Journal of Histotechnology最新文献

Histological preparation via paraffin embedding is the gold standard method for evaluating tissue structure and composition, whether it is originated from biopsy or engineered in vitro. Quite often, deformation and shrinkage occur during the histological preparation, which are difficult to predict and qualify. The present study investigates the morphometric changes in bioprinted hydrogels composed of alginate and gelatine, common tissue engineering materials, focusing on three morphologies: full slabs, porous slabs, and porous cubes. These structures underwent key histological steps, including fixation, processing (dehydration, clearing, and infiltration with melted paraffin), embedding, and slicing, to evaluate their shrinkage behavior. Shrinking factors were systematically measured, showing that processing had the most significant effect (34-40% shrinking), followed by fixation (20-28% shrinking). Porous structures exhibited greater shrinkage compared to full slabs due to their internal geometry. Additionally, anisotropic behavior was observed in porous cubes, with different shrinking factors in the XY plane (horizontal) and Z direction (vertical), leading to an overall volumetric shrinking factor of 81.3%. The results demonstrated the critical influence of hydrogel structure on deformation and emphasized the need for tailored histological protocols to maintain structural fidelity. While this study focused on hydrogels alone, future work will incorporate cellularized bioengineered tissues to evaluate the impact of cell-mediated remodeling and extracellular matrix deposition on histological outcomes. This research offers a framework for optimizing histological preparation in bioengineered tissues, enabling more accurate assessment of their structure and function for regenerative medicine applications.

There are several variations of laboratory procedures to preserve and demonstrate monosodium urate (MSU) crystals in tissue for the diagnosis of gout. MSU is water soluble and washed away in most staining solutions, so these procedures generally necessitate two slides; one which is silver stained and one which is left unstained to confirm the negative birefringence of MSU when viewed under polarized light. The modified Gomori's methenamine silver (GMS) for urates is an underutilized procedure which provides sensitive, high-contrast staining of MSU crystals. Additionally, we have observed that MSU deposits are preserved in the stained sections, allowing for examination by polarized light and subsequent diagnosis with only one slide. We have found this modification of the GMS stain to be the most sensitive and efficient method for the diagnosis of gout histologically due to the procedure's relatively short duration as well as the sharp black staining of MSU crystals and preservation of MSU birefringence on a single slide.

The claustrum is a sheet-like layer of gray matter situated between the external and extreme capsules of the mammalian brain. This structure was first described by the French physician and anatomist Vicq d'Azyr in 1786. The claustrum's phylogeny, ontogeny and functional characteristics have long been the subject of debate and considerable investigative efforts. However, despite such efforts (or perhaps as a result thereof), significant disparities and discrepancies remain, most notably in the context of the claustrum's afferent and efferent connections. For the purpose of this study, we sought to focus our efforts on fronto-claustral and occipito-claustral connections. Twelve healthy, adult male cats, each weighing ~ 3.5 kg, were studied, seven of which underwent electrolytic lesions of the frontal cortex (A3, A4, and a portion of A6), and five of the occipital cortex (A17, A18, A21). From three to six days after lesioning, subjects were euthanized in accordance with ethical norms. After the brains were removed and blocked, samples of the claustrum were taken and prepared for electron microscopy. Three to six days after lesions of the frontal cortex, we observed an abundance of degenerative boutons in the dorsal claustrum. The vast majority of boutons exhibited the characteristic signs of dark degeneration, whereas only 10% appeared to have undergone light degeneration. Similar results were seen in the dorsal claustrum over the same period of time following lesions of the visual cortex. These results suggest that the dorsal claustrum receives at least two types of connections - separately and concurrently - from the frontal and occipital cortices.

Due to the prevalence of hematoxylin and eosin (H&E) staining in routine histological preparations, understanding the factors that impact stain color characteristics is vital to attain consistently high-quality stains. In the last decade, increased use of digital pathology and image analysis (specifically by optical density [OD] measures) has provided new ways of assessing staining precision. This paper combines data from two studies that tracked H&E staining quality in both nuclear and cytoplasmic components of 12 tissues (11 human and 1 porcine) by OD after overuse of H&E staining reagents from 5 vendors. Both studies showed a decrease in eosin stain intensity by OD and visual inspection (by a histologist) with reagent overuse. This trend varied in degree by tissue type and reagent vendor. Nonetheless, staining quality for H&E staining from all vendors and for all organs remained acceptable (but not always optimal) for microscopic evaluation by the College of American Pathologists (CAP) and National Society for Histotechnology (NSH) staining criteria when stained sections were reviewed by a board-certified veterinary pathologist.

The application of Clear Gorilla Glue® household adhesive to microscope slides has been found to enhance the mounting and retention of traditionally difficult tissue types, notably bone and tooth specimens. Improvement in end results were observed across H&E staining, immunohistochemistry, and in situ hybridization.

With an increasing concentration of microplastics (MPs) in every biome, laboratories with a focus on creating histology slides from resin-embedded specimens could be partially responsible for expanding the emission of microscopic resinous particles into the environment. With current research elucidating harmful health impacts from MPs, releasing them incautiously is arguably unethical and, in the near future, plausibly illegal. The Orthopedic Bioengineering Research Laboratory (OBRL) is in Colorado, a state known not only for its natural beauty but also for its increasing number of legislative amendments aimed at reducing plastic pollution. The histology department of the OBRL has chosen to self-regulate due to the importance of protecting health and the environment. Because virtually every molecule of plastic ever created is still in existence, a greater need for MP research and mitigation has become apparent. Remediation is specifically important due to findings indicating negative impacts on neurodevelopment, neuronal organelle function, mental health, and the increased risk of dementia.

The retinal pigment epithelium (RPE) is associated with the emergence and development of diabetic retinopathy. Interestingly, a previous clinical study observed that the atrophy of RPE cells surrounding the optic disc in type 1 diabetic children were significantly less pronounced compared to normal children, contradicting current reports. In order to explore the molecular mechanisms behind this contradictory phenomenon, we conducted a series of experiments and reached the following results. First, RPE cells proliferation increased in a glucose concentration-dependent manner in vitro, accompanied by elevated Brachyury and CTGF protein expression, but decreased overall cell viability. Secondly, in vitro experiments and diabetes mouse models confirmed that insulin promoted RPE cell proliferation in high glucose concentrations by activating ERK1/2 phosphorylation. Furthermore, insulin down-regulated the expression of Brachyury and CTGF proteins, possibly reducing high-glucose‒induced damage to RPE cells. In conclusion, the effect of insulin treatment on the proliferation of RPE cells was significantly more significant than that of hyperglycemia, which may be related to the activation of Erk1/2 or reduction of RPE cell damage by inhibiting the occurrence of EMT.

Build-up of static electricity frequently hampers proper production and handling of serial paraffin sections produced on a microtome, a problem that is exacerbated in modern, fully climatized laboratories. In this context, we review a number of suggested remedies for static electricity, concerning increase of humidity, in one way or the other, and various kinds of anti-static devices. An excellent solution, the shockless one-point ionizing bar that we have implemented in our laboratory, albeit not new, is presented here in detail.

Bladder cancer diagnosis is challenged by invasive monitoring and workflow inefficiencies impacting diagnostic reliability. This prospective study enrolled 150 patients (January 2020-December 2022) and evaluated a novel liquid-based immunocytochemistry platform, coupled with integrated machine learning, for detecting urothelial carcinoma in voided urine. All 150 cytology slides met the adequacy threshold of ≥2,644 urothelial cells and showed preserved cytomorphology. Eight cases of papillary urothelial neoplasm of low malignant potential (PUNLMP) were set aside a-priori, yielding an analytic cohort of 142 patients (115 urothelial-carcinoma, 27 benign) for performance analysis. hTERT (sensitivity 92.2%, specificity 66.7%), GATA-3 (67.0%, 88.9%), and CK17 (89.6%, 66.7%). In multi-marker analysis, sensitivity reached 100% (95% CI 96.8-100) when any marker was positive, whereas specificity reached 100% (95% CI 87.3-100) when all three markers were positive. The workflow-optimized platform standardizes specimen preparation and multi-marker interpretation, offering a robust foundation for urine-based bladder-cancer diagnostics. Larger, multi-center validation studies are warranted to refine specificity estimates and facilitate laboratory integration. This study demonstrates that addressing fundamental workflow challenges in bladder cancer diagnostics before implementing artificial intelligence creates more effective diagnostic tools. By prioritizing specimen integrity and standardization through a novel liquid immunocytochemistry platform, exceptional diagnostic performance was achieved with 100% sensitivity and specificity under defined marker parameters across various cancer stages. This workflow-first approach to integrating machine learning with advanced biomarker analysis offers a model for developing clinically practical diagnostic innovations that can reduce reliance on invasive monitoring procedures while improving detection accuracy.

Formalin-fixed paraffin embedded tissues are a mainstay in contemporary histology and pathology practice and represent the most readily available and widely used sample format that has guided early studies and set standards for a new field of spatial biology. However, fixed tissues are not always suitable for all molecular applications and detection of certain molecular targets can be suppressed by these practices. There is a greater diversity of analysis techniques that are commercially available to researchers today, and pathologists and core services are now engaging with groups that prefer fresh-frozen samples more than ever before. Some of these analysis techniques can be characterized as 'label-free' and access unique classes of molecular targets in situ. Our team has developed tools and procedures to facilitate a synergy between the realm of label-free spatial biology and the histology laboratory for biomedical discovery programs. Herein, we discuss the development of 3D printed cassette holders for use in a variety of commercially available cryostats. This apparatus was tested using fresh frozen tissue slices embedded in hydroxypropyl methylcellulose polyvinylpyrrolidone for subsequent analyses. Based on our tests, we propose this protocol as an option to better preserve sample history during the lifetime of a specimen and curtail the decomposition of any lower abundance endogenous chemical moieties for spatial 'omics' analysis using mass spectrometry.