymphadenopathy suggestive of non-Hodgkin's lymphoma (NHL) or sarcoidosis was noted. Both conditions require alternative therapeutic and prognostic considerations to PCNSL. A regime of intrathecal and adjuvant systemic chemotherapy led to transient clinical improvement prior to the patient's demise through overwhelming sepsis and multiorgan failure. Post mortem findings confirmed synchronous PCNSL with mediastinal lymph node sarcoidosis.

Genome wide expression profiling identifies specific deregulated pathways in meningioma.

Genome-wide expression signatures improve the understanding of tumor biology. We performed expression profiling of 24 meningioma including 8 of each WHO grade and 2 dura controls analyzing 55.000 transcripts including 18.300 known genes. We compared expression in meningioma vs. dura, expression of low grade (WHO I) vs. higher-grade (WHO II and WHO III) tumors and expression of meningothelial and syncytial meningioma vs. fibroblastic meningioma. Overall expression was significantly decreased in meningioma compared to dura and in meningothelial and syncytial compared to fibroblastic meningioma. Gene expression was exemplarily confirmed by immunohistochemistry using independent samples. Applying our statistical gene set analysis toolkit "GeneTrail", we identified significantly deregulated biochemical pathways using Kyoto encyclopedia of genes and genomes and Transpath databases. Kyoto encyclopedia of genes and genomes pathways with decreased expression in meningioma included cell adhesion molecules (p<0.0001) and cytokine-cytokine receptor interactions (p<0.0001). Pathways with increased expression included several metabolic pathways. Extended expression profiling by a novel statistical gene set enrichment identified pathways that have previously not been associated with meningioma.

Gliomatosis cerebri: growing evidence for diffuse gliomas with wide invasion.

Recent evidence suggests that there are no features that sufficiently distinguish gliomatosis cerebri (GC) from diffuse gliomas. They show the same age and sex distribution, clinical signs, prognostic factors and harbor similar genetic abnormalities. Furthermore, GC can progress secondarily to a higher grade of malignancy. The 2007 WHO classification itemizes GC among astrocytic tumors. Nevertheless, an oligodendroglial differentiation can be observed in up to 40%. These data implicate that treatment guidelines might follow those for diffuse gliomas in general. However, the large extension usually limits surgery to biopsies. The role of whole brain radiotherapy is unclear because no impact on survival could be demonstrated. At least some patients might benefit from chemotherapy (temozolomide).

Intra-individual comparison of p-[123I]-iodo-L-phenylalanine and L-3-[123I]-iodo-alpha-methyl-tyrosine for SPECT imaging of gliomas.

OBJECTIVES: Radioactive amino-acids accumulate in gliomas even with an intact blood-brain-barrier. L-3-[(123)I]-iodo-alpha-methyl-tyrosine (IMT) is well established for SPECT imaging of gliomas. Recently, we introduced p-[(123)I]-iodo-L-phenylalanine (IPA) for the characterisation of brain lesions. This study compares both tracers in glioma patients. METHODS: Eleven patients with gliomas (1 WHO grade 1, 5 grade 2, 1 grade 3, 2 grade 4 gliomas, 1 unconfirmed upgrading and 1 post-therapeutic non-neoplastic lesion) underwent SPECT imaging with IPA (early and delayed acquisitions at 30 min and 3 h) and IMT (early only). Maximum tumour-to-brain ratios (TBR) were calculated using region-of-interest analysis to assess uptake of IMT and IPA. Imaging results were compared to histopathological findings. RESULTS: Early TBRs of IMT and IPA were strongly correlated (r = 0.828, p = 0.002). TBRs were higher for IMT than IPA (1.95+/-0.50 versus 1.79+/-0.42; p < 0.05), but independent from tumour cell density (p > 0.1). Visual interpretation by different observers was more concordant for IMT-SPECT than IPA-SPECT (kappa 1.0 versus 0.774). No differences in early TBRs were observed between low-grade and high-grade gliomas for IMT (1.97+/-0.53 versus 2.21+/-0.44, p > 0.5) or IPA (1.70+/-0.23 versus 2.21+/-0.56, p = 0.167) with a trend to higher TBRs in low-grade tumours for IMT (p = 0.093). In contrast to the known wash-out of IMT, we observed persistent accumulation of IPA in gliomas. CONCLUSIONS: IPA shows lower TBRs than IMT, especially in low-grade tumours, so IMT should be preferred for the delineation of low-grade gliomas by SPECT imaging. Due to its prolonged retention, however, IPA remains promising for therapeutic use in gliomas after labelling with I-131.