Three-dimensional virtual histology in unprocessed resected tissues with photoacoustic remote sensing (PARS) microscopy and optical coherence tomography (OCT).

Histological images are critical in the diagnosis and treatment of cancers. Unfortunately, current methods for capturing these microscopy images require resource intensive tissue preparation that may delay diagnosis for days or weeks. To streamline this process, clinicians are limited to assessing small macroscopically representative subsets of tissues. Here, a combined photoacoustic remote sensing (PARS) microscope and swept source optical coherence tomography system designed to circumvent these diagnostic limitations is presented. The proposed multimodal microscope provides label-free three-dimensional depth resolved virtual histology visualizations, capturing nuclear and extranuclear tissue morphology directly on thick unprocessed specimens. The capabilities of the proposed method are demonstrated directly in unprocessed formalin fixed resected tissues. The first images of nuclear contrast in resected human tissues, and the first three-dimensional visualization of subsurface nuclear morphology in resected Rattus tissues, captured with a non-contact photoacoustic system are presented here. Moreover, the proposed system captures the first co-registered OCT and PARS images enabling direct histological assessment of unprocessed tissues. This work represents a vital step towards the development of a rapid histological imaging modality to circumvent the limitations of current histopathology techniques.

Pitfalls and Caveats in Applying Chromogenic Immunostaining to Histopathological Diagnosis.

Chromogenic immunohistochemistry (immunostaining using an enzyme-labeled probe) is an essential histochemical technique for analyzing pathogenesis and making a histopathological diagnosis in routine pathology services. In neoplastic lesions, immunohistochemistry allows the study of specific clinical and biological features such as histogenesis, behavioral characteristics, therapeutic targets, and prognostic biomarkers. The needs for appropriate and reproducible methods of immunostaining are prompted by technical development and refinement, commercial availability of a variety of antibodies, advanced applicability of immunohistochemical markers, accelerated analysis of clinicopathological correlations, progress in molecular targeted therapy, and the expectation of advanced histopathological diagnosis. However, immunostaining does have various pitfalls and caveats. Pathologists should learn from previous mistakes and failures and from results indicating false positivity and false negativity. The present review article describes various devices, technical hints, and trouble-shooting guides to keep in mind when performing immunostaining.

Digital Pathology Initiatives and Experience of a Large Academic Institution During the Coronavirus Disease 2019 (COVID-19) Pandemic.

CONTEXT.­: Pathology practices have begun integrating digital pathology tools into their routine workflow. During 2020, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic, causing a global health crisis that significantly affected the world population in several areas, including medical practice, and pathology was no exception. OBJECTIVE.­: To summarize our experience in implementing digital pathology for remote primary diagnosis, education, and research during this pandemic. DESIGN.­: We surveyed our pathologists (all subspecialized) and trainees to gather information about their use of digital pathology tools before and during the pandemic. Quality assurance and slide distribution data were also examined. RESULTS.­: During the pandemic, the widespread use of digital tools in our institution allowed a smooth transition of most clinical and academic activities into remote with no major disruptions. The number of pathologists using whole slide imaging (WSI) for primary diagnosis increased from 20 (62.5%) to 29 (90.6%) of a total of 32 pathologists, excluding renal pathology and hematopathology, during the pandemic. Furthermore, the number of pathologists exclusively using whole slide imaging for primary diagnosis also increased from 2 (6.3%) to 5 (15.6%) during the pandemic. In 35 (100%) survey responses from attending pathologists, 21 (60%) reported using whole slide imaging for remote primary diagnosis following the Centers for Medicare and Medicaid Services waiver. Of these 21 pathologists, 18 (86%) responded that if allowed, they will continue using whole slide imaging for remote primary diagnosis after the pandemic. CONCLUSIONS.­: The pandemic served as a catalyst to pathologists adopting a digital workflow into their daily practice and realizing the logistic and technical advantages of such tools.

Evolution and new frontiers of histology in bio-medical research.

Histology refers to the study of the morphology of cells within their natural tissue environment. As a bio-medical discipline, it dates back to the development of first microscopes which allowed to override the physical visual limitation of the human eye. Since the first observations, it was understood that cell shape predicts function and, therefore, shape alterations can identify and explain dysfunction and diseases. The advancements in morphological investigation techniques have allowed to extend our understanding of the shape-function relationships close to the molecular level of organization of tissues, as well as to derive reliable data not only from fixed, and hence static, biological samples but also living cells and tissues and even for extended time periods. These modern approaches, which encompass quantitative microscopy, precision microscopy, and dynamic microscopy, represent the new frontier of morphology. This article summarizes how the microscopy techniques have evolved to properly face the challenges of biomedical sciences, thus transforming histology from a merely qualitative discipline, which played an ancillary role to traditional "major" sciences such as anatomy, to a modern experimental science capable of driving knowledge progress in biology and medicine.

MR Imaging-Histology Correlation by Tailored 3D-Printed Slicer in Oncological Assessment.

3D printing and reverse engineering are innovative technologies that are revolutionizing scientific research in the health sciences and related clinical practice. Such technologies are able to improve the development of various custom-made medical devices while also lowering design and production costs. Recent advances allow the printing of particularly complex prototypes whose geometry is drawn from precise computer models designed on in vivo imaging data. This review summarizes a new method for histological sample processing (applicable to e.g., the brain, prostate, liver, and renal mass) which employs a personalized mold developed from diagnostic images through computer-aided design software and 3D printing. Through positioning the custom mold in a coherent manner with respect to the organ of interest (as delineated by in vivo imaging data), the cutting instrument can be precisely guided in order to obtain blocks of tissue which correspond with high accuracy to the slices imaged. This approach appeared crucial for validation of new quantitative imaging tools, for an accurate imaging-histopathological correlation and for the assessment of radiogenomic features extracted from oncological lesions. The aim of this review is to define and describe 3D printing technologies which are applicable to oncological assessment and slicer design, highlighting the radiological and pathological perspective as well as recent applications of this approach for the histological validation of and correlation with MR images.

Histo-genomics: digital pathology at the forefront of precision medicine.

The toughest challenge OMICs face is that they provide extremely high molecular resolution but poor spatial information. Understanding the cellular/histological context of the overwhelming genetic data is critical for a full understanding of the clinical behavior of a malignant tumor. Digital pathology can add an extra layer of information to help visualize in a spatial and microenvironmental context the molecular information of cancer. Thus, histo-genomics provide a unique chance for data integration. In the era of a precision medicine, a four-dimensional (4D) (temporal/spatial) analysis of cancer aided by digital pathology can be a critical step to understand the evolution/progression of different cancers and consequently tailor individual treatment plans. For instance, the integration of molecular biomarkers expression into a three-dimensional (3D) image of a digitally scanned tumor can offer a better understanding of its subtype, behavior, host immune response and prognosis. Using advanced digital image analysis, a larger spectrum of parameters can be analyzed as potential predictors of clinical behavior. Correlation between morphological features and host immune response can be also performed with therapeutic implications. Radio-histomics, or the interface of radiological images and histology is another emerging exciting field which encompasses the integration of radiological imaging with digital pathological images, genomics, and clinical data to portray a more holistic approach to understating and treating disease. These advances in digital slide scanning are not without technical challenges, which will be addressed carefully in this review with quick peek at its future.

Estudio de la evolución en la actividad en un Departamento de Anatomía Patológica de un hospital de tercer nivel en la última década (2007-2016) / Study of the evolution in the activity of a Department of Pathology from a third level hospital in the last decade (2007-2016)

Objetivo: Estudiar la evolución de variables de interés de un Departamento de Anatomía Patológica de un hospital de tercer nivel durante la última década. Valorar el impacto del traslado hospitalario en la actividad (año 2014). Material y método: Estudio retrospectivo observacional en el que se analizaron las muestras registradas (biopsias, citologías, PAAF, autopsias, intraoperatorias) así como las técnicas complementarias (IHQ, histoquímica, IF y FISH) y la cartera de servicios, durante la década 2007-2016. Para el análisis estadístico se compararon los quinquenios 2007-2011 y 2012-2016. Resultados: Las siguientes variables han sido estadísticamente significativas: citologías (34055,8±1994,0 vs. 26590,4±2938,3; p=0,002), autopsias (156,2±27,3 vs. 122,0±14,78; p=0,039), inmunohistoquímica (17855,4±3424,2 vs. 28559,2±4734,7; p=0,003), histoquímica (11117,8±2300,9 vs. 6225,0±1330,5; p=0,003) e inmunofluorescencia (610,2±185,3 vs. 1205,4±154,0; p=0,001). Se han identificado correlaciones estadísticas de interés entre las variables. En el año 2014 se observó que las variables de mayor peso específico (biopsias, citologías, IHQ e histoquímica) en la carga asistencial del Departamento mostraron un descenso medio del 12,5%. Se ha identificado un incremento generalizado en el catálogo de muestras disponibles. El número de anticuerpos (78,7%), la histoquímica (38,7%) y FISH (400%) fueron los que mayor aumento mostraron. Conclusión: Se han identificado variaciones relevantes del volumen de actividad, así como en la cartera de servicios, especialmente en las técnicas orientadas a mejorar la precisión diagnóstica (IHQ e IF); y un descenso importante en el número de citologías, autopsias e histoquímica. En el año 2014 se ha observado un descenso de más del 12% en las principales variables del estudio

Objective: To study the evolution of variables of interest in a department of pathology from a third level hospital during the last decade and to evaluate the impact on these of the hospital relocation in 2014. Material and method: Retrospective observational study in which the recorded samples (biopsies, cytology specimens, FNA, autopsies, intraoperative) as well as the complementary techniques (IHC, Histochemistry, IF and FISH) and portfolio of services were analyzed during the years 2007-2016 inclusive. For the statistical analysis, the five-year periods 2007-2011 and 2012-2016 were compared. Results: The following variables were statistically significant: cytology (34055.8±1994.0 vs 26590.4±2938.3, p=0.002), autopsies (156.2±27.3 vs 122.0±14.78, p=0.039), immunohistochemistry (17855.4±3424.2 vs 28559.2±4734.7, p=0.003), histochemistry (11117.8±2300.9 vs 6225.0±1330.5, p=0.003) and immunofluorescence (610.2±185.3 vs. 1205.4±154.0, p=0.001). Statistical correlations of interest among variables have been identified. In 2014, it was observed that the variables of greater specific weight (biopsies, cytology, IHQ and histochemistry) in the work load of the Department showed an average decrease of 12.5%. A generalized increase in the panel of available samples has been identified, the largest increase being seen in the number of antibodies (78.7%), histochemistry (38.7%) and FISH (400%). Conclusion: Relevant variations in work volume, as well as the service portfolio, have been identified, especially in the techniques aimed at improving diagnostic accuracy (IHQ and FI), and a significant decrease in the number of cytology specimens, autopsies and histochemistry. In the year 2014 a decrease of more than 12% in the main variables of the study was observed

Control de calidad de la citología ginecológica: programa de calidad de la Sociedad Española de Citología. Resultados de la segunda ronda / The Spanish Society of Cytology: quality control program of gynecological cytology

Introducción. Las recomendaciones del cribado de cáncer de cérvix en España incluyen la participación en programas de control de calidad externos a los laboratorios de citología. La Sociedad Española de Citología (SEC) ha iniciado un programa de control de calidad de la citología ginecológica (CG). Objetivo. Presentar y analizar los resultados de la segunda ronda del control de calidad de la SEC. Material y métodos. Se incluyeron casos procesados mediante citología en medio líquido. Se escanearon las laminillas mediante la plataforma Aperio. Se seleccionaron 23 muestras procedentes de un banco de casos con al menos un 75% de acuerdo entre 4 expertos citopatólogos. Los diagnósticos de los casos para estudio incluyeron: uno negativo, 15 lesiones de bajo grado (4 ASCUS y 11 LSIL) y 7 lesiones de alto grado (uno ASCH y 6 HSIL). La CML correspondía a ThinPrep® en 16 casos y a SurePath® en 7. Se realizó el estudio de la correlación diagnóstica interobservador. Resultados. Participaron 16 hospitales. Las concordancias medias fueron: global 70,6% y por tipo de lesión 63,1%. En negativo 71,9%, en ASCUS 56,2%, en LSIL 69,5% y en HSIL 82,8%. Los casos discordantes correspondían con mayor frecuencia a negativos y a ASCUS. Se observó discordancia severa (HSIL/ASCH frente a negativo) en un 4,4% de los casos. Conclusiones. Nuestros resultados son similares a los descritos en la literatura, encontrando muy escasas discordancias severas (AU)

Introduction. In Spain, the guidelines for cervical cancer screening include a recommendation to enroll in external quality control programs. The Spanish Society of Cytology (SEC) has initiated its own quality control program of gynecological cytology (QCPGC). Aim. To describe and discuss the results of the second round of SEC¿s QCPGC. Material and method. The cases are selected by a group of expert cytologists. The cases with an agreement of 75% of four cytopathologists were used. The cases were scanned with Aperio. The scanned cases not available were excluded. We included a total of 23 cases, 1 negative, 15 low grade lesions (4 ASCUS and 11 LSIL) and 7 high grade lesions (1 ASCH and 6 HSIL). Sixteen cases were studied with ThinPrep™ platform and in 7 cases the SurePath™ platform was used. Results. Sixteen hospitals participated. The global mean concordance was 70.6%. The mean concordance in the type of lesion was 63.1%. The concordance was 71.9% in negative diagnoses, 56.2% in ASCUS, 69.5% in LSIL and 82.8% in HSIL The discordant cases were diagnosed more frequently as negative and ASCUS. 4.4% of cases had major discordances (HSIL or ASCH versus negatives). Conclusions: Our results are similar to those reported in the literature, with very little severe discordance. The method of exchanging slides does not allows continuous training, since the review of discordant cases can not be made. Therefore, methodological corrections are contemplated for future rounds (AU)

Comparison of different histological protocols for the preservation and quantification of the intestinal mucus layer in pigs.

The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production. The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness. Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution. The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution. After dehydration, methacarn fixed tissues were embedded in paraffin wax. Both sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness. Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared. In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area. Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements. Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness. The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness. Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon. Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples. In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs. Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness. The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs.

Disease activity and mucosal healing in inflammatory bowel disease: a new role for histopathology?

Histologic evaluation of disease activity in the setting of inflammatory bowel disease is gaining interest within the gastroenterology community. Recent data suggests that histologic measurements of inflammation in ulcerative colitis are more sensitive at detecting disease activity and perform better than endoscopic measurements in predicting clinical outcomes. Histologic measurements are also increasingly used in ulcerative colitis clinical trials to assess response to new therapies. Histologic measurements of disease activity are less well studied in Crohn's disease, but are gaining attention. Current published treatment algorithms in inflammatory bowel disease do not take into consideration histologic activity; however, this may change in the near future. In order for histologic measurements to be included in clinical decision-making, validated, reliable, and responsive histologic scoring systems are needed. In this review, the recent literature on the significance of histologic activity in both ulcerative colitis and Crohn's disease is summarized. Histologic scoring systems are also briefly discussed.

A beginner's guide to tissue clearing.

The last decade has seen a proliferation of tissue clearing methods that render large biological samples transparent and allow unprecedented three-dimensional views of enormous volumes of tissue. For a scientist wondering whether these methods will be useful to address their research problems, it can be bewildering to sort through the ever-increasing number of papers introducing new clearing methods. Here, I provide a concise summary for the novice describing what tissue clearing is, which research problems it can be applied to, how to decide on a clearing method, and where the field is headed in the future.

Variation in Brain Morphology of Intertidal Gobies: A Comparison of Methodologies Used to Quantitatively Assess Brain Volumes in Fish.

When correlating brain size and structure with behavioural and environmental characteristics, a range of techniques can be utilised. This study used gobiid fishes to quantitatively compare brain volumes obtained via three different methods; these included the commonly used techniques of histology and approximating brain volume to an idealised ellipsoid, and the recently established technique of X-ray micro-computed tomography (micro-CT). It was found that all three methods differed significantly from one another in their volume estimates for most brain lobes. The ellipsoid method was prone to over- or under-estimation of lobe size, histology caused shrinkage in the telencephalon, and although micro-CT methods generated the most reliable results, they were also the most expensive. Despite these differences, all methods depicted quantitatively similar relationships among the four different species for each brain lobe. Thus, all methods support the same conclusions that fishes inhabiting rock pool and sandy habitats have different patterns of brain organisation. In particular, fishes from spatially complex rock pool habitats were found to have larger telencephalons, while those from simple homogenous sandy shores had a larger optic tectum. Where possible we recommend that micro-CT be used in brain volume analyses, as it allows for measurements without destruction of the brain and fast identification and quantification of individual brain lobes, and minimises many of the biases resulting from the histology and ellipsoid methods.

The Histochemistry and Cell Biology pandect: the year 2014 in review.

This review encompasses a brief synopsis of the articles published in 2014 in Histochemistry and Cell Biology. Out of the total of 12 issues published in 2014, two special issues were devoted to "Single-Molecule Super-Resolution Microscopy." The present review is divided into 11 categories, providing an easy format for readers to quickly peruse topics of particular interest to them.

Neurogénesis endógena tras el daño cerebral traumático y su modulación mediante terapia celular / Endogenous neurogenesis after traumatic brain injury and its modulation by cell therapy

Objetivo: Estudiar la neurogénesis inducida por una lesión traumática cerebral y su modulación mediante terapia celular. Material y métodos: Se realizó un modelo de lesión cerebral traumática en ratas Wistar adultas causando un daño cerebral grave. Transcurridos dos meses de la lesión, se administraron intralesionalmente células madre estromales (CME) alogénicas obtenidas de médula ósea, o suero fisiológico. Se estudió histológicamente la zona subventricular (ZSV) de cada animal al objeto de valorar la neurogénesis endógena espontánea tras la lesión y su posible modificación por la administración intracerebral de CME. Igualmente, se valoró la modificación de los déficit funcionales tras el tratamiento. Resultados: Se detectó un aumento en la neurogénesis endógena en el grupo tratado con CME respecto del control. Este hallazgo estuvo asociado a una progresiva mejoría en la respuesta motora y sensorial en el grupo de animales que recibieron CME comparado con el grupo de animales control. Conclusiones: La eficacia de la terapia celular para revertir los déficit funcionales secundarios a una lesión traumática cerebral parece correlacionarse con un incremento de la neurogénesis endógena a nivel de la SVZ (AU)

Objective: To study the neurogenesis induced by traumatic brain injury and its modulation by cell therapy. Material and methods: We performed a model of traumatic brain injury in adult Wistar rats, causing severe brain damage. After 2 months of injury, allogeneic bone marrow stromal cells (BMSC) or saline were administered intralesionally. We studied histologically the subventricular zone (SVZ) of each animal, in order to assess endogenous neurogenesis after traumatic brain injury and its possible modulation by intracerebral administration of BMSC. Furthermore, we studied the modification of neurological deficits after BMSC administration. Results: We detected a rise in endogenous neurogenesis in the group treated with BMSC with respect to the control. This finding was associated with a progressive improvement in motor and sensory response in the group of animals that received BMSC compared with control animals. Conclusion: The efficacy of cell therapy using BMSC appears to correlate with an increase of endogenous neurogenesis at SVZ level (AU)

Preparing pathology for personalized medicine: possibilities for improvement of the pre-analytical phase.

With the introduction of new biological agents for cancer treatment enabling 'personalized medicine', treatment decisions based on the molecular features of the tumour are more common. Consequently, tissue evaluation in tumour pathology is becoming increasingly based on a combination of classical morphological and molecular analysis. The results of diagnostic tests rely not only on the quality of the method used but, to a large extent, also on the quality of specimens, which is dependent on the pre-analytical procedures and storage. With the introduction of predictive immunohistochemical and molecular tests in clinical pathology, improvement and standardization of pre-analytical procedures has become crucial. The aim of this review is to increase awareness with regard to tissue handling and for standardization of the pre-analytical phase of a diagnostic process. In addition, several processing steps in tissue handling that need to be improved in order to obtain the quality needed for modern molecular medicine will be discussed. Optimal, standardized procedures are crucial if a high standard of test results is to be achieved, which is what each patient deserves.

Dyes from a twenty-first century perspective.

In June 2008, the Biological Stain Commission sponsored A Seminar on Dyes and Staining the purpose of which was twofold: first, to show that very useful information applicable to biomedical dyes and staining is available from unrelated disciplines and second, to summarize modern thinking on how dyes, solvents, and tissues interact to produce selective staining. In this introduction to the papers from the symposium, we acknowledge that biomedical dye research has declined as newer technologies have gained importance. We should point out, however, that dyes and staining still are vitally important. Moreover, needs abound for innovative studies concerned with dye analysis, synthesis, and mode of action. Concepts and tools from unrelated fields hold promise for significant breakthroughs in many areas of interest.

Histology safety: now and then.

Histology safety usually focuses on general laboratory issues, but this article concentrates on the hazards affecting the individual histotech and their evolution in the last half a century. Using the information from a survey especially designed for the occasion, the hazards were divided into 4 groups, and their prevalence was expressed as percentages for national and foreign laboratories. All the laboratories received a "safety index" (SI) with an average value of 0.77 +/- 0.11 for 63 national laboratories and 0.69 +/- 0.13 for 22 foreign laboratories, these 2 averages being statistically different (P < .02). The historical evolution of the SI required answering the same questionnaire retrospectively, and so it was done for 17 laboratories with an SI average of 0.27 +/- 0.12 for 1955/1989 and 0.77 +/- 0.13, almost 3 times larger for 1990/2007, with improvement of all safety issues. The technological, organizational, and regulatory advances before 1989 showed an unremarkable effect on the SI, and the only circumstance considered as the driving force behind the almost triple increment of the SI during 1990/2007 was the awareness that the AIDS epidemic instilled in the minds and consciences of the medical laboratory personnel in general. Even after almost tripling the average SI value in 2007, national histology laboratories obtained a grade average of "C+" only, leaving room for improvement.