Enhancing Specific Fluorescence In Situ Hybridization with Quantum Dots for Single-Molecule RNA Imaging in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma. Owing to its high signal-to-noise ratio, QD split-FISH is a fast, inexpensive, and sensitive method for quantifying mRNA expression in FFPE tumor tissues, making it suitable for biomarker imaging and diagnostic pathology.

A novel case of cutaneous, nasal and systemic fusariosis in a goat.

The clinical findings associated with nasal, cutaneous and systemic fusariosis in a 3-year-old billy Boer goat are summarised. The clinical features, treatment, postmortem findings and laboratory diagnostics are reported and discussed in the context of existing knowledge on mycoses of small ruminants. The goat presented primarily for respiratory signs (inspiratory dyspnoea) with unilateral left-sided mucopurulent nasal discharge, and multifocal variably ulcerative and necrotic cutaneous nodules. Histopathology of nasal and cutaneous biopsies revealed necrotising pyogranulomatous inflammation with intralesional septate hyphal elements that correlated with culture of Fusarium oxysporum. The patient continued to deteriorate clinically during treatment with oxytetracycline and meloxicam, with the addition of sodium iodide and potassium iodide, and was humanely euthanased. Postmortem examination revealed multifocal nodular lesions throughout the kidneys, abdominal lymph nodes and lungs. These lesions were consistent with those identified antemortem from which F. oxysporum was cultured. Although treatment was unsuccessful, to the author's knowledge, no instance of rhinofacial or systemic caprine infection with Fusarium spp. has been documented in the veterinary literature, making this the first recognised instance of this form of infection in small ruminant species.

Carcinoma Cuniculatum of the Maxilla Arising From Oroantral Fistula: A Report of an Extremely Rare Case.

Carcinoma cuniculatum (CC) is extremely rare in the maxilla. Here, we report a case of CC arising from an oroantral fistula (OAF). The patient was a 70-year-old Japanese man who was followed up for a non-closing OAF. Although there were no findings based on an intraoral examination, follow-up contrast-enhanced computed tomography and magnetic resonance imaging showed a 22-mm mass in the maxilla close to the OAF. Histologically, cystic and endophytic papillary proliferation of squamous epithelium with abundant keratinization mimicking rabbit burrows occupied the alveolar bone. This tumor was directly connected to the atypical proliferation of the covering epithelium of the OAF. The tumor cells showed mild cytological atypia and a few mitoses. Finally, the patient was diagnosed with CC arising from an OAF. CC is often misdiagnosed; nonetheless, the unique endophytic, branching, and tunnel-like structure is a hallmark of this tumor. We present the first well-documented case of CC arising from an OAF, discuss its diagnostic features, and highlight its differences from other common benign and malignant pathological entities.

The 5th edition of WHO classification of tumors of endocrine organs: changes in the diagnosis of follicular-derived thyroid carcinoma.

The 5th edition of the World Health Organization (WHO) classification of endocrine tumors was released in 2022. Several novelties have been introduced concerning the nomenclature and histopathological diagnosis of follicular-derived thyroid neoplasms. Tumor types have been sharply classified according to prognostic risk categories into benign tumors, low-risk neoplasms and malignant neoplasms. A grading system for differentiated thyroid carcinomas has been implemented with the aim of improving the stratification of tumors. Particular attention has been paid to the molecular profile of well-differentiated histotypes. In this review, the main changes introduced by the latest edition of the WHO system are presented. The practical effects on the diagnostic pathology of thyroid tumors, along with the clinical implications expected with the new classification scheme, are critically discussed.

The Role of Histopathology in Ruminant Diagnostics.

Histopathology remains an important tool for ruminant disease diagnostic investigations. Some ruminant diseases require histopathology to make a definitive diagnosis. Clinical history, proper tissue sampling and handling, and proper fixation all increase the efficiency of a histopathologic examination and the likelihood of an accurate diagnosis. This article discusses some of the main organ systems of ruminants and highlights common ruminant diseases encountered by diagnosticians where histopathology is particularly important. Where applicable, correlative gross lesions, special considerations regarding tissue sampling, and histologic report interpretation are discussed.

Common and Not-So-Common Pathologic Findings of the Gastrointestinal Tract of Rhesus and Cynomolgus Macaques.

Rhesus and cynomolgus macaques are the most frequently used nonhuman primate (NHP) species for biomedical research and toxicology studies of novel therapeutics. In recent years, there has been a shortage of laboratory macaques due to a variety of competing factors. This was most recently exacerbated by the surge in NHP research required to address the severe acute respiratory syndrome (SARS)-coronavirus 2 pandemic. Continued support of these important studies has required the use of more varied cohorts of macaques, including animals with different origins, increased exposure to naturally occurring pathogens, and a wider age range. Diarrhea and diseases of the gastrointestinal tract are the most frequently occurring spontaneous findings in macaques of all origins and ages. The purpose of this review is to alert pathologists and scientists involved in NHP research to these findings and their impact on animal health and study endpoints, which may otherwise confound the interpretation of data generated using macaques.

An Autopsy Case of Idiopathic Rhabdomyolysis in 1979: Immunoperoxidase Detection of Myoglobin Casts in Formalin-Fixed, Paraffin-Embedded Sections of the Kidney.

In 1979, the author in his younger days experienced an autobiographical case of idiopathic rhabdomyolysis. The heme casts in formalin-fixed, paraffin-embedded sections of the kidney were immunoreactive for myoglobin. In these days, the immunoperoxidase technique had been utilized as a research seed by using paraformaldehyde-fixed frozen sections. The precious experience prompted the young author of his younger days to apply the immunoperoxidase method to diagnostic pathology using formalin-fixed paraffin-embedded sections. A brief history of early development of chromogenic immunostaining in diagnostic pathology in Japan is summarized.

Artificial Intelligence in Digital Pathology: What Is the Future? Part 2: An Investigation on the Insiders.

Motivation: This study deals with the introduction of artificial intelligence (AI) in digital pathology (DP). The study starts from the highlights of a companion paper. Objective: The aim was to investigate the consensus and acceptance of the insiders on this issue. Procedure: An electronic survey based on the standardized package Microsoft Forms (Microsoft, Redmond, WA, USA) was proposed to a sample of biomedical laboratory technicians (149 admitted in the study, 76 males, 73 females, mean age 44.2 years). Results: The survey showed no criticality. It highlighted (a) the good perception of the basic training on both groups, and (b) a uniformly low perceived knowledge of AI (as arisen from the graded questions). Expectations, perceived general impact, perceived changes in the work-flow, and worries clearly emerged in the study. Conclusions: The of AI in DP is an unstoppable process, as well as the increase of the digitalization in the health domain. Stakeholders must not look with suspicion towards AI, which can represent an important resource, but should invest in monitoring and consensus training initiatives based also on electronic surveys.

Morphological Characteristics of Alveolar and Cystic Echinococcosis Lesions in Human Liver and Bone.

Among echinococcoses diseases of human interest, two have a global public health impact: cystic and alveolar echinococcosis caused by Echinococcus granulosus sensu lato and Echinococcus multilocularis, respectively. Cystic and alveolar echinococcosis are neglected infectious diseases epidemiologically and are clinically vastly different with distinct microscopic features. Because of the rareness of these zoonotic diseases, pathologists have limited diagnostic experience in the analysis of the lesions caused by Echinococcus tapeworms. Here, we describe the main microscopic features to be considered to characterize these lesions: laminated layer, central necrosis, growth pattern, and delineation from adjacent tissue. Moreover, immunohistology using monoclonal antibodies is of great diagnostic help in reaching a definitive diagnosis by identifying the laminated body and small particles of E. multilocularis (spems) and small particles of E. granulosus (spegs).

Improving Generalization of Deep Learning Models for Diagnostic Pathology by Increasing Variability in Training Data: Experiments on Osteosarcoma Subtypes.

BACKGROUND: Artificial intelligence has an emerging progress in diagnostic pathology. A large number of studies of applying deep learning models to histopathological images have been published in recent years. While many studies claim high accuracies, they may fall into the pitfalls of overfitting and lack of generalization due to the high variability of the histopathological images. AIMS AND OBJECTS: Use the model training of osteosarcoma as an example to illustrate the pitfalls of overfitting and how the addition of model input variability can help improve model performance. MATERIALS AND METHODS: We use the publicly available osteosarcoma dataset to retrain a previously published classification model for osteosarcoma. We partition the same set of images into the training and testing datasets differently than the original study: the test dataset consists of images from one patient while the training dataset consists images of all other patients. We also show the influence of training data variability on model performance by collecting a minimal dataset of 10 osteosarcoma subtypes as well as benign tissues and benign bone tumors of differentiation. RESULTS: The performance of the re-trained model on the test set using the new partition schema declines dramatically, indicating a lack of model generalization and overfitting. We show the additions of more and moresubtypes into the training data step by step under the same model schema yield a series of coherent models with increasing performances. CONCLUSIONS: In conclusion, we bring forward data preprocessing and collection tactics for histopathological images of high variability to avoid the pitfalls of overfitting and build deep learning models of higher generalization abilities.

Disseminated melanized fungal infection due to Cladosporium halotolerans in a dog coinfected with canine adenovirus-1 and canine parvovirus-2.

This report presents the pathologic findings associated with disseminated infection due to Cladosporium halotolerans in a dog that was simultaneously infected with canine adenovirus-1 (CAdV-1) and canine parvovirus-2 (CPV-2). A 12-year-old, mixed breed dog, with a clinical history of neurological manifestations was submitted for routine autopsy due to poor prognosis. The principal pathologic findings were mycotic necrotizing nephritis, hepatitis, and splenitis with embolic dissemination to the brain resulting in mycotic necrotizing meningoencephalitis, ventriculitis, choroid plexitis, and obstructive hydrocephalus associated with intralesional and intravascular septate pigmented fungi. PCR and sequencing of the ITS region of fungi revealed that the intralesional fungal organisms had 82% nucleotide identity with members of the Cladosporium sphaerospermum complex of organisms. However, a PCR assay and sequencing of the beta tubulin gene confirmed that the organism identified in this dog had 100% nucleotide sequence identity with C. halotolerans. Using immunohistochemistry, intralesional antigens of CAdV-1 were identified within the epithelial cells of the liver and lungs; there was positive immunolabeling for CPV-2 antigens in degenerated cardiomyocytes. These findings confirmed the active participation of C. halotolerans in the development of disseminated cladosporiosis in this dog and represent a rare occurrence of concomitant infection with CAdV-1 and CPV-2.

Practical applications of DNA genotyping in diagnostic pathology.

INTRODUCTION: Among various human tissue identity testing platforms, short tandem repeat (STR) genotyping has emerged as the most powerful and cost-effective method. Beyond forensic applications, tissue identity testing has become increasingly important in modern medical practice, in areas such as diagnostic pathology. Areas covered: A brief overview of various molecular/genetic techniques for identity testing is provided. This includes restriction fragment length polymorphism, single nucleotide polymorphism array and STR genotyping by multiplex PCR. Diagnostic applications of STR genotyping are covered in greater details: genotyping diagnosis of gestational trophoblastic disease, resolving tissue specimen mislabeling or histologic contaminant or 'floaters', bone marrow engraftment/chimerism analysis and interrogation of the primary source of malignancy in patients receiving organ donation. Four clinical cases are then presented to further illustrate these important clinical applications along with discussion of the interpretation, limitations, and pitfalls of STR genotyping. Expert commentary: STR genotyping is currently the most applicable method of identity testing and has extended its role well into the practice of diagnostic pathology with novel and powerful applications beyond forensics.

Pathologic, histochemical, and immunohistochemical findings in pulmonary and encephalitic cryptococcosis in a goat.

We describe the pathologic, histochemical, and immunohistochemical findings associated with pulmonary and encephalitic cryptococcosis in a 3-y-old, mixed-breed, nanny goat from central-west Brazil. The goat had progressive neurologic signs over 30 d; cryptococcosis was diagnosed antemortem by cytologic evaluation of cerebrospinal fluid. Treatment was initiated, but the animal died spontaneously shortly thereafter. Grossly, there was a large space-occupying gelatinous mass (cryptococcoma) in the left lung and smaller masses in the cerebral temporal and frontal cortex, thalamus, hippocampus, basal nuclei, and mesencephalon with consequent internal hydrocephalus. Histologic evaluation revealed marked granulomatous cryptococcal pneumonia and meningoencephalitis. Intralesional narrow-necked budding cryptococcal yeasts were identified on special stains (Mayer mucicarmine and Grocott methenamine silver) in sections of lung and brain. Immunohistochemistry utilizing a panel of monoclonal antibodies that selectively label capsules of Cryptococcus spp. was consistent with C. neoformans var. grubii.

Distinct Histopathological Features at Diagnosis of Very Early Onset Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Children with very early onset inflammatory bowel disease [VEO-IBD] represent a unique cohort, often with a severe phenotype that is refractory to conventional medications, and some cases have underlying primary immunodeficiencies. Previous work has identified distinct histopathological patterns in the gastrointestinal tract in patients with primary immunodeficiencies. The aim of this study is to characterise the diagnostic histological findings in patients with VEO-IBD as compared with older onset paediatric IBD, and determine if there are unique pathological changes that can shed light on the driving forces of the disease, particularly immunodeficiencies. METHODS: Clinical retrospective chart review, including disease characteristics and endoscopic findings, was performed on all included subjects. Two paediatric pathologists reviewed biopsies from diagnostic upper endoscopies and colonoscopies of subjects with very early onset inflammatory bowel disease and older onset inflammatory bowel disease, to evaluate for the presence of 11 histological features previously associated with inflammatory bowel disease and primary immunodeficiencies. RESULTS: The diagnostic gastrointestinal biopsies of subjects with very early onset inflammatory bowel disease differed from those in older onset paediatric IBD, demonstrated by increased frequency of apoptosis, severe chronic architectural changes, small intestine villous blunting, and eosinophils in the crypts, lamina propria, and surface epithelium. CONCLUSIONS: The diagnostic biopsies of children with very early onset inflammatory bowel disease can identify characteristic features that may be important in guiding the diagnostic work-up in this population.

IMMUNOHISTOCHEMICAL AND MOLECULAR EVIDENCE OF PUTATIVE NEORICKETTSIA INFECTION IN COATIS ( NASUA NASUA) FROM SOUTHERN BRAZIL.

The pathologic, molecular, and immunohistochemical findings associated with Neorickettsia helminthoeca are described in coatis ( Nasua nasua). Tissue sections (small intestine, lungs, kidney, liver, and spleen) of coatis ( n = 3) that died at the Bela Vista Biological Refuge, Foz do Iguaçu, Paraná, southern Brazil were routinely processed from histopathology. Selected formalin-fixed paraffin-embedded (FFPE) tissue sections of the small intestine, lungs, and spleen were used in an immunohistochemical (IHC) assay designed to identify the antigens of N. helminthoeca. Additionally, FFPE tissue sections of the small intestine were used to demonstrate antigens of canine parvovirus-2 (CPV-2) by IHC. Histopathology revealed chronic enteritis in all coatis. Parasitic enteritis was diagnosed in two coatis; one of these contained examples of a trematode within the lumen of the small intestine and the ovum of a trematode encysted in the intestinal mucosa. Other significant pathologic findings included interstitial pneumonia ( n = 2) and pyogranulomatous splenitis ( n = 1). Positive immunolabeling for N. helminthoeca was identified within macrophages of the small intestine and reticuloendothelial cells within the germinal centers of the spleen of all coatis; the intestinal trematode was N. helminthoeca IHC-positive. All pulmonary sections revealed negative immunolabeling for N. helminthoeca. Furthermore, the antigens of CPV-2 were not identified in the intestine of any coati. These findings indicate that these coatis were infected by N. helminthoeca, but since clinical and gross pathological findings were not recorded, it is uncertain if this pathogen produced clinical disease in this canid host; therefore, coatis may be asymptomatic or dead-end hosts for this organism.

Nonproliferative and Proliferative Lesions of the Rat and Mouse Endocrine System.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Next-Generation Sequencing in Diagnostic Pathology.

Interrogation of tissue informs on patient management through delivery of a diagnosis together with associated clinically relevant data. The diagnostic pathologist will usually evaluate the morphological appearances of a tissue sample and, occasionally, the pattern of expression of a limited number of biomarkers. Recent developments in sequencing technology mean that DNA and RNA from tissue samples can now be interrogated in great detail. These new technologies, collectively known as next-generation sequencing (NGS), generate huge amounts of data which can be used to support patient management. In order to maximize the utility of tissue interrogation, the molecular data need to be interpreted and integrated with the morphological data. However, in order to interpret the molecular data, the pathologist must understand the utility and the limitations of NGS data. In this review, the principles behind NGS technologies are described. In addition, the caveats in the interpretation of the data are discussed, and a scheme is presented to "classify" the types of data which are generated. Finally, a glossary of new terminology is included to help pathologists become familiar with the lexicon of NGS-derived molecular data.