Histochemical Staining Methods Research Articles

Preparation and Sectioning of Paraffin-Embedded Tissue for Histology and Histochemistry.

Cutting thin sections of plant tissues enables observation of internal structures and inner cell layers, and it facilitates various histochemical staining methods. Paraffin embedding gives the tissues a uniform stiffness that is sufficient for sectioning with a microtome. Here, we describe the procedures for preparation of paraffin-embedded tissues and the techniques for sectioning with a microtome. This method is applicable to various types of tissues, including rice shoot apex and immature inflorescence.

Сharacterization of the immune microenvironment's cellular composition and its influence on gene expression during metaplastic changes of the gastric mucosa epithelium

Rationale. Intestinal metaplasia of gastric mucosa epithelium in chronic atrophic gastritis is considered by most authors as a precancerous condition, but it is potentially reversible. The study of the regulation mechanisms of metaplastic epithelial changes development may become the key in understanding the process of carcinogenesis and prevention of cancer development. Objective. To determine whether there is a relationship between microenvironment and development of metaplasia of gastric mucosa epithelium of patients with chronic atrophic gastritis by assessing gene expression and cellular composition of immune infiltrate. Materials and Methods. A retrospective cohort study, the alternative hypothesis of which is that the composition of the gastric mucosa immune microenvironment differs between cases with and without metaplastic changes in the epithelium. Biopsy specimens of the mucosa (n = 38) obtained during endoscopic examination from five sites (2 from the antrum, 2 from the body, 1 from the corner) of the stomach of patients with chronic atrophic gastritis of unspecified etiology, as well as the results of RNA sequencing isolated from biopsy specimens of patients with chronic gastritis obtained from the NCBI open database (n = 12) served as the material for the study. Histological, histochemical staining methods, immunohistochemical study were applied during the study. Morphometric, statistical and bioinformatic analyses were performed. Results. It was found that the proportion of macrophages, T-cytotoxic lymphocytes and plasmocytes was increased in the samples with metaplastic changes of the gastric mucosa epithelium. A correlation between T-cytotoxic lymphocytes and the chance of metaplasia development was found. It was found that changes in the number of T-helper cells, T-cytotoxic lymphocytes, T-regulatory cells and plasmocytes are associated with changes in the expression of five genes most specific for intestinal-type epithelium. Conclusion. The significant difference in the composition of the immune microenvironment between samples with and without metaplastic changes in the mucosal epithelium indicates the potential influence of immune cells on the development of metaplasia and progression of the pathological process along the Correa cascade. One of the mechanisms of regulation of metaplasia development by microenvironment may be their influence on gene expression as an epigenetic factor.

Current state of in situ gene expression studiesin animal tissues

Morphological studies of farm animals are most often conducted using simple preparation and staining techniques. The study of the processes of embryogenesis, postembryonic features of the development of organs and tissues, as well as the effect of various substances remains to be elucidated not only using histochemical and immunohistochemical staining methods, but also using RNA in situ hybridization and transcriptome in situ sequencing. Aspects of many cellular and tissue processes for cattle, pigs and chickens in the context of comparative physiology have not yet been studied. The high productivity of farm animals is associated with the intensive functioning of all organs and systems of the body. The influence of the agricultural management of farm animals and its consequences on the development of an organism in ontogeny deserve a separate area of research from the point of in situ gene expression. Despite rapid development of transcriptome sequencing technologies, which result in the discovery of new candidate genes for many processes, RNA in situ hybridization remains the gold standard for their validation. This review briefly presents modern techniques and their modifications for studying in situ gene expression. Transcriptome studies that have been implemented in cattle, pigs and chickens as model organisms include: in situ RNA hybridization using ZZ-probes, tyramide signal amplification, hybridization chain reaction, digoxigenin-labeled probes, RT-PCR, single cell transcriptome sequencing, in situ transciptome sequencing. This paper is a review of the results of studies on cattle, pigs and chickens. The results of research in this area are relevant for understanding the features of adaptation mechanisms at the transcriptomic level in highly productive animals under industrial conditions in order to search for new markers of valuable agricultural traits. It should be noted that in literature there are very few studies using RNA in situ hybridization, despite the availability and simplicity of the method.

Advancing tomato crop protection: Green leaf volatile-mediated defense mechanisms against Nesidiocoris tenuis plant damage

Although Nesidiocoris tenuis is highly effective as a biological control agent, it can also damage tomato plants due to its zoophytophagous behavior. When N. tenuis pierces the stems and petioles of tomato plants with its stylets, it triggers callose deposition and subsequent cell death, resulting in blocked nutrient transport, floral abortions, or wilting of tender shoots. Recently, it has been shown that exposure of tomato plants to the green leaf volatile (Z)-3-hexenyl propanoate [(Z)-3-HP] activates defensive mechanisms, including the regulation of genes involved in the synthesis and degradation of callose. In this study, conducted under greenhouse conditions, we tested the hypothesis that damage caused by N. tenuis could be reduced by exposing tomato plants to (Z)-3-HP through polymeric dispensers. Tomato plants exposed to (Z)-3-HP and non-exposed control plants were inoculated with N. tenuis. Nesidiocoris tenuis established in both groups with no significant differences between the two treatments. However, as hypothesized, the damage caused by N. tenuis was significantly lower in the plants exposed to (Z)-3-HP. Gene expression analysis of salicylic, jasmonic, and abscisic acids, along with histochemical staining methods, was used to compare the defensive responses of tomato plants infested solely with N. tenuis versus those infested with N. tenuis and exposed to (Z)-3-HP. Our findings confirm the influence of (Z)-3-HP exposure on differential defensive activation between treatments and reduced callose deposition in (Z)-3-HP-exposed plants. These results pave the way for improved management of N. tenuis by enhancing the plant's defenses based on inter-plant communication.

Особенности выявления костных включений в мясных изделиях микроструктурными методами.

Mechanically separated poultry meat (MSPM), being a valuable food raw material, is widely used in the meat industry. Therefore, its detection in finished products is an urgent task in controlling their quality and composition. The article presents the results of the application of histochemical staining methods to identify bone fragments as the main indicator of the presence of MSPM in meat products. To ensure accuracy and first of all, comparability of results of histological investigations, it is important that all laboratories adhere to the principles of the microscopic laboratory practice and use the uniform methodology for preparation of histological specimens and interpretation of obtained results.

Investigation of Histological and Histochemical Features of Mouse Placenta in Different Periods of Pregnancy

This study aimed to investigate the histological and histochemical properties of mouse placenta samples from different periods of pregnancy. For this purpose, mouse placenta samples were collected on the fourth, tenth, and seventeenth days of pregnancy and blocked in paraffin. Serial sections of 5μ thickness were taken at 50μ intervals. Histological and histochemical staining methods were applied to the sections. As a result, histological and histochemical characteristics of mouse placenta from different periods of pregnancy were determined.

Phenotypic and molecular characterisation of Xanthomonas axonopodis pv. punicae from pomegranate leaves

The present study has sought to provide insight into the blight-inciting phytopathogen Xanthomonas axonopodis pv. punicae, causing a devasting outbreak in pomegranate cultivation. Yellow-coloured circular with glistening featured colonies of five isolates of X. axonopodis pv. punicae have been phenotypically characterized and the identity of the isolates was confirmed by specific gyrB and 16S rRNA gene sequences. Results of BLAST analysis of PBL1 and PBL2 showed 99.64% and 99.76% identity with X. axonopodis pv. punicae strain 1(GenBank: KY354098.1) and strain 15 IARI (GenBank: JN036628.1) respectively. Further phylogenetic analysis revealed the strong association of X. axonopodis pv. punicae isolates with other Xanthomonas pathovars such as X. phaseoli pv. manihotis, X. citri pv. punicae and X. citri pv. mangiferaeindicae. An attempt was made to find the optimal growth media for bacterial proliferation and it was evident that NGA served as an excellent medium to support the multiplication of each isolate with a significant number of colonies (P < 0.05). The medium order per the best CFU/ml was NGA > YDCA > YGCA > NSA. The drug susceptibility test gained a lot of attention since most plant pathogens acquired resistance to antibiotics. Both streptomycin and amoxicillin could act as potentially effective drugs in inhibiting the growth of X. axonopodis pv. punicae (P 0.05). The H2O2 accumulation during the pathogen attack in the infected leaves was located by an endogenous peroxidase-dependent in situ histochemical staining method. Brown precipitate visible at a different colour intensity demonstrated the efficiency of PAMPs to trigger plant immunity to produce H2O2. The present study was the first report on the isolation and characterization of X. axonopodis pv. punicae isolated from blight-infected Pomegranate leaves from the mentioned regions of Tamil Nadu, India.

Cloning and functional analysis of the DXR gene and promoter region in Osmanthus fragrans var. semperflorens.

The precise biological function and activity of the deoxylulose-5-phosphate reductoisomerase (DXR) gene and its promoter in Osmanthus fragrans var. semperflorens remain unclear, even though OfDXR is known as the crucial enzyme involved in plant terpenoid synthesis. This study aimed to shed light on the role and activity of the OfDXR gene and its promoter in O. fragrans var. semperflorens by employing RACE-PCR and Hi-TAIL-PCR techniques for the cloning of the gene and promoter sequence from the petal tissue. Subsequently, genetic transformation and histochemical staining methods were utilized to analyze their function and activity. The OfDXR gene exhibited a DNA sequence length of 5241 bp, encompassing 12 exons and 11 introns. The corresponding cDNA sequence of the OfDXR gene was 1629 bp, encoding 474 amino acid residues. Expression analysis revealed that the OfDXR gene was predominantly active in the petals during the early full blooming stage. Overexpression of the OfDXR gene in Arabidopsis plants at the primary or full blooming stage led to an augmentation in the total terpenoid content. Furthermore, the promoter sequence of the OfDXR gene spanned a length of 1174 bp and contained conserved regulatory/response elements, demonstrating functional activity. These findings indicate that the OfDXR gene plays a pivotal role in terpenoid synthesis, while its promoter exhibits robust activity.

Пролиферативный потенциал жировой ткани при сахарном диабете 2 типа и суперинвазионном описторхозе

INTRODUCTION: The article presents the results of studying the proliferative activity of adipocytes and of neoangiogenesis in adipose tissue in healthy individuals, in those who died with type 2 diabetes mellitus and with superinvasive opisthorchiasis. The parasite is the original factor that triggers productive processes due to the mutation of numerous proliferative genes. Stem cells are initiated with their subsequent differentiation along the lines of various differons. AIM: to identify proliferative processes and processes of neoangiogenesis of adipose tissue in type 2 diabetes mellitus and superinvasive opisthorchiasis. MATERIALS AND METHODS: sectional material (n = 58): group 1 — healthy individuals (n = 10), group 2 — deceased with type 2 diabetes mellitus from accidental causes (n = 22), group 3 — persons with superinvasive opisthorchiasis died by violence, with duration of invasion more than 10 years (n = 26). The material was subjected to routine histological examination, including histochemical and immunohistochemical staining methods. Further, microscopy was carried out with subsequent statistical analysis. RESULTS: reduction of the proliferative activity of adipose tissue cells in type 2 diabetes mellitus and increase in the parameters in opisthorchiasis invasion compared with healthy individuals were revealed; in diabetes mellitus, the hypertrophic variant of adipose tissue prevailed, the volumetric density of blood vessels decreased, while in patients with superinvasive opisthorchiasis, lipolysis and angiogenesis increased. CONCLUSION: predictors of type 2 diabetes mellitus were assumed to be low proliferative activity of adipocytes, low volumetric density of blood vessels and hypertrophic variant of adipose tissue. With high mitotic activity and hyperplastic variant of adipose tissue, like in superinvasive opisthorchiasis, a protective effect is formed, which can be considered in the context of a predictor factor of type 2 diabetes mellitus.

The sawfish (Rhinopristiformes, Pristidae) rostrum displayed in the “Basilica Santuario del Carmine Maggiore” in Naples, Italy: A long story of legends and taxonomic errors

Abstract Although historically widespread in most of the shallow and warm waters of the world, the presence of sawfishes in the Mediterranean Sea is still a debated question. For some authors, they never inhabited this basin except as vagrants, while for other authors both Pristis pristis and Pristis pectinata were present in the Mediterranean Sea but were extirpated in the 1960s–1970s. The sawfish rostrum kept in the Basilica Santuario del Carmine Maggiore (Naples, Italy), and considered by some authors the first record of sawfish in the Mediterranean was studied using a combination of morphological, genetic, radiocarbon dating and histochemical staining methods to validate the taxonomic identification, estimate its age and assess its geographical origin. Results indicate that the rostrum does not belong to P. pristis as reported by previous authors, but instead possesses morphological and genetic characters typical of P. pectinata. In addition, the radiocarbon age shows that the rostrum is more recent than previously believed, dating it back to the mid-nineteenth century, and genetic and meristic results cast doubt on its presumed Mediterranean origin. This study demonstrates that historical records should always be critically evaluated before using them to draw any far-reaching conclusion about species’ past ecology and/or biogeography, and that future studies using historical information and specimens should adopt an integrative taxonomy approach similar to the one used here.

Histochemical effects of brodifacoum on rat spleen

In this study, the histochemical effects of Brodifacoum, an anticoagulant used against rodents, on the spleen are examined under a light microscope using CD4 and CD8 histochemical staining methods. A single dose of 0.2 mg Brodifacoum was dissolved in Dimethyl Sulfoxide (DMSO) and was given orally to mature male rats. Spleen samples were collected under ether anesthesia after 24 h, 72 h, 14 days, and 30 days from the rats in the experimental groups and after 14 days from the rats in the control group. In this light microscope study, it was observed that the capsule, white pulp, and red pulp zones in the rat spleen were constructed normally and as their natural structures primary and secondary follicles (germinal center) they were few, and CD4 and CD8 lymphocytes were spherically structured. In the 24 h spleens of the rats, the diameters of germinal centers were expanded and deterioration of the structure of CD4 and CD8 cells was observed. Related to the increase in time (72 h and 14 days) it was determined that primary follicles increased in number and the diameters of germinal centers expanded. In addition to this, after30 days, the rate of CD4:CD8 of the brodifacoum applied rat spleens were approximately the rate of the control group, and the improvement of the structures of the cells was reported as an effect of regeneration. As a result of this study, it was found that Brodifacoum caused immunohistochemical abnormalities in the rat spleen, affected the morphological structure of CD4 and CD8 T lymphocytes and created an immune response in rats. It is thought that the obtained results will be a source for the studies on Brodifacoum.

Three-dimensional in vitro maturation of rabbit oocytes enriched with sheep decellularized greater omentum.

BackgroundTo prevent ovarian hyperstimulation syndrome, in vitro maturation (IVM) allows the oocytes for infertility treatment without hormone therapy. Although many oocytes matured during IVM, some deficiencies in the culture conditions lead to inhibition of the growth and development of the cumulus cells and the oocyte nuclear and cytoplasmic maturation.ObjectivesThe challenge of improving the oocyte culture conditions prompted us to use greater omentum (GOM), full of growth factors and proteins, as a rich supplement to the base culture medium.MethodsCumulus‐oocyte complexes were recovered from rabbits and divided into 3D and 2D conditions cultured for 12 and 24 h. In 3D cultures, the oocytes embedded in alginate containing FBS decellularized GOM. Corresponding supplements were also added in 2D conditions—maturation of the oocytes evaluated by Aceto‐Orcein, TEM, and RT‐PCR for MAP2K1 and Cdk2.ResultsDNA quantification, Hoechst, and H&E staining confirmed cell depletion from GOM, and SEM showed the preservation of ultra‐architecture after decellularization. Histochemical staining methods showed appropriate extracellular matrix preservation. ELISA assessment showed retention of VEGF content. MTT assessment indicated decellularized GOM was non‐toxic. Both Aceto‐Orcein assessment and ultra‐structure study of the oocytes showed that supplementation of 2D or 3D cultures with decellularized omentum promoted oocyte maturation. Expression of MAP2K1 and Cdk2 also increased in the presence of GOM.ConclusionsGOM supplementation has a beneficial impact on oocyte maturation, probably due to the presence of growth factors and proteins.

TOPOGRAPHIC RELATIONSHIPS OF MORPHOLOGICALLY HETEROGENEOUS ORGANS IN REACTIONS TO DEPLETED URANIUM

Annotation: Researches of the effect of radionuclides on the human body remain relevant up to date. Their effect of penetrating the body is characterized by involvement of numerous systems of human body. The causal significance of the effects of depleted uranium exposure has not been specifically defined, however, people who live in zones of armed conflict, as well as armed forces personnel, had health problems of "unknown origin", and with remote symptomatic etiology. The risk of possible exposure is also may appear when manufacture of nose spikes for armor piercers and atomic energetics products. In view of these perspectives, depleted uranium, which used in various types of weapons, has been considered as the cause of complaints. Acute uranium intoxication is appeared in polytropic effect that is apparently depended on the resistance of the body. Getting into the internal milieu, depleted uranium exhibits radiation effect of exposure followed by alpha particles radiation. Long-term consequences after oral incorporation of depleted uranium with drinking water are matters of particular interest. The study purpose is defined by assessing of morphofunctional state of the trachea and thyroid gland, united by topographic proximity and different by morphological heterogeneity, upon one month after a single oral incorporation of an aqueous solution of depleted uranium oxides. General histological and histochemical staining methods on thin paraffin slices have been performed for studying on morphological and functional state of the trachea and thyroid gland The results of the morphological and statistical analysis of the trachea wall upon staining with hematoxylin and eosin revealed changes in the structural components that determined deformation of its functionality a month after oral incorporation of an aqueous solution of depleted uranium oxides. Morphological diagnosis was characterized by a change thickness of the epithelial layer, its multi-rowed structure, the cellular composition of the epithelium, and the loss of special organelles. Modifying effect of depleted uranium is also extended to the glandular epithelium with exposure of secretory segments hypertrophy and excretory ducts. There was revealed the diffuse lymphocyte infiltration of the tracheal membranes and formation of lymph nodules. Histochemical analysis of hormone formation in the thyroid gland has demonstrated a change in the ratio of follicles according to the degree of iodization of colloid amino acids with accurate predomination of non-iodized amino acids of irregular shape with accurate smaller diameter against target parameters, with flat or low cubic epithelium, indicating a decrease of gland functionality. The bioeffect of depleted uranium, independently from organ implementation and tissue organization, was prolonged indicating a functional inability.

In Vivo Biocompatibility Investigation of an Injectable Calcium Carbonate (Vaterite) as a Bone Substitute including Compositional Analysis via SEM-EDX Technology.

Injectable bone substitutes (IBS) are increasingly being used in the fields of orthopedics and maxillofacial/oral surgery. The rheological properties of IBS allow for proper and less invasive filling of bony defects. Vaterite is the most unstable crystalline polymorph of calcium carbonate and is known to be able to transform into hydroxyapatite upon contact with an organic fluid (e.g., interstitial body fluid). Two different concentrations of hydrogels based on poly(ethylene glycol)-acetal-dimethacrylat (PEG-a-DMA), i.e., 8% (w/v) (VH-A) or 10% (w/v) (VH-B), were combined with vaterite nanoparticles and implanted in subcutaneous pockets of BALB/c mice for 15 and 30 days. Explants were prepared for histochemical staining and immunohistochemical detection methods to determine macrophage polarization, and energy-dispersive X-ray analysis (EDX) to analyze elemental composition was used for the analysis. The histopathological analysis revealed a comparable moderate tissue reaction to the hydrogels mainly involving macrophages. Moreover, the hydrogels underwent a slow cellular infiltration, revealing a different degradation behavior compared to other IBS. The immunohistochemical detection showed that M1 macrophages were mainly found at the material surfaces being involved in the cell-mediated degradation and tissue integration, while M2 macrophages were predominantly found within the reactive connective tissue. Furthermore, the histomorphometrical analysis revealed balanced numbers of pro- and anti-inflammatory macrophages, demonstrating that both hydrogels are favorable materials for bone tissue regeneration. Finally, the EDX analysis showed a stepwise transformation of the vaterite particle into hydroxyapatite. Overall, the results of the present study demonstrate that hydrogels including nano-vaterite particles are biocompatible and suitable for bone tissue regeneration applications.

Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE.

Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.

RETRACTED ARTICLE: Exosome-delivered BMP-2 and polyaspartic acid promotes tendon bone healing in rotator cuff tear via Smad/RUNX2 signaling pathway

Statement of Retraction Lei Han, Hong Liu, Huajun Fu, Yugen Hu, Weili Fang & Junsheng Liu - 2022, Exosome-delivered BMP-2 and polyaspartic acid promotes tendon bone healing in rotator cuff tear via Smad/RUNX2 signaling pathway, Bioengineered 13(1), doi: 10.1080/21655979.2021.2019871 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Experimental study on the proliferating activity and differentiation of renal stem cells in superinvasive opisthorchiasis

AIM: This study aimed to identify the replication potential of the kidneys in different forms of opisthorchiasis in laboratory animals.&#x0D; MATERIALS AND METHODS: An experiment was conducted on 60 Syrian male hamsters. The first group was set as the control (n = 10), the second group (n = 25) was infected with metacercariae (Opisthorchis felineus), and the third group (n = 25) was a model of a superinvasive form of opisthorchiasis infection with 50 O. felineus larvae and repeated infection with 50 metacercariae in 14 and 25 days. The hamsters were withdrawn from the experiment on days 7, 15, and 30 via an overdose of narcosis and decapitation. The kidneys were isolated and histologically examined through histochemical and immunohistochemical staining methods. Microscopy was conducted, and results were statistically analyzed.&#x0D; RESULTS: The quantitative characteristics, proliferation tendencies, and differentiation of regional stem cells were identified. In the cortical and medullary substance of the kidneys, CD117, Oct4, and CD34 markers were expressed, and CD31-positive stem cells further differentiated to progenitor cells. Epithelial structures developed in the form of tubules. In the glomeruli, vasculogenesis occurred, and the number of vascular loops increased.&#x0D; CONCLUSION: O. felineus secretome initiated the activation of stem cells in the renal tubules and pericytes of a microcirculatory network. The transitional epithelium of the renal pelvis and the initial parts of the ureter proliferated. Under the action of the secretome of parasites, stem cells proliferated directly in glomerular loops.

Histochemical Techniques in Plant Science: More Than Meets the Eye

Histochemistry is an essential analytical tool interfacing extensively with plant science. The literature is indeed constellated with examples showing its use to decipher specific physiological and developmental processes, as well as to study plant cell structures. Plant cell structures are translucent unless they are stained. Histochemistry allows the identification and localization, at the cellular level, of biomolecules and organelles in different types of cells and tissues, based on the use of specific staining reactions and imaging. Histochemical techniques are also widely used for the in vivo localization of promoters in specific tissues, as well as to identify specific cell wall components such as lignin and polysaccharides. Histochemistry also enables the study of plant reactions to environmental constraints, e.g. the production of reactive oxygen species (ROS) can be traced by applying histochemical staining techniques. The possibility of detecting ROS and localizing them at the cellular level is vital in establishing the mechanisms involved in the sensitivity and tolerance to different stress conditions in plants. This review comprehensively highlights the additional value of histochemistry as a complementary technique to high-throughput approaches for the study of the plant response to environmental constraints. Moreover, here we have provided an extensive survey of the available plant histochemical staining methods used for the localization of metals, minerals, secondary metabolites, cell wall components, and the detection of ROS production in plant cells. The use of recent technological advances like CRISPR/Cas9-based genome-editing for histological application is also addressed. This review also surveys the available literature data on histochemical techniques used to study the response of plants to abiotic stresses and to identify the effects at the tissue and cell levels.

In Silico Multi-Compartment Detection Based on Multiplex Immunohistochemical Staining in Renal Pathology.

With the rapid advancement in multiplex tissue staining, computer hardware, and machine learning, computationally-based tools are becoming indispensable for the evaluation of digital histopathology. Historically, standard histochemical staining methods such as hematoxylin and eosin, periodic acid-Schiff, and trichrome have been the gold standard for microscopic tissue evaluation by pathologists, and therefore brightfield microscopy images derived from such stains are primarily used for developing computational pathology tools. However, these histochemical stains are nonspecific in terms of highlighting structures and cell types. In contrast, immunohistochemical stains use antibodies to specifically detect and quantify proteins, which can be used to specifically highlight structures and cell types of interest. Traditionally, such immunofluorescence-based methods are only able to simultaneously stain a limited number of target proteins/antigens, typically up to three channels. Fluorescence-based multiplex immunohistochemistry (mIHC) is a new technology that enables simultaneous localization and quantification of numerous proteins/antigens, allowing for the possibility to detect a wide range of histologic structures and cell types within tissue. However, this method is limited by cost, specialized equipment, technical expertise, and time. In this study, we implemented a deep learning-based pipeline to synthetically generate in silico mIHC images from brightfield images of tissue slides-stained with routinely used histochemical stains, in particular PAS. Our tool was trained using fluorescence-based mIHC images as the ground-truth. The proposed pipeline offers high contrast detection of structures in brightfield imaged tissue sections stained with standard histochemical stains. We demonstrate the performance of our pipeline by computationally detecting multiple compartments in kidney biopsies, including cell nuclei, collagen/fibrosis, distal tubules, proximal tubules, endothelial cells, and leukocytes, from PAS-stained tissue sections. Our work can be extended for other histologic structures and tissue types and can be used as a basis for future automated annotation of histologic structures and cell types without the added cost of actually generating mIHC slides.

The spectrum of pathogens in 187 cases of pulmonary fungal disease diagnosed by histopathology-a retrospective analysis

Objective: To investigate the spectrum of pathogens causing lung fungal disease diagnosed by histopathology through histochemical special staining, compared to the fungal culture results, and to further evaluate the diagnostic value of histochemical special staining in pulmonary fungal disease. Methods: We performed a retrospective analysis of 187 cases of pulmonary fungal disease diagnosed by histopathology in Peking Union Medical College Hospital from 2001 to 2015 (including 92 cases with pulmonary resection or open lung biopsy, 33 with percutaneous lung biopsy and 62 ones with fiberoptic bronchoscopic lung biopsy). All cases were treated with hexamine silver, PAS, mucus carmine and acid-fast staining in addition to conventional HE staining. The clinical records and the fungal culture results were reviewed. Results: There were 103 male and 84 female patients, aged from 12 to 70 years [average (48±14) years]. There were 85 cases(45.5%) of pulmonary aspergillosis(including 60 cases of invasive infection and 25 cases of aspergilloma), 51 cases(27.3%) of pulmonary cryptococosis, 6 cases (3.2%)of pulmonary mucormycosis, 3 cases(1.6%) of pulmonary histoplasmosis, 3 cases (1.6%)of pulmonary candidiasis, and 2 cases (1.1%) of pneumocystosis, while in the remaining 37 cases (19.8%) the pathogens could not be clearly classified by microscopy due to limited tissue or degeneration. Among the 88 patients with pulmonary fungal disease diagnosed by histopathology from 2011 to 2015, 35 ones (39.9%) were detected by fungal culture (including lung biopsy, intraoperative swab, blood, bronchoalveolar lavage fluid and sputum, etc.). The diagnostic results of 18 cases were completely consistent between histopathological examination and fungal culture (18/35, 51.4%), while 13 cases (13/35, 37.1%) were diagnosed by histopathology but no fungi were cultured, and in 3 cases (3/35,8.6%) the culture was positive for fungi which could not be classified clearly by histopathology. In another case the pathogen was found to be Cryptococcus histopathologically but the lavage culture grew"candida", but the patient's blood cryptococcal antigen was positive. Conclusions: Among patients with histopathological diagnosis of pulmonary fungal disease, pulmonary aspergillosis was the most common, followed by pulmonary cryptococcosis, pulmonary mucormycosis, pulmonary histoplasmosis, pulmonary candidiasis and pneumocystosis. A small number of cases could not be classified by histopathology through histochemical special staining. There was a high consistency in discovering fungal pathogens between pathological histochemical special staining and culture method, but 37% pulmonary fungal disease diagnosed by histopathology were culture negative. In practice, the role of histochemical special staining in diagnosing pulmonary fungal disease should be paid more attention.