Quality control in diagnostic immunohistochemistry: integrated on-slide positive controls.

Standardization in immunohistochemistry is a priority in modern pathology and requires strict quality control. Cost containment has also become fundamental and auditing of all procedures must take into account both these principles. Positive controls must be routinely performed so that their positivity guarantees the appropriateness of the immunohistochemical procedure. The aim of this study is to develop a low cost (utilizing a punch biopsy-PB-tool) procedure to construct positive controls which can be integrated in the patient's tissue slide. Sixteen frequently used control blocks were selected and multiple cylindrical samples were obtained using a 5-mm diameter punch biopsy tool, separately re-embedding them in single blocks. For each diagnostic immunoreaction requiring a positive control, an integrated PB-control section (cut from the appropriate PB-control block) was added to the top right corner of the diagnostic slide before immunostaining. This integrated control technique permitted a saving of 4.75% in total direct lab costs and proved to be technically feasible and reliable. Our proposal is easy to perform and within the reach of all pathology labs, requires easily available tools, its application costs is less than using external paired controls and ensures that a specific control for each slide is always available.

High-quality cell block preparation from scraping of conventional cytology slide: a technical report on a modified cytoscrape cell block technique.

BACKGROUND: Immunocytochemistry (ICC) on formalin-fixed paraffin embedded cell blocks is an ancillary tool commonly recruited for differential diagnoses of fine needle aspiration cytology (FNAC) samples. However, the quality of conventional cell blocks in terms of adequate cellularity and evenness of distribution of cytologic material is not always satisfactory for ICC. We introduce a modified agarose-based cytoscrape cell block (CCB) technique that can be effectively used for the preparation of cell blocks from scrapings of conventional FNAC slides. METHODS: A decoverslipped FNAC slide was mounted with a small amount of water. The cytological material was scraped off the slide into a tissue mold by scraping with a cell scraper. The cytoscrape material was pelleted by centrifugation and pre-embedded in ultra-low gelling temperature agarose and then re-embedded in conventional agarose. The final agarose gel disk was processed and embedded in paraffin. RESULTS: The quality of the ICC on the CCB sections was identical to that of the immunohistochemical stains on histological sections. By scrapping and harvesting the entirety of the cytological material off the cytology slide into a compact agarose cell button, we could avoid the risk of losing diagnostic material during the CCB preparation. CONCLUSION: This modified CCB technique enables concentration and focusing of minute material while maintaining the entire amount of the cytoscrape material on the viewing spot of the CCB sections. We believe this technique can be effectively used to improve the level of confidence in diagnosis of FNAC especially when the FNAC slides are the only sample available.

Increasing specificity in imaging mass spectrometry: high spatial fidelity transfer of proteins from tissue sections to functionalized surfaces.

Imaging mass spectrometry (IMS) is a technique in full expansion used in many clinical and biological applications. A common limitation of the technology, particularly true for protein analysis, is that only the most abundant and/or more easily ionizable molecules are typically detected. One approach to overcome this limitation is to transfer proteins contained within tissue sections onto functionalized surfaces with high spatial fidelity for IMS analysis. In this case, only proteins with an affinity for the surface will be retained whereas others will be removed. The chemical nature of the surface is therefore critical. The research work presented herein proposes a high spatial fidelity transfer method for proteins from thin tissue sections onto a nitrocellulose surface. The method employs a home-built apparatus that allows the transfer process to be performed without any direct physical contact between the section and the transfer surface while maintaining physical pressure between the surfaces to help protein migration. The performance of this system was demonstrated using mouse liver and kidney sections. Serials sections were also collected either to be stained with hematoxylin and eosin (H&E) to assess the spatial fidelity of the transfer process or to be directly analyzed as a control sample to differentiate the signals detected after transfer. IMS results showed a high spatial fidelity transfer of a subset of proteins. Some of the detected proteins were poorly observed or not observed with conventional direct tissue analysis, demonstrating an increase in detection sensitivity and specificity with the newly developed method.

Electrochemical removal of metallic implants from Technovit 9100 New embedded hard and soft tissues prior to histological sectioning.

Solid metallic implants in soft or hard tissues are serious challenges for histological processing. However, metallic implants are more frequently used in e.g. cardiovascular or orthopaedic therapies. Before clinical use, these devices need to be tested thoroughly in a biological environment and histological analysis of their biocompatibility is a major requirement. To allow the histological analysis of metallic implants in tissues especially in calcified hard tissues, we describe a method for embedding these tissues in the resin Technovit 9100 New and removing the metallic implants by electrochemical dissolution. With the combination of these two processes, we are able to achieve 5 µm thick sections from soft or hard tissues with a superior preservation of tissue architecture and especially the implant-tissue interface. These sections can be stained by classical stainings, immunohistochemical and enzymehistochemical as well as DNA-based staining methods.

Evaluation of a completely automated tissue-sectioning machine for paraffin blocks.

Tissue sectioning automation can be a resourceful tool in processing anatomic pathology specimens. The advantages of an automated system compared with the traditional manual sectioning rely on the consistency of the final sectioned material translated into invariable thickness, uniform orientation during serial sectioning and less tissue sectioning artifacts. This technical note presents the design of an automated tissue-sectioning device and compares the sectioned specimens with normal manual tissue sectioning performed by experienced histology technician.

A role of three-dimensional (3D) reconstruction in the classification of lung adenocarcinoma.

BACKGROUND: Three-dimensional (3D) reconstruction from paraffin embedded sections has been considered laborious and time-consuming. However, the high-resolution images of large object areas and different fields of view obtained by 3D reconstruction make one wonder whether it can add a new insight into lung adenocarcinoma, the most frequent histology type of lung cancer characterized by its morphological heterogeneity. OBJECTIVE: In this work, we tested whether an automated tissue sectioning machine and slide scanning system could generate precise 3D reconstruction of microanatomy of the lung and help us better understand and define histologic subtypes of lung adenocarcinoma. METHODS: Four formalin-fixed human lung adenocarcinoma resections were studied. Paraffin embedded tissues were sectioned with Kurabo-Automated tissue sectioning machine and serial sections were automatically stained and scanned with a Whole Slide Imaging device. The resulting stacks of images were 3D reconstructed by Mirax Panoramic View software. RESULTS: Two of the four specimens contained the islands of tumor cells detached in alveolar spaces that had not been described in any of the existing adenocarcinoma classifications. 3D reconstruction revealed the details of spatial distribution and structural interaction of the tumor that could hardly be observed by 2D light microscopy studies. The islands of tumor cells extended into a deeper aspect of the tissue, and were interconnected with each other and with the main tumor with a solid pattern that was surrounded by the islands. The finding raises the question whether the islands of tumor cells should be classified into a solid pattern in the current classification. CONCLUSION: The combination of new technologies enabled us to build an effective 3D reconstruction of resected lung adenocarcinomas. 3D reconstruction may help us refine the classification of lung adenocarcinoma by adding detailed spatial/structural information to 2D light microscopy evaluation.

Flash freezing of Mohs micrographic surgery tissue can minimize freeze artifact and speed slide preparation.

BACKGROUND: In Mohs micrographic surgery, excised tissue is traditionally prepared for cryotomy by freezing in the cryostat's refrigerated chamber. Any delay may cause drying artifact and tissue autolysis and affect slide turn around time (TAT). Flash freezing is used in frozen section processing of general pathology specimens to expedite TAT and enhance tissue histology by minimizing ice crystal formation (freeze artifact). DESIGN: This was a pilot quality improvement study to compare flash freezing of Mohs sections with the traditional method of freezing in the cryostat. Mohs layers divided into at least two sections (one set) were enrolled. One half was flash frozen in an isopentane histobath (-56 to -62°C); the other half was frozen in the cryostat (-27 to -30°C). RESULTS: Forty-one sets were enrolled. Average cryostat and histobath freeze times (range) were 144 seconds (90-240 seconds) and 22 seconds (15-40 seconds), respectively. Laboratory technicians felt that it was easier to achieve smooth, wrinkle-free sections in histobath frozen tissue in 90% of tissue sets. Physicians favored histology from flash frozen specimens (range 65-85%) over the traditional method of cryostat freezing. CONCLUSION: Flash freezing in a histobath produced a more rapidly opacified (frozen) specimen ready for cryotomy, expediting slide TAT. Tissue histology also demonstrated better quality and minimized freeze artifact.

Practical Approaches for Cryo-FIB Milling and Applications for Cellular Cryo-Electron Tomography.

Cryo-electron tomography (cryo-ET) is a powerful technique to examine cellular structures as they exist in situ. However, direct imaging by TEM for cryo-ET is limited to specimens up to ∼400 nm in thickness, narrowing its applicability to areas such as cellular projections or small bacteria and viruses. Cryo-focused ion beam (cryo-FIB) milling has emerged in recent years as a method to generate thin specimens from cellular samples in preparation for cryo-ET. In this technique, specimens are thinned with a beam of gallium ions to gradually ablate cellular material in order to leave a thin, electron-transparent section (a lamella) through the bulk material. The lamella can be used for high-resolution cryo-ET to visualize cells in 3D in a near-native state. This approach has proved to be robust and relatively simple for new users and exhibits minimal sectioning artifacts. In this chapter, we describe a general approach to cryo-FIB milling for users with prior cryo-EM experience, with extensive notes on operation and troubleshooting.

Microdevice to capture colon crypts for in vitro studies.

There is a need in biological research for tools designed to manipulate the environment surrounding microscopic regions of tissue. In the current work, a device for the oriented capture of an important and under-studied tissue, the colon crypt, has been designed and tested. The objective of this work is to create a BioMEMs device for biological assays of living colonic crypts. The end goal will be to subject the polarized tissue to user-controlled fluidic microenvironments in a manner that recapitulates the in vivo state. Crypt surrogates, polymeric structures of similar dimensions and shape to isolated colon crypts, were used in the initial design and testing of the device. Successful capture of crypt surrogates was accomplished on a simple device composed of an array of micron-scale capture sites that enabled individual structures to be captured with high efficiency (92+/-3%) in an ordered and properly oriented fashion. The device was then evaluated using colon crypts isolated from a murine animal model. The capture efficiency attained using the fixed biologic sample was 37+/-5% due to the increased variability of the colon crypts compared with the surrogate structures, yet 94+/-3% of the captured crypts were properly oriented. A simple approach to plug the remaining capture sites in the array was performed using inert glass beads. Blockage of unfilled capture sites is an important feature to establish a chemical gradient across the arrayed crypts. A chemical concentration gradient (Cluminal/Cbasal>10) was demonstrated across the arrayed crypts for over 8 h. Finally unfixed colon crypts were demonstrated to be effectively captured by the micromesh array and to remain viable on the capture sites at 5 h after mouse sacrifice. The present study demonstrates the feasibility and potential for rationally microengineered technologies to address the specific needs of the biologic researcher.

Tracking with virtual slides: a tool to study diagnostic error in histopathology.

AIMS: To determine the reasons for diagnostic error by virtual slides which allow unsupervised study of diagnosis and error. METHODS AND RESULTS: Software was developed to produce visualizations of the diagnostic track followed by pathologists as they viewed virtual slides. These showed the diagnostic path in four dimensions (x, y, time and zoom), areas studied for >1000 ms, and included pathologists' comments about the areas viewed. The system was used to study two trainee and two expert pathologists diagnosing 60 Barrett's oesophageal biopsy specimens. Comparisons of the diagnostic tracks showed the reason for errors. Forty-six cases had an expert consensus diagnosis. The trainees made errors in 21 and 15 cases, respectively, of which 11 and nine were clinically significant. Errors were made across the whole spectrum of diagnoses from negative to intramucosal carcinoma. Detailed examination of the tracks showed that in all errors there was incorrect interpretation of information; in three errors there was an additional failure to identify diagnostic features. CONCLUSIONS: Tracking with virtual slides is a useful tool in studying diagnosis and error, which has the potential for use in training and assessment.

A mechanized device for mounting histological tissue sections.

BACKGROUND: Traditional methods for mounting tissue sections onto slides are suboptimal as the amount of labor required quickly multiplies with increasing number of samples. Methods to accelerate the tissue mounting process while reducing the associated risk of tissue damage are needed. NEW METHOD: We designed and 3D printed a mechanized device with an inclined platform used to mount tissue sections onto slides in buffer solution. The main advantage of this design is to reduce the time required for mounting sections as well as minimize the possibility of damaging delicate or thin tissue sections. RESULTS: Using our device, we illustrate and describe in detail the steps required to mount smaller coronally cut mouse brain sections, as well as bigger tangentially cut ferret brain sections. This method's efficiency was assessed by comparing the time required to mount an entire slide of ferret brain sections using our method and the conventional method. Using our device reduced the tissue mounting time by 60%. COMPARISON WITH EXISTING METHOD(S): Compared to existing conventional tissue mounting methods, our device is a simple and user friendly alternative that substantially reduces the time required to mount tissue sections while preserving tissue section quality. CONCLUSIONS: Using our device can streamline histological processing and prove to be especially useful for a variety of tissue types as the platform was designed to accommodate different size microscope slides, and thus use for varying tissue section sizes.

Ex vivo kidney slice preparations as a model system to study signaling cascades in kidney epithelial cells.

Several model systems have been used to study signaling cascades in kidney epithelial cells, including kidney histology after systemic treatments, ex vivo isolated tubule perfusion, epithelial cell lines in culture, kidney micropuncture, and ex vivo kidney slices. We and others have found the ex vivo kidney slice method useful to study the signaling cascades involved in the regulation of kidney transport proteins. In this chapter we describe our adaptations to this classic method for the study of the regulation of kinases and endocytosis in rodent kidney epithelial cells. Briefly, slices are obtained by sectioning of freshly harvested rat or mouse kidneys using a Stadie-Riggs tissue slicer. Alternatively, a vibratome can be used to obtain slices at a more consistent and finer thickness. The harvested kidney and kidney slices are kept viable in either cell culture media or in buffers that mimic physiological conditions equilibrated with 5% CO2 at body temperature (37°C). These buffers keep the slices viable during hours for incubations in the presence/absence of different pharmacological agents. After the incubation period the slices can be used for biochemistry experiments by preparing tissue lysates or for histological evaluation after fixation. Moreover, the fixed slices can be used to evaluate changes in subcellular trafficking of epithelial proteins or endosomes via immunolabeling followed by confocal microscopy. The resulting micrographs can then be used for systematic quantification of protein- or compartment-specific changes in subcellular localization under each condition.

Isolation and Generation of Osteoblasts.

This chapter describes the isolation, culture, and staining of osteoblasts. The key advantages of this assay are that it allows direct measurement of bone matrix deposition and mineralization, as well as yielding good quantities of osteoblasts at defined stages of differentiation for molecular and histological analysis. An additional focus of this chapter will be the culture of osteoblasts from less conventional animal species.

Histomorphometry in Rodents.

Bone histomorphometry remains an important tool with which to study the pathophysiology of bone disease and the cellular mechanism by which treatments work. Here we review the methods for embedding, sectioning, staining, and analysis of bone sections in rodents.

Transmission Electron Microscopy of Bone.

Electron microscopic analysis of mineralized tissues like bone and dentin is essential for understanding of cell-cell/cell-matrix interactions, and the three-dimensional organization of these tissues. This chapter describes a few methods to process mineralized tissues obtained from different sources for ultrastructural analysis by transmission electron microscopy.

Application of Electron Microscopes in Nanotoxicity Assessment.

In this chapter, we highlight the applications of electron microscopes (EMs) in nanotoxicity assessment. EMs can provide detailed information about the size and morphology of nanomaterials (NMs), their localization in cells and tissues, the nano-bio interactions, as well as the ultrastructural changes induced by NMs exposure. Here, we share with the readers how we prepare the tissue sample, and the different types of EMs used among the nanotoxicologists. It is possible to deploy conventional EMs along or in combination with other analytical techniques, such as electron energy loss spectroscopy (EELS), energy dispersive X-ray spectroscopy (EDS or EDX), and TEM-assisted scanning transmission X-ray microscopy (STXM), toward further elemental and chemical characterization. Appropriate images are inserted to illustrate throughout this chapter.

Analysis of Bone Architecture in Rodents Using Micro-Computed Tomography.

This chapter describes the use of micro-computed tomography scanning for analyzing bone structure, focussing on rodent bone. It discusses sample preparation, the correct setup of the scanner, the impact of some of the important scanner settings and new applications.

Analysis of primary cilia in renal tissue and cells.

Primary cilia are singular, sensory organelles that extend from the plasma membrane of most quiescent mammalian cells. These slender, microtubule-based organelles receive and transduce extracellular cues and regulate signaling pathways. Primary cilia are critical to the development and function of many tissue types, and mutation of ciliary genes causes multi-system disorders, termed ciliopathies. Notably, renal cystic disease is one of the most common clinical features of ciliopathies, highlighting a central role for primary cilia in the kidney. Additionally, acute kidney injury and chronic kidney disease are associated with altered primary cilia lengths on renal epithelial cells, suggesting ciliary dynamics and renal physiology are linked. Here we describe methods to examine primary cilia in kidney tissue and in cultured renal cells. We include immunofluorescence and scanning electron microscopy to determine ciliary localization of proteins and cilia structure. Further, we detail cellular assays to measure cilia assembly and disassembly, which regulate cilia length.

Quantifying autophagic flux in kidney tissue using structured illumination microscopy.

Kidney disease is estimated to affect 15% of the world's population. Autophagy is a key homeostatic pathway in eukaryotic cells, which has been linked to numerous pathological states. In the kidney, autophagy has been shown to modulate both acute and chronic injuries. Despite the importance of autophagy in kidney disease, few techniques to precisely monitor autophagic flux in kidney tissue are available. Here we describe an improved technique to quantify autophagic flux using an RFP-GFP-LC3 reporter mouse and super-resolution microscopy. Using structured illumination microscopy, we can resolve individual autophagosomes within kidney tubular cells. We describe the preparation of slides, staining, imaging and data processing. 3D surface rendering is utilized to categorize and quantify autophagosomes by number, size, fluorescence and autophagic flux in response to ischemia.