Estrategia y actividades de aprendizaje en la enseñanza práctica de Histología Médica en la UCO

La organización y estrategia docente que seguimos, desde el comienzo del nuevo plan de estudios del grado de Medicina en la Universidad de Córdoba, en la enseñanza práctica de la Histología Médica ha ido modificándose desde la realización de experiencias piloto iniciadas en el curso 2005-06 hasta el momento actual. Se presentan y se justifican las diferentes actividades prácticas que actualmente venimos desarrollando (práctica de laboratorio, introducción al diagnóstico histológico, sesiones de microscopía y prácticas de diagnóstico diferencial), la estrategia y metodología docente que se lleva a cabo para conseguir el máximo de eficacia en el aprendizaje y los sistemas de evaluación aplicados para cada una de las actividades. Presentamos los resultados obtenidos en la valoración –grado de satisfacción–realizada por parte de los alumnos en los dos últimos cursos académicos y para las dos asignaturas de Histología Médica (I y II) que configuran esta disciplina en nuestro plan de estudios

Diatoms in forensic analysis: A practical approach in rats

A diagnosis of drowning is a challenge in legal medicine, as there is generally a lack of pathognomonic findings indicative of drowning. Diatom analysis has been considered very supportive for a diagnosis of drowning, although the test is still controversial for some investigators. We assessed diatoms association with drowning in the peripheral tissues of drowned rats and the effects of the drowning medium on the diatom yield. A modified acid digestion method was optimised for diatom recovery in water and rat tissues. Eighteen adult Wistar rats were employed for the study, subdivided into six groups of three rats. Groups 1, 3 and 5 were drowned in seawater, lake water, or river water respectively, while groups 2, 4 and 6 were controls (immersed after death in seawater, lake water or river water respectively). Water samples were taken from the sea, lake and river in Málaga and Córdoba (Spain) for the purposes of diatomological mapping and drowning of the rats. Diatoms were successfully recovered from all water samples and matched with tissues of the drowned rats. There were significant differences in diatom numbers between control and test samples for all the tissues studied, as well as within test samples. Histological investigations conducted on lung samples obtained from drowned rats provided complementary and valuable information. This study demonstrates the feasibility of the diatom test as a reliable method for the diagnosis of drowning, especially if adequate precautions are taken to avoid contamination, and if interpretation of the analysis is performed in light of other complementary investigations

Relations among capillaries, skeletal muscle fibers and satellite cells during regeneration

Introducción: En este estudio se analizan las modificaciones en las relaciones topográficas de los capilares con las fibras musculares y células satélites durante la fase de remodelación de la regeneración muscular. Material y métodos: Los músculos tibial anterior de ratas Wistar, lesionados por inyección intramuscular de miotóxico, fueron analizados a los 5, 7 y 20 días postlesión en microscopía de luz y electrónica. En cortes semifinos teñidos con azul de toluidina fueron analizadas la densidad capilar, densidad de fibras e índice capilar/fibra; sobre micrografías electrónicas se analizaron las distancias de difusión entre capilares-fibras musculares regenerativas y capilares-células satélites, así como el número de células satélites junto a un capilar. Resultados: A los 5 días postlesión la densidad capilar y la densidad de fibras se encontraron significativamente incrementadas así como las distancias de difusión entre los capilares con las fibras regenerativas y células satélites; además sólo un 47% de las células satélites se situaban junto a un capilar. A partir del día 7 postlesión todos los parámetros se aproximaron a los valores controles, normalizándose a los 20 días postlesión. Conclusión: Estos hallazgos indican que al inicio de la fase de remodelación la mayor densidad capilar no parece estar ligada a mayores necesidades de oxigenación de las fibras musculares regenerativas y células satélites Estos resultados están en buena relación con recientes estudios que señalan que bajas concentraciones de oxígeno favorecen la miogénesis.Introduction: This study analyzes the modifications in topographic relations among capillaries, skeletal muscle fibers and satellite cells during the remodeling phase of muscle regeneration. Material and methods: Tibial anterior muscles of Wistar rats were injured by intramuscular myotoxic injection and analyzed 5, 7 and 20 days postlesion under light and electron microscopy. Capillary density, fiber density, and capillary/fiber index were analyzed in toluidine blue semithin sections; diffusion distances between capillaries-regenerative muscle fibers and capillaries-satellite cells, as well as the number of satellite cells beside a capillary, were analyzed on electron micrographics. Results: At day 5 postlesion capillary density and fiber density DF are significantly increased as well as diffusion distances of capillaries in relation to regenerative fibers and satellite cells; moreover only the 47% of satellite cells are beside a capillary. From day 7 postlesion, all the parameters approach normal values, becoming normal at day 20 postlesion. Conclusion: These findings show that high capillary density at the beginning of the remodeling phase is not related to an increase in the oxygenation requirement of regenerative muscle fibers and satellite cells. These results are in good relation with recent studies that point out that low oxygen concentrations contribute to myogenesis

Ultrasonographic and Histological Correlation after Experimental Reconstruction of a Volumetric Muscle Loss Injury with Adipose Tissue

Different types of scaffolds are used to reconstruct muscle volume loss injuries. In this experimental study, we correlated ultrasound observations with histological findings in a muscle volume loss injury reconstructed with autologous adipose tissue. The outcome is compared with decellularized and porous matrix implants. Autologous adipose tissue, decellularized matrix, and a porous collagen matrix were implanted in volumetric muscle loss (VML) injuries generated on the anterior tibial muscles of Wistar rats. Sixty days after implantation, ultrasound findings were compared with histological and histomorphometric analysis. The muscles with an autologous adipose tissue implant exhibited an ultrasound pattern that was quite similar to that of the regenerative control muscles. From a histological point of view, the defects had been occupied by newly formed muscle tissue with certain structural abnormalities that would explain the differences between the ultrasound patterns of the normal control muscles and the regenerated ones. While the decellularized muscle matrix implant resulted in fibrosis and an inflammatory response, the porous collagen matrix implant was replaced by regenerative muscle fibers with neurogenic atrophy and fibrosis. In both cases, the ultrasound images reflected echogenic, echotextural, and vascular changes compatible with the histological findings of failed muscle regeneration. The ultrasound analysis confirmed the histological findings observed in the VML injuries reconstructed by autologous adipose tissue implantation. Ultrasound can be a useful tool for evaluating the structure of muscles reconstructed through tissue engineering

Reconocimiento de estructuras histológicas en muestras histopatológicas

Uno de nuestros objetivos en Histología es enseñar a los alumnos a describir, reconocer e interpretar imágenes microscópicas que les permitan, de una forma sistematizada, llegar a un diagnóstico histológico. Presentamos el diseño de una nueva actividad práctica con una orientación más novedosa y aplicativa, basada en el trabajo en pequeños grupos y la utilización de recursos on line. Empleando imágenes histopatológicas en las que la estructura normal ha quedado muy alterada, los alumnos deben de discriminar entre las zonas normales y anormales, describir y reconocer los elementos “residuales” células y tisulares normales. La práctica se llevó a cabo en una de las Aulas multimedia de la Facultad de Medicina y Enfermería de la UCO que se encuentran completamente equipadas para el trabajo en pequeños grupos y con infraestructura y equipamiento para el empleo de las TIC’s. Cada grupo trabaja de forma independiente empleando todos los recursos disponibles, emitiendo informes completos en un tiempo limitado y que deben ser consensuados y firmados por todos. Los alumnos/as fueron encuestados en relación a la utilidad, complementariedad y adquisición de las competencias CE22, CT12 CT10 y CT 19. Los resultados avalan que esta orientación aplicativa de la histología al relacionarla con la histopatología representa un elemento motivador para los alumnos favoreciendo su interés por lo que observan al microscopio, al mismo tiempo que se potencian la adquisición de destrezas y habilidades necesarias en su formación como médicos.Proyecto nº 2015-2-3010 financiado por el III PLAN DE INNOVACIÓN Y MEJORA EDUCATIVA. Curso 2015/ 2016. UC

Myoinvasive pattern as a prognostic marker in low-grade, early-stage endometrioid endometrial carcinoma

Low-grade and early Federation for Gynecology and Obstetrics (FIGO) stage endometrioid endometrial carcinomas (EEC) have an excellent prognosis. However, approximately 10% of patients develop recurrence, which cannot be correctly predicted at diagnosis. We evaluated myoinvasive patterns as a prognostic factor of relapse in low-grade, early-stage EEC. Two-hundred and fifty-eight cases were selected according to the following inclusion criteria: (i) endometrioid endometrial carcinomas, (ii) grade 1 or 2 with (iii) FIGO stage I or II, and (iv) clinical follow-up. Slides were reviewed to annotate the myoinvasive pattern present in each case (infiltrative glands, microcystic, elongated and fragmented –MELF-, broad front, adenomyosis-like and adenoma malignum). Microsatellite instability was studied by immunoexpression of mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6). There were 29 recurrences (11.2%) among the 258 cases analysed. A predominant broad front myoinvasive pattern was significantly associated with tumour relapse (p = 0.003). The presence of a pattern of infiltrative glands (p = 0.001) and microsatellite instability (p = 0.004) were associated with lower disease-free survival, without having an impact on overall survival. Our observations suggest the potential value of the pattern of myoinvasion as a prognostic factor in low-grade, early-stage endometrioid endometrial carcinomaThis research was funded by the Instituto de Salud Carlos III (ISCIII) (PI17/01723), co-financed by the European Development Regional Fund ‘A way to achieve Europe’ (FEDER)

Near-peer Teaching in Histology Laboratory

Background: Near-peer teaching is an educational method based on being taught by one or more students who are more advanced in one specific area of the same curriculum. The aim of this study was to analyze outcomes and medical students’ reactions to near-peer teaching in Histology Laboratory session. Methods: Histology Laboratory session was firstly designed as a practical session driven by academic staff, while in our new approach was driven by Histology intern students, which are upper year students in Medicine curriculum. Our near-peer teaching was evaluated using a multiple choice test when half of students had attended the session, the results of which were compared with those from traditional teaching. A reaction evaluation survey was also administered at the end of the course. Results: Multiple choice test results did not showed statistical differences between near-peer and traditional teaching strategies. Results from the reaction evaluation were mostly positive, especially with regard to feeling comfortable in the session taught by intern students and how intern students managed to transmit the information properly.Conclusion: Near-peer teaching in Histology Laboratory practical session is an effective alternative teaching method, with outcomes equivalent to traditional design, and highly valued by undergraduate medical student

The association between the tumor immune microenvironments and clinical outcome in low-grade, early-stage endometrial cancer patients

Endometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low-grade, early-stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out tomeasure CD8, CD68, FOXP3, PD-1,and PD-L1markers, aswell as cytokeratin (CK), on tissuemicroarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering.Most samples (69%) belonged to theimmune-desert subtype, characterized by lowimmune cell densities. Tumor-infiltrating lymphocyte (TIL)-rich samples (4%) displayed high CD8+ T-cell infiltration, as well as a high percentage of CD8/PD-1+ cells. Immune-exclusion samples (19%) displayed the lowest CD8+ infiltration combined with high PD-L1 expression levels in CK+ tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8+/PD-1+ andCK/PD-L1+ cells.FOXP3andmacrophage-rich phenotypes (3%and 4% of total samples) displayed relatively high levels of FOXP3+ regulatory T-cells and CD68+ macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune-exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single-marker immunohistochemistry (IHC) performed on whole tissue sections. TIL-rich tumors demonstrated increased CD8+ T-cells by IHC, while immune-exclusion tumors displayed a lack of CD8+ T-cells and frequent expression of PD-L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low-grade, early-stage endometrial carcinomasCEA and IgM were funded by Fundación La Marató de TV3. This project was supported by grants from Partners of Choice Network from AstraZeneca and by the Instituto de Salud Carlos III (ISCIII) (PI17/01723 and PI21/00920), co-financed by the European Regional Development Fund ‘A way to achieve Europe’ (FEDER). We thank Marco Cassano (Lunaphore Technologies) for his help in writing the manuscrip

Clinicopathological features and prognostic significance of CTNNB1 mutation in low-grade, early-stage endometrial endometrioid carcinoma

Low-grade and early-stage endometrioid endometrial carcinomas (EECs) have an overall good prognosis but biomarkers identifying patients at risk of relapse are still lacking. Recently, CTNNB1 exon 3 mutation has been identified as a potential risk factor of recurrence in these patients. We evaluate the prognostic value of CTNNB1 mutation in a single-centre cohort of 218 low-grade, early-stage EECs, and the correlation with beta-catenin and LEF1 immunohistochemistry as candidate surrogate markers. CTNNB1 exon 3 hotspot mutations were evaluated by Sanger sequencing. Immunohistochemical staining of mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6), p53, beta-catenin, and LEF1 was performed in representative tissue microarrays. Tumours were also reviewed for mucinous and squamous differentiation, and MELF pattern. Nineteen (8.7%) tumours harboured a mutation in CTNNB1 exon 3. Nuclear beta-catenin and LEF1 were significantly associated with CTNNB1 mutation, showing nuclear beta-catenin a better specificity and positive predictive value for CTNNB1 mutation. Tumours with CTNNB1 exon 3 mutation were associated with reduced disease-free survival (p = 0.010), but no impact on overall survival was found (p = 0.807). The risk of relapse in tumours with CTNNB1 exon 3 mutation was independent of FIGO stage, tumour grade, mismatch repair protein expression, or the presence of lymphovascular space invasion. CTNNB1 exon 3 mutation has a negative impact on disease-free survival in low-grade, early-stage EECs. Nuclear beta-catenin shows a higher positive predictive value than LEF1 for CTNNB1 exon 3 mutation in these tumoursThis research was funded by the Instituto de Salud Carlos III (ISCIII) (PI17/01723), co-financed by the European Development Regional Fund ‘A way to achieve Europe’ (FEDER

Spatial distribution of CD3- and CD8-positive lymphocytes as pretest for POLE wild-type in molecular subgroups of endometrial carcinoma.

INTRODUCTION Over the years, the molecular classification of endometrial carcinoma has evolved significantly. Both POLEmut and MMRdef cases share tumor biological similarities like high tumor mutational burden and induce strong lymphatic reactions. While therefore use case scenarios for pretesting with tumor-infiltrating lymphocytes to replace molecular analysis did not show promising results, such testing may be warranted in cases where an inverse prediction, such as that of POLEwt, is being considered. For that reason we used a spatial digital pathology method to quantitatively examine CD3+ and CD8+ immune infiltrates in comparison to conventional histopathological parameters, prognostics and as potential pretest before molecular analysis. METHODS We applied a four-color multiplex immunofluorescence assay for pan-cytokeratin, CD3, CD8, and DAPI on 252 endometrial carcinomas as testing and compared it to further 213 cases as validation cohort from a similar multiplexing assay. We quantitatively assessed immune infiltrates in microscopic distances within the carcinoma, in a close distance of 50 microns, and in more distant areas. RESULTS Regarding prognostics, high CD3+ and CD8+ densities in intra-tumoral and close subregions pointed toward a favorable outcome. However, TCGA subtyping outperforms prognostication of CD3 and CD8 based parameters. Different CD3+ and CD8+ densities were significantly associated with the TCGA subgroups, but not consistently for histopathological parameter. In the testing cohort, intra-tumoral densities of less than 50 intra-tumoral CD8+ cells/mm2 were the most suitable parameter to assume a POLEwt, irrespective of an MMRdef, NSMP or p53abn background. An application to the validation cohort corroborates these findings with an overall sensitivity of 95.5%. DISCUSSION Molecular confirmation of POLEmut cases remains the gold standard. Even if CD3+ and CD8+ cell densities appeared less prognostic than TCGA, low intra-tumoral CD8+ values predict a POLE wild-type at substantial percentage rates, but not vice versa. This inverse correlation might be useful to increase pretest probabilities in consecutive POLE testing. Molecular subtyping is currently not conducted in one-third of cases deemed low-risk based on conventional clinical and histopathological parameters. However, this percentage could potentially be increased to two-thirds by excluding sequencing of predicted POLE wild-type cases, which could be determined through precise quantification of intra-tumoral CD8+ cells