Search

Of all the histopathological forms, papillary thyroid carcinoma (PTC) is the most common histological subtype of thyroid malignancy, representing about 80-90% of all thyroid malignancies. Until now, histopathological studies have described a number of variants of papillary thyroid carcinoma: classic, follicular, solid, encapsulated, diffuse sclerosing, tall-cell, micro papillary, etc. A total of one hundred fifty two cases of thyroidectomy were done during the study period of which forty five cases were thyroid neoplasms. Detailed clinical history and investigations related to the case were obtained and noted. The specimens were fixed in 10% formalin, grossed according to the standard grossing protocol and then processed by paraffin embedding. Sections were cut serially at a thickness of 4-5 microns. Multiple sections were taken from each case and routine Haematoxylin and Eosin staining was done. The findings were noted in proforma. Of the 34 PTC classic and its variants, PTC classic were 16 (47.1%) and variants were 18 (52.9%). Out of 18 PTC variants, FVPTC were 12 (66.6%), EFVPTC and micro papillary carcinoma were 3 (16.7%) each. [ABSTRACT FROM AUTHOR]

Introduction: Menorrhagia is one of the commonest clinical conditions for which patients seek advice in the gynecological outpatient department. Aim and objectives of study: To determine the prevalence of various histopathological changes, patterns and lesions of endometrium associated with menorrhagia in the women of different age groups; to describe the morphological features of various lesions of endometrium; to assess demographic pattern of various types of endometrium lesions in this part of our country and to do clinicopathological correlation and give diagnosis based on it. Materials and methods: This was a prospective, non-interventional and observational hospital based crosssectional one year study carried out between January 2020 to June 2021 in Histopathology Department of Dhiraj Hospital on 52 cases of D & C and hysterectomy specimens. All the specimens were fixed in 10% formalin were received in the department of Pathology and processed and embedded in paraffin wax. Multiple serial sections of 4-5 microns thickness were obtained from the paraffin block and then stained with H & E. Detailed study of the sections was done by light microscopy. Results: 52 cases of menorrhagia were studied clinically and histopathologically. In present study, large proportion of patients with menorrhagia showed proliferative endometrial pattern (38.46%) followed by secretory phase (34.62%). Hyperplastic pattern was seen in 13.46% of patients. Non hyperplastic endometrial pattern was more common than hyperplastic pattern. Simple hyperplasia was more commonly seen than complex and atypical hyperplasia. Overall incidence of atypical hyperplasia was low (28.57%) and atrophic endometrium seen in 5.7% of cases. Endometrial polyp was seen in 3.84% cases and endometrial carcinoma was seen in 3.84% cases. Conclusion: Histopathological study of endometrium is very useful to reveal different endometrial patterns in menorrhagia and to distinguish between anovulatory and ovulatory causes. It is also useful for the diagnosis of endometrial hyperplasia and endometrial carcinoma. [ABSTRACT FROM AUTHOR]

Introduction: An ideal clearing agent should remove alcohol from the tissue specimens to enable the penetration of paraffin wax which provides sturdiness to the tissue specimen. Xylene has toxic effects and is still used because of its accurate clearing property and maintaining the staining quality. Natural alternatives to xylene provide a less toxic environment for histopathological technicians. Aim: The study is to evaluate the accuracy of clarified butter as xylene substitute in routine histopathological tissue processing procedure. Materials and Methods: 40 paired soft tissue specimens were obtained from the Department of Oral pathology and microbiology and subjected to routine histopathological tissue processing with xylene and ghee as clearing agents. Post processing, the tissue specimens were immersed in paraffin wax overnight, embedded, sectioned and stained using hematoxylin and eosin. Histopathological criteria namely nuclear staining, cytoplasmic staining, artefacts and background staining were evaluated and the scores were tabulated and statistical analysis was done using SPSS version 20 by IBM. Results: Our study results showed that tissue specimens processed in xylene and ghee had similar nuclear staining. In cytoplasmic staining, tissue specimens processed in ghee had superior results than xylene processing. Artefacts were present more in xylene processing than ghee processing. Background staining was more appreciated in xylene processed tissue specimens than ghee processing. Conclusion: Substituting xylene with ghee showed better results as a clearing agent in routine histopathological tissue processing. Ghee as a clearing agent is non toxic, cost effective compared to xylene and a safer alternative. [ABSTRACT FROM AUTHOR]

Objective. To evidence of the presence of two species of monogeneans of the genus Cichlidogyrus that cause damage to gill tissue, in tilapia Oreochromis spp. grown in Ecuador. Materials and methods. The gills were placed in a Petri dish with boiling water so that the parasites relax, and later they were fixed in 4% formalin. The samples were checked under a stereoscope microscope. The parasites were counted and preserved in 70% ethanol for their taxonomic identification according to their morphological characteristics. The infection parameters (prevalence, mean abundance and mean intensity) were calculated for each species of parasite. For the description of histological damages, fragments of parasitized gills were fixed in 10% neutral formalin and processed by the histological paraffin embedding technique. Results. Cichlidogyrus sclerosus showed highest infection parameters, prevalence of 65%, mean abundance of 24.24 ± 68.62 and mean intensity of 37.62 ± 82.93. Cychlidogyrus dossoui was observed in a prevalence of 4.5%, mean abundance of 0.13 ± 0.66 and mean intensity of 3 ± 1.41. Both species caused hyperplasia in lamellae. Conclusions. This study constitutes the first report of infestation by Cichlidogyrus sclerosus and C. dossoui in tilapia culture systems of Ecuador. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of mycosis fungoides (MF) and its mimickers is a major diagnostic challenge in era of Dermatopathology. Objective: To draw a diagnostic stepwise approach to minimize this challenge focusing on the benign (unusual) mimickers of infectious etiology. Patients/methods: This retrospective study included 94 paraffin blocks of patients with clinical suspicious of MF or its mimickers, during the period from Jan 2019-July 2021. The hematoxylin and eosin (H&E) stained sections and their associated clinical presentations were reviewed. Ancillary studies were performed upon the preliminary clinical diagnosis of each case. Deeper serial sections with selected special stains were done for benign mimickers. Primary (CD3/CD4/CD7/CD68) and secondary (CD20/CD8/CD30) panels of immunohistochemistry (IHC) markers were performed for most of the cases. Results: A wide spectrum of MF mimickers was identified, our cases were categorized into 3 groups: the first is classic MF (24) and its variants (5), the second is benign mimickers [subgrouped into infectious (31) & non infectious (21)] and the third is heterogeneous group; (other lymphomas/parapsoriasis) (13). A suggested stepwise diagnostic approach with selected IHC panels characterizes each group. Conclusion: A constellation of diagnostic clinical data, diagnostic histopathological clues and the suggested stepwise approach minimize the misdiagnosis of classic MF and conclusively identify the infectious mimickers. [ABSTRACT FROM AUTHOR]

Bovine leukemia virus (BLV) is an important pathogen associated with enzootic bovine leukosis. In this study, we performed PCR and sequencing analysis to characterize BLVgp51 sequences from formalin-fixed paraffin-embedded (FFPE) specimens made from 1974 to 2000 and successfully obtained BLV proviral genome sequences from 94% of the analyzed samples. Furthermore, from these samples, we reconstructed eight full-length and nearly fulllength BLVgp51 sequences. These sequences were classified as BLV genotype 1, implying that genotype1 has already been circulating in Japan since the 1970s. In our results, the proviral DNA was detected in the 1970s, 1980s, and 1990s in the same manner, indicating that the detection of BLV proviral genome depends on storage conditions rather than storage period. The sequences obtained in this study provide direct insights into BLV sequences before 2000, which serves as a good calibrator for inferring ancient BLV diversity. [ABSTRACT FROM AUTHOR]

Objective: Alzheimer's disease (AD) is the most common form of dementia, can be created in experimental models by using toxins. Possible therapeutic effects of secondary metabolites Rosmarinic (RA) and Oleanolic acids (OA) obtained from Salvia species (sage) were investigated using histopathological and immunohistochemical tecniques in experimental Alzheimer's-like dementia model induced by scopolamine. Study Design: Male BALB/c mice (n: 48) of 3-4 weeks old, divided into 6 groups; control received only saline(i.p.) for 21 days, scopolamine group received 3 mg/kg of scopolamine (i.p.) between 8-21th days. Scopolamine + RA group received 5 mg/kg RA (i.p.) between days 0 and 21 and 3 mg/kg of scopolamine (i.p.) between 8-21th days. Scopolamine + OA group received 5 mg/kg of OA (i.p.) between days of 0-21 and 3 mg/kg of scopolamine (i.p.) between 8-21th days. RA group received 5 mg/kg RA (i.p.) between 0-21th days. OA group received 5 mg/kg of OA (i.p.) between 0-21th days. Animals were sacrificed under anesthesia, brain tissues were excised and placed in 10% formaldehyde, embedded in paraffin wax. 5 µm sections were cut, stained for histopathological examinations. Immunoreactivity for Beta-amyloid accumulation was observed. ImageJ 153 software was used to analyze IHC figures. Positive signaling for DAB density was calculated, ANOVA test with the post hoc dunnett's or tukey test were applied, p<0.05 was considered statistically significant. Results: In scopolamine+RA treated group, tissue degeneration was less compared to the scopolamine group. Scopolamine+OA group revealed signs of pyknosis in neurons. The amyloid beta immunoexpression was positive in scopolamine group partially positive in scopolamine+OA group but, negative in RA treated scopolamine group. Intensity of signal in scopolamine group was statistically increased compared to control (p=0.0102). Intensity of signal was reduced in Scopolamine+RA and Scopolamine+OA groups, was statistically significant (p=0.0281 and p=0.0362, respectively). Conclusion: We suggest that pretreatment of RA and OA decreased beta amyloid formation and ameliorated tissue structure but, further studies with different methods are needed to be commercially available. [ABSTRACT FROM AUTHOR]

Purpose: Micro computed tomography (micro-CT) can provide detailed information about the internal structure of materials. This study aimed to demonstrate the diagnostic value of micro-CT in formalin fixed paraffin embedded pulmonary adenocarcinomas by correlating the micro-CT findings of tumoral and non-tumoral areas with hematoxylin and eosin (HE) sections. Methods: Paraffin blocks obtained from three adenocarcinomas were scanned with micro-CT. Ten regions of interest (ROIs) from adenocarcinoma and 11 ROIs from pulmonary parenchyma (ROI-C and ROI-N, respectively) areas were compared regarding the various structural parameters. Results: All parameters were significantly different regarding the tumoral and non-tumoral ROIs. The percent object volume, structure thickness, structure linear density, connectivity and connectivity density were higher in ROI-Cs (p < 0.000, p < 0.000, p = 0.001, p < 0.000, and p < 0.000 respectively); whereas intersection surface and structure model index were higher in ROI-Ns (p < 0.000 and p < 0.000). The open porosity percentage was higher in ROI-Ns (68.86 + 2.96 vs 48.29 + 5.11, p < 0.000) and the closed porosity percentage was higher in ROI-Cs (2.29 + 0.55 vs 0.57 + 0.17 p < 0.000). Conclusions: The tumoral and non-tumoral areas in paraffin blocks can be distinguished from each other, using the quantitative and qualitative information obtained by micro-CT. Making this distinction with quantitative data obtained from micro-CT can therefore be the basis of creating artificial intelligence algorithms in the future. [ABSTRACT FROM AUTHOR]

Sheeps are economically beneficial to human population and are considered important in uplifting the rural economy of India. Moniezia expansa, a common cestode parasite of sheep, inhabits the ileium portion of small intestine of host. In view of its common occurrence and necessity for assessment of the nature and damage caused in the form of various alterations in morphology and physiology of host due to the presence of parasites, the study of histopathology and histochemical observations of protein, glycogen and lipid in the intestine of sheep infected with M.expansa (test) and its comparision with the intestine of uninfected sheep (control) is important. For this, paraffin wax sections of 6-8 µ were prepared from the test and control intestines and were stained with haematoxylin and eosin for histopathological observations. For histochemical studies, various tests for protein, glycogen and lipids adopted from Pearse, 1968 were performed. Histopathological studies revealed that infected intestine obstructed with M. expansa to have pale patchy thickenings containing several rows of numerous blistersort of structures at the site of parasite attachment. Test sections showed the alteration in structure of villi in mucosal layer, superficial parts of villi are greatly reduced and degenerated and showed necrosis. Epithelial lining, goblet cells, crypts of Lieberkuhn were not observed. Lamina propria is oedematous showing infiltration of lymphocytes, plasma cells and macrophages. Lamina muscularis was disorganised. Submucosa and tunica muscularis was reduced in thickness. The histochemical distribution of protein, glycogen and lipid in the intestine of sheep infected with Moniezia expansa and its comparison with that of control (uninfected intestine) has been studied for the first time. The study of protein revealed that the superficial degenerated part of villi, submucosa and tunica muscularis layer showed comparatively less protein and the oedematous lamina propria showed more protein in test sections than in that of control sections. The glycogen concentration in tunica mucosa and submucosa in test sections is found to be less than in that of control sections. Distribution of lipid showed low concentration in superficial villi part and slightly higher concentration in lamina propria, lamina muscularis and tunica muscularis in test sections than in that of control sections. Thus, it reveals that the presence of parasite in host deprive it of many essential nutrients and alters intestinal morphology. [ABSTRACT FROM AUTHOR]

Triclosan (TCS) 5-chloro-2-(2, 4-dichloro phenoxy) phenol is a manmade broad-spectrum antimicrobial agent that is widely used in personal care, household goods and other applications. The present study was carried out to analyse the histopathological changes of liver and intestine of freshwater fish Channa striatus, which was exposed to 1/10th of LC50 of triclosan for 28 days. The median lethal concentration of triclosan in Channa striatus was estimated by using Probit analysis which was 0.602ppm. After each experimental period, the liver, and intestine were dissected out and fixed immediately in 10 % buffered formalin. After embedding in paraffin wax, 5 micron thick sections were cut and stained with H & E stains. In the liver, triclosan caused central vein congestion, inflammation, oedema, degeneration and necrosis of hepatocytes. Intestine anomalies included degeneration and necrosis, destruction of villi and serosa layer, and clogged blood capillaries. The present study revealed that Triclosan causes degenerative changes in various vital organs of Channa striatus and severity of histological changes were dose-related. [ABSTRACT FROM AUTHOR]

Background: Clearing in histopathological tissue processing should be able to make the tissues translucent and clear for the tissues to be visible under light microscopy and should render the clearing agent to be miscible with the dehydrant and the impregnation wax in the preceding and following processing steps. Xylene is a gold standard clearing agent but increasing concerns about the potential carcinogenicity, implementing eco-friendly agents in routine histopathology is necessary. Aim: The aim of the study is to assess the clearing ability of Cedarwood oil as an alternative to Xylene in routine tissue processing. Materials & Methods: The study was carried out in the Department of Oral Pathology and Microbiology. Formalin fixed 50 tissue samples of size 3-7mm were taken and subsequent dehydration done with acetone and alcohol. The dehydrated tissue is later processed using 90ml of Cedarwood oil with few drops of Xylene and Thymol. After clearing the tissues were subjected to impregnation and embedded in paraffin wax, later which sections were made and stained using H & E stain. Results: The results of our study on comparison showed better outcome in tissues processed with cedarwood oil than xylene. Statistical Significant correlation was observed in nuclear staining (p value = 0.001) ; cytoplasmic staining (p value = 0.08) and background staining (p value = 0.045) indicating a positive results on using cedarwood oil as clearing agent. Conclusion: The cedarwood oil can be considered as a safer natural alternative to xylene in laboratories. The cedarwood oil is eco - friendly and easily available with enhanced tissue processing qualities. [ABSTRACT FROM AUTHOR]

The experiment included the use of 12 female, white pregnant rats, divided into two groups: control group (6) pregnant women injected with physiological saline solution (Nacl 0.9%) by intraperitoneal injection (IP) starting from the beginning of pregnancy to the end of the experiment, to 3 females sacrificed at the time of implantation, which is from the sixth day of pregnancy and 3 females on the tenth day of pregnancy. The treatment group included 12 pregnant women injected with dexamethasone with a concentration of 0.2 mg/kg BW from the first day of pregnancy to the end of the experiment. This group was divided into 3 females who were sacrificed during the induction period, which is on the 6th day of pregnancy and 3 females on the 10th day of pregnancy. Liver samples were placed in 10% formalin solution. Samples were passed according to the method of tissue technology for a landfill in paraffin wax. The 5-micron-cut tissue was stained with haemotoxylin - eosin (H&E). On the sixth and the tenth day of gestation, the control and treatment group showed that the cause of the expansion of Sinusoids and the congestion in the central veins and the morphological changes that occurred in the liver tissue resulted from the side effects of dexamethasone. [ABSTRACT FROM AUTHOR]

Whilst adequate for most existing pathological tests, formalin is generally considered a poor DNA preservative and use of alternative fixatives may prove advantageous for molecular testing of tumour material; an increasingly common approach to identify targetable driver mutations in lung cancer patients. We collected paired PAXgene® tissue‐fixed and formalin‐fixed samples of block‐sized tumour and lung parenchyma, Temno‐needle core tumour biopsies and fine needle tumour aspirates (FNAs) from non‐small cell lung cancer resection specimens. Traditionally processed formalin fixed paraffin wax embedded (FFPE) samples were compared to paired PAXgene® tissue fixed paraffin‐embedded (PFPE) samples. We evaluated suitability for common laboratory tests (H&E staining and immunohistochemistry) and performance for downstream molecular investigations relevant to lung cancer, including RT‐PCR and next generation DNA sequencing (NGS). Adequate and comparable H&E staining was seen in all sample types and nuclear staining was preferable in PAXgene® fixed Temno tumour biopsies and tumour FNA samples. Immunohistochemical staining was broadly comparable. PFPE samples enabled greater yields of less‐fragmented DNA than FFPE comparators. PFPE samples were also superior for PCR and NGS performance, both in terms of quality control metrics and for variant calling. Critically we identified a greater number of genetic variants in the epidermal growth factor receptor gene when using PFPE samples and the Ingenuity® Variant Analysis pipeline. In summary, PFPE samples are adequate for histopathological diagnosis and suitable for the majority of existing laboratory tests. PAXgene® fixation is superior for DNA and RNA integrity, particularly in low‐yield samples and facilitates improved NGS performance, including the detection of actionable lung cancer mutations for precision medicine in lung cancer samples. [ABSTRACT FROM AUTHOR]

Background: Inflammation, Infections, occupational diseases and neoplastic lesions are common in lungs. In Autopsy internal organs including lungs are studied to decide cause of death and figure out prevalence of various lung lesions. So, prophylactic prevention plan can be prepared for prevention of various lung lesions induced mortality and reducing need for invasive biopsy as well. Aim & objective: To find out frequency of various lung lesions in relation to age and sex and analyze histopathological spectrum of lung lesions. Material & Methods: Non interventional, record based cross sectional, retrospective autopsy study was done on 139 cases of lung autopsy samples at department of pathology of tertiary care hospital, Bhavnagar, Gujarat for 2 years on samples received from January 2016 to January 2018.Lungs were fixed in 10% formalin & processed. Paraffin wax embedding was done & sections stained with H&E stain. Gross and microscopic examination of samples done and diagnosis was done.All findings were recorded and tabulated. Conclusion: Pneumonia is most common observed pathological lung lesion in our study which suggest that infections of lungs are common cause for mortality. Therefore, we suggest effective implementation of measures to prevent hospital acquired pneumonia may reduce mortality.Smoking was associated in nearly 70% patients. Autopsy study of such lung lesions can provide vision to plan preventive strategy to reduce mortality due to lung pathology. [ABSTRACT FROM AUTHOR]

The aim of this study is to determine C. parvum in small intestinal samples of lambs (up to 30 days old) with enteritis. For this aim, fresh intestinal smears prepared from 60 lambs (euthanized due to diarrhea) and stained with Modified Ziehl- Neelsen (MZN). In addition to these 60 fresh samples, 85 different small intestinal samples obtained from dead lambs (total 145 small intestinal samples) were embedded in paraffin for histopathology, immunofluorescence (IF) and immunohistochemistry (IHC). Slides were stained according to protocols (MZN, hematoxylin-eosin, IHC and IF). In MZN staining, 13.3% of smear samples were found positive for Cryptosporidium spp. Besides, C. parvum positivity was detected in the rate of 13.8% in IF and IHC stains. In the histopathologic examination, desquamation and necrosis of intestinal epithelium, villus atrophy, Cryptosporidium spp. along the surface of villus epithelium, hyperemia, mononuclear cellular infiltration, mitosis in crypt epithelium, intraepithelial lymphocytes, dilatation in the lymphatic canal and Eimeria spp. in some cases were observed in varying degree. In conclusion, IF, IHC and MZN stains were found effective in revealing cryptosporidiosis in lambs. [ABSTRACT FROM AUTHOR]

This study was carefully designed to determine the section compression of paraffin embedded sections. Two sections (one with thickness 5 µm and one 10 µm set by microtome) were cut from each of 2 sets of 12 testicular tissue (adult rats) blocks and stained with hematoxylin. Using scanned images, the area and the vertical (along the sectioning direction) and horizontal diameters of the block face were measured and compared with those of the unstained, stained or coverslipped section. Using the coverslipped section, the vertical and horizontal diameters of round spermatid nuclear profiles and the actual thickness of section were measured with light microscopy. Overall, the area of the coverslipped section was reduced by 5.5%-8.6% (on average) in comparison with that of the block face, with 69.5%-84.4% of the reduction being contributed by section compression in the process of section cutting, mounting and drying. The vertical (linear) compression of section, the primary cause of section area compression, was 5.9%-8.9%. The vertical compression of nuclear profiles was 1.5%-2.3% in 2 sets of sections and 5.2%-5.7% in other sections, indicating a non-uniform compression of structures within some sections depending on procedures of section drying. The measured mean thickness of sections decreased by 3.1%-5.0%. [ABSTRACT FROM AUTHOR]

The BIOMED‐2 PCR protocols targeting IGH and IGK genes may be useful for detecting clonality in Hodgkin lymphoma (HL). The clonality detection rates, however, have not been very high with these methods using paraffin‐embedded tumor sections. We previously described the usefulness of the semi‐nested BIOMED‐2 IGH assay in B‐cell malignancies. In this study, we devised a novel semi‐nested BIOMED‐2 IGK assay. Employing 58 cases of classical HL, we carried out the standard BIOMED‐2, BIOMED‐2 followed by BIOMED‐2 re‐amplification, and BIOMED‐2 followed by semi‐nested BIOMED‐2, all targeting IGH and IGK, using paraffin‐embedded tissues. In both IGH and IGK assays, semi‐nested assays yielded significantly higher clonality detection rates than the standard assays and re‐amplification assays. Clonality was detected in 13/58 (22.4%) classical HL cases using the standard IGH/IGK assays while it was detected in 38/58 (65.5%) cases using semi‐nested IGH/IGK assays. The detection rates were not associated with the HL subtypes, CD30‐positive cell density, CD20‐positive cell density, or Epstein–Barr virus (EBV) positivity. In conclusion, tumor clonality was detected in nearly two‐thirds of classical HL cases using semi‐nested BIOMED‐2 IGH/IGK assays using paraffin tumor sections. These semi‐nested assays may be useful when the standard IGH/IGK assays fail to detect clonality in histopathologically suspected HLs. [ABSTRACT FROM AUTHOR]

Fungal rhinosinusitis (FRS) is commonly caused by various Aspergillus species (spp) and Mucorales fungi, and the treatment and prognosis of cases differ depending on the causative fungus. The present study describes a novel immunohistochemical method that has high sensitivity and specificity for distinguishing between these two types of fungi in patients with FRS. Three groups were included in the study. Group A included formalin‑fixed paraffin‑embedded blocks of 51 nasal tissue specimens of patients with FRS (27 Aspergillus spp and 24 Mucorales) that were continuously obtained from the Department of Pathology of Tongren Hospital in Beijing as the experimental group and 34 cultures (26 Aspergillus spp and 8 Mucorales) of FRS that were randomly selected from the bacterial laboratory of Tongren Hospital in Beijing to verify the staining results of the paraffin‑embedded blocks. Formalin‑fixed paraffin‑embedded blocks of 10 esophageal cancer specimens were included in Group B as the positive control group. All specimens in Groups A and B were stained with interferon‑γ (IFN‑γ) antibody. Group C consisted of the same specimens as described in Group A, however, when performing the immunohistochemical assay, IFN‑γ antibody was replaced by PBS and this served as the negative control group. The differences in IFN‑γ immunohistochemical staining between Aspergillus spp and Mucorales were analyzed. Staining of IFN‑γ in paraffin‑embedded samples was positive in 92.6% (25/27) of specimens in which Aspergillus spp were the causative pathogen, which was significantly higher compared with specimens in which Mucorales was causative (P<0.001), with only 4.2% (1/24) of specimens staining positive for IFN‑γ. Immunohistochemical staining of cell cultures was 100% positive for Aspergillus spp, whereas all Mucorales were negative. Thus, the results of the current study indicated that IFN‑γ antibody immunohistochemical staining may be used as a novel diagnostic tool to distinguish between Aspergillus spp and Mucorales when identifying the causative agent in FRS, providing a useful supplementary test to the current immunohistochemical methods in the clinical diagnosis of FRS. [ABSTRACT FROM AUTHOR]

The concerns about the presence of microplastics (MPs) in marine ecosystems have widely increased in the past years. This is reflected in a growing number of studies addressing the effects of exposure to these materials in indigenous, farmed and even laboratory marine animals subjected to toxicity-oriented bioassays. There have been, however, many constraints in the detection of MPs in biological tissues, as routine histological techniques tend to degrade these materials, which are especially sensitive to organic solvents. This issue hinders the application of standard histopathological procedures based on convenient paraffin wax-embedding protocols, with consequences for biomonitoring and bioassay procedures. The method described here was developed and validated for the detection of polystyrene microplastics in biological tissue processed for paraffin-based histology. The strategy was developed and tested from whole-soft body sections of marine mussels that internalised the MPs following dedicated bioassays. The protocol is based on the replacement of xylenes with isopropanol for the purpose of intermediate infiltration and deparaffinization. Special modifications for staining, mounting and archiving are needed and are detailed as well. The protocol is shown to be a highly cost- and time-effective procedure compatible with formalin-based fixatives plus standard sectioning and staining, yielding complete preservation of MPs and optimal tissue conditioning. The method also produced excellent results with pre-stained MPs, with fluorochromes included, altogether providing excellent localisation of polystyrene MPs in paraffin-processed biological tissue. [ABSTRACT FROM AUTHOR]

Worldwide use of formalin-fixed paraffin-embedded blocks (FFPE) is extensive in diagnosis and research. Yet, there is a lack of optimized/standardized protocols to process the blocks and verify the quality and presence of the targeted tissue. In the context of an international study on head and neck cancer (HNC)—HPV-AHEAD, a standardized protocol for optimizing the use of FFPEs in molecular epidemiology was developed and validated. First, a protocol for sectioning the FFPE was developed to prevent cross-contamination and distributed between participating centers. Before processing blocks, all sectioning centers underwent a quality control to guarantee a satisfactory training process. The first and last sections of the FFPEs were used for histopathological assessment. A consensus histopathology evaluation form was developed by an international panel of pathologists and evaluated for four indicators in a pilot analysis in order to validate it: 1) presence/type of tumor tissue, 2) identification of other tissue components that could affect the molecular diagnosis and 3) quality of the tissue. No HPV DNA was found in sections from empty FFPE generated in any histology laboratories of HPV-AHEAD consortium and all centers passed quality assurance for processing after quality control. The pilot analysis to validate the histopathology form included 355 HNC cases. The form was filled by six pathologists and each case was randomly assigned to two of them. Most samples (86%) were considered satisfactory. Presence of >50% of invasive carcinoma was observed in all sections of 66% of cases. Substantial necrosis (>50%) was present in <2% of samples. The concordance for the indicators targeted to validate the histopathology form was very high (kappa > 0.85) between first and last sections and fair to high between pathologists (kappa/pabak 0.21–0.72). The protocol allowed to correctly process without signs of contamination all FFPE of the study. The histopathology evaluation of the cases assured the presence of the targeted tissue, identified the presence of other tissues that could disturb the molecular diagnosis and allowed the assessment of tissue quality. [ABSTRACT FROM AUTHOR]

In 2009, in the Tepeaca Valley, Puebla, it was found a cyst-forming nematode of the genus Heterodera associated with carrot. At this time, it is unknown the distribution in some producing municipalities, its life cycle and the histological changes on infected plant roots. Between 2008 and 2016, 28 carrot fields were sampled in six municipalities of the Tepeaca Valley. The roots were separated and white females were searched, the cysts were extracted from soil. Carrot cv. Mexicana were grown in naturally infested soil in the greenhouse, the roots were stained with fuchsine lacto glycerol acid to know the development time of each cycle phase. To describe the histological changes, the infected roots were imbibed in paraffin and sections stained with fast green-fuchsine for analysis. Heterodera sp. was found in 93 % of the plots and completed its cycle in 73 days at 20-25 °C. The histological alterations show the presence of feeding sites (syncytia) with dense and granular cytoplasm, with hypertrophied nuclei, dissolution and thickening of cell walls located mainly in the cortex and vascular cylinder. There was also the presence of lignificate areas with hyperplasia and hypertrophy. [ABSTRACT FROM AUTHOR]

Mouse models of colitis are used for efficacy testing of novel therapeutic compounds. A commonly used endpoint for colitis studies is the microscopic scoring of lesions on whole colon samples, either manually or by automated image analysis platforms. A comprehensive scoring system must evaluate the complete and continuous mucosal surface of the entire colon, so areas prone to show pathology are visible on a single hematoxylin and eosin (H&E)-stained slide. Slide preparation requires rolling the colon into a Swiss roll configuration in a biopsy cassette, fixing the tissue in formalin, embedding in paraffin, and sectioning sagittally through the center of the sample. Technical challenges often arise during the collection, embedding, and sectioning of samples, and errors lead to artifacts that compromise microscopic evaluation. Artifacts typically present as overlap of colon coils are termed ‘tissue folds’; segments of tissue absent from the slide is termed ‘tissue loss.’ We have identified key processing steps prone to contribute to these two artifacts and developed a method using the HistoPress embedding tool (American Master Tech) that significantly reduces their occurrence. [ABSTRACT FROM PUBLISHER]

Background: This is the first report of anisakiasis in a Croatian patient, evidenced from an archival paraffin-embedded and hematoxylin-eosin stained tissue section. Anisakiasis has been only suspected in the country based on previously detected anti-Anisakis IgE seroprevalence in the healthy coastal population, as well as an acute case where pathohistological and serological findings suggested the diseases, but the migrating larva has not been retrieved.Case Presentation: Seventy years-old female, operated in 1998 for pulmonary carcinoma, was admitted to the General hospital Šibenik, Croatia in 2003, because of gastric pain and nausea that lasted for couple of days. She was showing good general condition, full mobility and lucidity, subfebrile status. Abdominal palpation inferred acute pain in paraumbilical and ileocecal region. Exploratory right pararectal laparotomy revealed a hardened, 5 cm-long structure, located intraluminally in the sigmoid colon, not perforating colon serosa. The process has been dissected and sent for patohistological diagnosis. Results showed a 2 mm-long whitish nematode spiralised in muscular layer of colon mucosa surrounded by granulomatous inflammation.Conclusion: After genomic DNA isolation of the nematode from the histological section, and amplification at the mitochondrial cytochrome oxidase 2 locus, etiological agent has been identified as Anisakis pegreffii. Used methodology suggests that screening of archival suspicious sections is feasible in order to study epidemiology of this zoonotic disease poorly recognised in Croatia. [ABSTRACT FROM AUTHOR]

Malignancies of the uterine body ranks first in the female genital system, followed by malignancies of the ovary and cervix. The aim of the study was to analyze the immunoexpression of E-cadherin on a number of 55 cases with histopathological diagnosis of endometrioid type of endometrial carcinoma. Immunohistochemical staining was performed on tissue fixed in 10% formalin and embedded in paraffin. We used E-cadherin monoclonal antibody (Dako, clone NCH-38) diluted as 1:50. We scored the intensity of membranous E-cadherin immunoexpression (0-absent, 1-weak, 2-moderate, 3-strong) and also the percentage of marked cells according to histological grade (G1-well differentiated, G2-moderately differentiated, G3-poorly differentiated). The positivity of E-cadherin showed a variable intensity. In cases with well and moderately differentiated carcinomas the intensity levels ranged between 1 and 3, whereas in poorly differentiated carcinomas intensity levels were either 0 or 1. Membranous positivity of malignant cells in the invasion front and in the center of the tumor showed a statistically significant association with histological grade of tumor (2 = 45.576. DF = 2, p <0.001). Regarding the tumoral stage, we observed a similar trend for E-cadherin immunoexpression, similar to that of the histological grade. Cell adhesion, as indicated by the positivity for E-cadherin, decreased in tumor advancing edges compared to the center of the tumors also with the increasing histological grade, demonstrating alltogether the possibility of using this marker as a prognostic factor in endometrioid endometrial carcinoma. [ABSTRACT FROM AUTHOR]

Background Recent research has investigated the use of autofluorescence ( AF) for distinguishing between normal and cancerous tissues according to different fluorescence characteristics. Aim To analyze if AF can help differentiate cancerous lesions from other nonneoplastic lesions, such as dermatitis, in each layer of the skin ex vivo. Methods Paraffin wax-embedded tissue samples were obtained from patients who were histopathologically diagnosed with squamous cell carcinoma ( SCC), psoriasis, chronic dermatitis (lichen simplex chronicus, prurigo nodularis) or acute dermatitis (atopic dermatitis). AF intensity was measured in four layers of the epidermis (corneal, granular, spinous and basal) and two layers of the dermis (papillary and reticular). Results AF was highest in all layers of psoriasis samples compared with all layers of all other groups. Higher AF values were seen in SCC compared with all skin layers of acute and chronic dermatitis; this finding was especially true in the corneal layer, papillary dermis and reticular dermis. Conclusions This ex vivo AF study provides basic data for future in vivo studies of AF as a noninvasive diagnostic tool. [ABSTRACT FROM AUTHOR]

Fusarium is an emerging pathogen with high levels of morbidity and mortality. A significant tissue response is observed in infected patients, and the condition has been associated with the production of toxic metabolites. The aim of the present study was to identify a major fraction of crude metabolic extract of Fusarium oxysporum and investigate its effects on the skin of healthy rats. Fraction F1 was obtained from the cultivation of F. oxysporum in Czapek-Dox. In the treatment groups, fraction F1 (0.05 mg/ ml) was injected intradermally, while (50 µl) 0.9% of saline solution was injected in the control groups. The animals were killed 3, 6, 12, and 24 h after inoculation. The skin was fixed for inclusion in paraffin to obtain histological sections and stained with hematoxylin and eosin, Sirius red, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Samples were analyzed using Fourier transform Raman spectroscopy. The tissue reactions were classified and compared over time and by treatment. In the treatment group, inflammatory reaction peaked at 6 h, being classified as moderate, with infiltrate composed mainly of neutrophils. terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining was negative. The area occupied by types I and III collagen in the treatment group increased over time. There was a change in the area occupied by amide I and the ratios of the -CH2 and -CH3 molecules. It can be argued that the fraction F1 contains elements that contribute to the invasion of Fusarium in the skin, destructurizing the organization of the extracellular matrix. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

To date the diagnosis of abdominal angiostrongyliasis (AA) depends on the histological identification of Angiostrongylus costaricensis (AC) in surgical specimens. However, microscopic evaluation is time consuming and often fails in identifying the parasite. We tested whether PCR might help in the diagnosis of AA by identifying parasite DNA in formalin-fixed paraffin-embedded (FFPE) tissue. We used primers based on DNA from Angiostrongilus cantonensis. Four groups of FFPE intestinal tissue were tested: (1) confirmed cases (n = 20), in which AC structures were present in the target tissue; (2) presumptive cases (n = 20), containing changes secondary to AC infection in the absence of AC structures; (3) negative controls (n = 3), consisting of normal colonic tissue; and (4) tissue affected by other parasitoses (n = 7), including strongyloidiasis, ascaridiasis, schistosomiasis, and enterobiasis. Most lesions of confirmed cases were located in small and/or large bowel (90%), as compared with presumptive cases, in which 70% of lesions were in appendix (P = 0.0002). When confronted with cases of other parasitoses, PCR showed sensitivity of 55%, specificity of 100% and positive predictive value of 100%. In presumptive cases PCR was positive in 4 (20%). All specimens from negative controls and other parasitoses were negative. In conclusion, the PCR technique showed intermediate sensitivity and optimal specificity, being clinically relevant when positive for abdominal angiostrongyliasis. It allowed a 20% gain in diagnosis of presumptive cases. PCR might help in the diagnosis of abdominal angiostrongyliasis, particularly when the pathologists are not experienced with such disease. [ABSTRACT FROM AUTHOR]

Histopathologic diagnosis of gastrointestinal (GI) tract cytomegaloviral (CMV) infection relies on hematoxylin and eosin (H&E)-stained tissue, along with the aid of immunohistochemistry (IHC). However, non-classic appearing inclusions or atypical IHC staining patterns remain an ongoing concern for pathologists. We reported the use of real-time polymerase chain reaction (qPCR) on nucleic acid extracted from paraffin-embedded, formalin-fixed tissue of GI biopsies from cases of CMV infection (n = 91) diagnosed by H&E and IHC. Seventy-nine biopsies, including normal colon biopsies (n = 35), active colitis (n = 25), and active duodenitis (n = 19), were used as negative controls. Of 91 CMV-positive biopsies diagnosed by histology, 88 tested positive by qPCR, with a sensitivity of 96.7%. Of 79 negative controls, 78 were negative and 1 positive by qPCR, resulting in a specificity of 98.7%. Of the cases that were positive for CMV by histopathology, there were an additional 40 biopsies taken from these patients either during the same or previous procedures, some taken just days prior, which were negative for CMV by histology. Interestingly, 22 (55%) of these biopsies tested positive by qPCR, which correlated well with additional clinical CMV results. By defining qPCR as the "gold standard" for a CMV result, histology (H&E and/or IHC) had a sensitivity and specificity of 79% and 97%, respectively. Eighteen biopsies were found negative by H&E and equivocal by IHC. Among them, 14 (78%) tested positive for CMV by qPCR, which also correlated well with additional clinical results. qPCR is a sensitive, specific, and rapid molecular tool that may be helpful to aid in early diagnosis of CMV infection on equivocal or clinically highly suspicious small GI biopsies. [ABSTRACT FROM AUTHOR]

Alaria alata is a trematode of carnivores from Europe. The mesocercarial stage was recently identified in wild boar meat from Europe. Previous histopathologic studies showed the presence of unidentified parasitic cysts within the tongues of raccoons from northern Germany. For identification of the parasite species, tissue samples of 105 raccoons originating from a National Park in northern Germany and from Berlin metropolitan area were collected. Histological examination of cryotome sections of frozen as well as paraffin-embedded tongues were used to identify parasite cysts. These were located in the connective and adipose tissue and in close proximity to small arterioles, suggesting a hematogenous spread of the parasite. Often, cysts were surrounded with mild infiltration by inflammatory cells. Additionally, mesocercariae were isolated from defrosted tongue samples of 11 raccoons. Molecular-biology assays confirmed the parasite species as A. alata. Except for one positive raccoon from Berlin City, all other positive raccoons originated from the sylvan Müritz National park, indicating an abundance of intermediate hosts in this area. Our results show that raccoons can act as paratenic hosts for A. alata and extend the broad host range of this parasite to a species introduced into Germany. [ABSTRACT FROM AUTHOR]

Background: The fast acquisition process of frozen sections allows surgeons to wait for histological findings during the interventions to base intrasurgical decisions on the outcome of the histology. Compared with paraffin sections, however, the quality of frozen sections is often strongly reduced, leading to a lower diagnostic accuracy. Deep neural networks are capable of modifying specific characteristics of digital histological images. Particularly, generative adversarial networks proved to be effective tools to learn about translation between two modalities, based on two unconnected data sets only. The positive effects of such deep learning-based image optimization on computer-aided diagnosis have already been shown. However, since fully automated diagnosis is controversial, the application of enhanced images for visual clinical assessment is currently probably of even higher relevance. Methods: Three different deep learning-based generative adversarial networks were investigated. The methods were used to translate frozen sections into virtual paraffin sections. Overall, 40 frozen sections were processed. For training, 40 further paraffin sections were available. We investigated how pathologists assess the quality of the different image translation approaches and whether experts are able to distinguish between virtual and real digital pathology. Results: Pathologists' detection accuracy of virtual paraffin sections (from pairs consisting of a frozen and a paraffin section) was between 0.62 and 0.97. Overall, in 59% of images, the virtual section was assessed as more appropriate for a diagnosis. In 53% of images, the deep learning approach was preferred to conventional stain normalization (SN). Conclusion: Overall, expert assessment indicated slightly improved visual properties of converted images and a high similarity to real paraffin sections. The observed high variability showed clear differences in personal preferences. [ABSTRACT FROM AUTHOR]

Objective: In 2009, International Federation of Gynecology and Obstetrics (FIGO) modified staging of vulvar cancer-the prognostic significance of the new classification relative to the prior system as well as to the commonly recognized prognostic factors has not been assessed. The aim of this study was to test prognostic ability of 2009 staging in a cohort of uniformly treated and staged cases with long-term follow-up. Methods: Pathologic characteristics were obtained by blind review of the original tissue samples. 76 patients who qualified for surgery on the basis of the same criteria, with full clinical history, were included in the study. The histological analyses were performed on 76 and 35 paraffin-embedded tissue samples from primary tumors and lymph nodes, respectively. Survival analyses included the Kaplan-Meier method, log-rank test and Cox proportional hazards model. Results: Univariate analysis has demonstrated that age ( p = 0.0170), lymph node metastasis ( p = 0.0393), tumor grade ( p = 0.0086) and FIGO1994 stage ( p = 0.001) were the significant prognostic factors for overall survival. Multivariate analysis has demonstrated that growing age (HR 2.25, 95 % CI 0.79-3.71, p = 0.0321), tumor grade (G1 vs. G2 and G3) (HR 1-3.11, 95 % CI 1.6-4.62, p = 0.0057) and FIGO1994 stage (HR 1.78, 95 % CI 0.55-3.01, p = 0.0061) are independent prognostic factors with respect to overall survival. Conclusions: The results indicate the prognostic advantage of the 1994 FIGO staging as it has become an independent prognostic factor in contrast to the new FIGO system. This should be tested in future larger cohort studies. Differentiation grade turned out to be a very valuable independent prognostic factor and should be incorporated as a routine component of the histopathologic reports in vulvar cancer. [ABSTRACT FROM AUTHOR]

We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (∼8 mm diameter) (n = 15) and whole control corneas (n = 7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC. [ABSTRACT FROM AUTHOR]

Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively. [ABSTRACT FROM AUTHOR]

Patients with ALK gene rearrangements often manifest dramatic responses to crizotinib, an ALK inhibitor. Accurate identification of patients with ALK-positive non-small cell lung cancer (NSCLC) is essential for the clinical application of ALK-targeted therapy. However, assessing EML4-ALK rearrangement in NSCLC remains challenging in routine pathology practice. The aim of this study was to compare the diagnostic accuracy of FISH, immunohistochemistry (IHC), and real-time quantitative RT-PCR (QPCR) methodologies for detection of EML4-ALK rearrangement in NSCLC and to appraise immunohistochemistry as a pre-screening tool. In this study, a total of 473 paraffin-embedded NSCLC samples from surgical resections and biopsies were analyzed by IHC with ALK antibody. ALK rearrangement was further confirmed by FISH and QPCR. ALK protein expression was detected in twenty patients (20/473, 4.2%). Of the 20 ALK-positive cases by IHC, 15 cases were further confirmed as ALK rearrangement by FISH, and 5 cases were not interpretable. Also, we evaluated 13 out of the 20 IHC-positive tissues by QPCR in additional to FISH, and found that 9 cases were positive and 2 cases were equivocal, whereas 2 cases were negative although they were positive by both IHC and FISH. The ALK status was concordant in 5 out of 8 cases that were interpretable by three methods. Additionally, none of the 110 IHC-negative cases with adenocarcinoma histology showed ALK rearrangements by FISH. Histologically, almost all the ALK-rearranged cases were adenocarcinoma, except that one case was sarcomatoid carcinoma. A solid signet-ring cell pattern or mucinous cribriform pattern was presented at least focally in all ALK-positive tumors. In conclusion, our findings suggested that ALK rearrangement was associated with ALK protein expression. The conventional IHC assay is a valuable tool for the pre-screening of patients with ALK rearrangement in clinical practice and a combination of FISH and QPCR is required for further confirmation. [ABSTRACT FROM AUTHOR]

Background: Intraoperative analysis of frozen tissue samples has been used to diagnose peri prosthetic joint infection in revision total knee arthroplasty, but the precision and reliability of this test throughout staged revision arthroplasty have not been well characterized. We undertook this study to determine the rate of discrepancy between frozen and permanent histopathologic samples in the diagnosis of peri prosthetic infection at both the index total knee arthroplasty revision procedure and the attempted reimplantation Methods: Seventy-six patients who underwent staged revision of a total knee arthroplasty for apparent infection had samples for frozen and permanent sections obtained at both the index revision procedure and subsequent reimplantation attempts. We investigated the rate of discrepancy between these frozen and permanent sections. The clinical and radio-graphic outcomes of patients with and without concordance between the frozen and permanent sections were reviewed. Results: Concordance was noted between 297 (97.7%) of the 304 frozen sections and the permanent sections. Seven discrepancies between diagnoses based on frozen and permanent sections were noted; however, additional samples in these patients were positive and management was not affected. Fewer discrepancies were found during the initial revision procedure (one of 92, 1%) compared with reimplantation (six of 212, 3%). The mean time to reimplantation was similar between patients with and without a discrepancy in diagnosis between the frozen and permanent sections (185 compared with 157 days). The rate of discrepancy between frozen and permanent histopathologic sections was low, and no discrepancy affected the final outcome of any patient. Conclusions: The rate of discrepancy between frozen and permanent histopathologic samples was low for both index revision and reimplantation procedures. In rare instances of discrepancy, management should be unaffected provided a sufficient number of samples were collected [ABSTRACT FROM AUTHOR]

Oncology research, and particularly breast pathology, is undergoing a revolution with the advent of molecular signatures. These tools are complementary to the classical clinicopathological parameters commonly used in routine. The intrinsic signature brings, via a new taxonomy of breast cancer, a fresh look at the tumor beyond the histopathological type, grade and classic parameters of the tumor burden. Other prognostic signatures are Mammaprint®, Oncotype Dx®, and MapQant Dx®. Some of these signatures are available for formalin-fixed paraffin-embedded tissues opening the door for an use in routine setting. However, pathological evaluation is still cornerstone for the optimal management of patients with breast cancer, with the back up of molecular signatures particularly for Hormone receptor positive, HER2 negative patients. Furthermore, true predictive signature are highly awaited. [ABSTRACT FROM AUTHOR]

Tapia G, Sanz C, Mate J L, Muñoz-Mármol A M & Ariza A (2012) Histopathology 60, 768-773 Improved clonality detection in Hodgkin lymphoma using the BIOMED-2-based heavy and kappa chain assay: a paraffin-embedded tissue study Aims: Although BIOMED-2 polymerase chain reaction (PCR) standardization protocols allow clonality detection in nearly 100% of non-Hodgkin B cell lymphomas, they have not been widely validated for Hodgkin lymphoma (HL). Our aim was to assess BIOMED-2 protocol sensitivity when using non-microdissected, formalin-fixed, paraffin-embedded (FFPE) tissue from HL cases. Methods and results: We studied 69 consecutive HL cases, of which 61 corresponded to classic HL (cHL) and eight to nodular lymphocyte-predominant HL (NLPHL). CD30-positive cell numbers (<10, 10-25 or >25 per ×200 field), background CD20-positive cell density (low or high) and tumour cell immunophenotype were evaluated. IGH and IGK clonality was assessed on FFPE tissue following BIOMED-2 protocols. Of the 58 assessable cHL cases, 15 (25.9%) exhibited IGH and/or IGK clonality; IGH clonality was shown by nine (15.5%) and IGK clonality by 12 (20.7%). Clonality detection rates in cHL improved as CD30-positive Reed-Sternberg (RS) cell density increased and CD20-positive B cell density decreased, although these correlations did not reach statistical significance. Of the eight NLPHL cases studied, none showed clonal rearrangement. Conclusions: Combined study of IGH and IGK rearrangement according to BIOMED-2 protocols improves clonality detection rate (up to 25% of cases) in HL, even when working on non-microdissected FFPE tissue. [ABSTRACT FROM AUTHOR]

Histotechnology is concerned with processing and preparing of the body tissue in such a manner as to enable a satisfactory study of it. Section cutting is an integral part of histology and histopathology. It is an art by itself requiring skill and knowledge on the part of technician or the person who needs to do section cutting. In the routine method of preparing paraffin sections, it is often encountered by the presence of artefacts like fine wrinkles or folds. Attempts have been made to remove the wrinkles by floating the sections in the warm water bath. However this method has not been able to remove all the wrinkles from the sections. We have designed a simple and reliable method, in which the paraffin sections were floated over the ethyl alcohol diluted with water (1:15) before they were placed in the water bath. Through this method, we have been able to get the wrinkle free sections of superior quality. The advantage of our method is that, it is easy to prepare the dilute alcohol and is cost effective. This method can be used by the histology and pathology technicians and the researchers. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the use of proliferating cell nuclear antigen index in the different histopathological variants of ameloblastoma, such as the follicular, plexiform, and unicystic types, and in ameloblastic carcinoma by immunohistochemical staining. The proliferating cell nuclear antigen index values of the variants of ameloblastomas and ameloblastic carcinomas are compared in order to determine the biological behavior of these tumors. Materials and Methods: For the present study, archival tissues that had been diagnosed as ameloblastoma and ameloblastic carcinoma were collected from the department of oral pathology. Specimens were embedded in paraffin wax and were sectioned at a thickness of 5 μm and stained with hematoxylin-eosin for reconfirming the histologic pattern. It was also stained immunohistochemically for anti-proliferating cell nuclear antigen antibody. Results: Positive proliferating cell nuclear antigen expression is seen as a light brown, granular stain. The proliferating cell nuclear antigen values of ameloblastic carcinoma were almost five times the value of ameloblastoma. Analysis of variance test, Fischer's exact test/variance ratio test, and Student's t-test were performed and the probability values were determined. Summary and Conclusion: This study showed that ameloblastic carcinoma had the maximum proliferative capacity. Among the variants of ameloblastoma, the plexiform variety had the maximum proliferative capacity, followed by the follicular and unicystic varieties. Altogether, these data indicate that proliferating cell nuclear antigen is related to the biological behavior and proliferation of tumor cells in the variants of ameloblastoma and ameloblastic carcinoma. [ABSTRACT FROM AUTHOR]

Abstract: In a prospective study, a large number of patients with basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) underwent surgery using three dimensional (3D) histology and were evaluated with respect to local recurrence. The excised tumours were treated using 3D-histology with a routine paraffin procedure until the surgical margins were clear of tumour. Prospective evaluation of recurrence-free survival and overall survival of 5227 primary BCCs in 3320 patients and 615 invasive primary SCCs in 600 patients was conducted in the form of a letter-based follow-up with feedback from the patients and the referring physicians. The mean follow-up period was 5 years. In the BCC collective, 36 out of 3320 patients developed local recurrence (1%, calculated as a percentage of treated BCCs: 0.7%). In the SCC collective, 20 local recurrences appeared (3%). The recurrence rate for SCCs with desmoplasia was 24%, whilst the recurrence rate for common types of SCC without desmoplasia was 1%. Surgery followed by 3D histology results in very low recurrence rates for BCC and SCC with no additional effort compared with the normal histopathological procedure. [Copyright &y& Elsevier]

Summary: In recent years opportunistic infections due to microsporidial organisms have become increasingly important in immunocompromised people. Infected animals could serve as reservoirs of such infections. A case of generalized encephalitozoonosis in a young kitten is reported. Diagnosis was established by histopathological, immunohistochemical and molecular biological investigations demonstrating characteristic lesions and DNA of Encephalitozoon cuniculi in formalin-fixed and paraffin wax-embedded tissue sections. Infections due to E. cuniculi are not common in cats, but a potential role of domestic cats in transmission of the infectious agent cannot be excluded. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate whether paraSogue ratio tests (PRT) using real-time PCR can accurately determine the DNA copy number (CN) using formalin fixed paraffin embedded (FFPE) tissue. Histopathol-ogy diagnostic archives are an enormous resource of FFPE tissue, but extracted DNA is of poor quality and may be unsuitable for CN assessment, thus representing a missed opportunity for studies of genetic association and somatic change in cancer in large cohorts of easily accessible samples. Assays with paralogues on chromosomes 18 and 20 (18∣20 PRT) and chromosomes 13 and X (13∣X PRT) were tested using archived FFPE pathology samples with known CN, including tonsil, placentae, and FFPE melanoma cell lines. The assay proved accurate over the dynamic range from 1:1 to 1:3 and gave precise results when repeated four times over several weeks. The precision of the assay was marginally reduced once the CT value for 10 ng of FFPE DNA increased above 30 cycles, reflecting importance of DNA quality. The assays distinguished changes in CN ratio with high sensitivity and specificity. The 13∣X PRT could detect cells with distinct genotypes microdissected from within the same FFPE sample. Therefore, PRTs are suitable for analyzing CN in FFPE tissues. [ABSTRACT FROM AUTHOR]

Abstract: Cross bridges are radial structures within the highly organized lamellar structure of the annulus fibrosus of the intervertebral disc that connect two or more non-consecutive lamellae. Their origin and function are unknown. During fetal development, blood vessels penetrate deep within the AF and recede during postnatal growth. We hypothesized that cross bridges are the pathways left by these receding blood vessels. Initially, the presence of cross bridges was confirmed in cadaveric human discs aged 25 and 53years. Next, L1–L2 intervertebral discs (n =4) from sheep ranging in age from 75days fetal gestation to adult were processed for paraffin histology. Mid-sagittal sections were immunostained for endothelial cell marker PECAM-1. The anterior and posterior AF were imaged using differential interference contrast microscopy, and the following parameters were quantified: total number of distinct lamellae, total number of cross bridges, percentage of cross bridges staining positive for PECAM-1, cross bridge penetration depth (% total lamellae), and PECAM-1 positive cross bridge penetration depth. Cross bridges were first observed at 100days fetal gestation. The overall number peaked in neonates then remained relatively unchanged. The percentage of PECAM-1 positive cross bridges declined progressively from almost 100% at 100days gestation to less than 10% in adults. Cross bridge penetration depth peaked in neonates then remained unchanged at subsequent ages. Depth of PECAM-1 positive cross bridges decreased progressively after birth. Findings were similar for both the anterior and posterior. The AF lamellar architecture is established early in development. It later becomes disrupted as a consequence of vascularization. Blood vessels then recede, perhaps due to increasing mechanical stresses in the surrounding matrix. In this study we present evidence that the pathways left by receding blood vessels remain as lamellar cross bridges. It is unclear whether the presence of cross bridges in the aging and degenerating intervertebral disc would be advantageous or detrimental, and this question should be addressed by future studies. [Copyright &y& Elsevier]

Summary: Formalin-fixed and paraffin wax-embedded (FFPE) tissue sections are usually used for histopathological and immunohistochemical analyses in prion diseases in animals and man. However, formalin fixation cross-links proteins, reducing disease-associated prion protein (PrPSc) immunolabelling. To detect PrPSc in animals naturally affected with bovine spongiform encephalopathy (BSE) and scrapie, we applied minimal pretreatment with sodium hydroxide (NaOH). This simple pretreatment, combined with enzymatic digestion using proteinase K (PK), was equally effective in the detection of PrPSc in FFPE tissue, and superior in terms of speed, compared with the usual autoclaving method. The most effective results, without any section loss, were obtained with 10μg/ml PK in phosphate buffered saline containing 0.1% Triton-X at room temperature for 10min and 150mM NaOH at 60°C for 10min. By this simple procedure, PrPSc was visualized in the brain of animals with BSE and scrapie using a range of anti-PrP primary antibodies. [ABSTRACT FROM AUTHOR]

Purpose: To assess the histopathologic changes in a postmortem sample derived from an eye donor with macular telangiectasia (MacTel) type 2 to gain further insight into the cause of the disease. Design: Clinicopathological case report. Participants: Postmortem tissue was collected from 5 different donors: 1 MacTel type 2 patient; 1 healthy control; 2 type 2 diabetic patients, 1 with retinopathy and 1 without retinopathy; and 1 patient with unilateral Coat''s disease. Methods: Macular pigment distribution in the posterior part of freshly dissected eyes was documented by macrophotography. Paraffin sections from both the macular and peripheral regions were assessed using antigen retrieval and immunohistochemistry to study the distribution of cell-specific markers. Blood vessels were visualized with antibodies directed against collagen IV and claudin 5; glial cells with antibodies against glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase (GS), and retinaldehyde binding protein (RLBP1, also known as CRALBP); microglia with an antibody against allograft inflammatory factor 1 (also known as Iba1); and photoreceptors with antibodies against rhodopsin and opsin. Using anatomic landmarks, the sections then were matched with the macular pigment distribution and a fluorescein angiogram of the patient that was obtained before the patient''s death. Main Outcome Measures: Presence and distribution of macular pigment and cell-specific markers. Results: Macular pigment was absent in the macula. Furthermore, abnormally dilated capillaries were identified in a macular region that correlated spatially with regions of fluorescein leakage in an angiogram that was obtained 12 years before death. These telangiectatic vessels displayed a marked reduction of the basement membrane component collagen IV, indicating vascular pathologic features. The presence of GFAP was limited to retinal astrocytes, and no reactive Müller cells were identified. Importantly, reduced immunoreactivity with Müller cell markers (vimentin, GS, and RLBP1) in the macula was observed. The area that lacked Müller cells corresponded with the region of depleted macular pigment. Conclusions: These findings suggest that macular Müller cell loss or dysfunction is a critical component of MacTel type 2, which may have implications for future treatment strategies. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. [Copyright &y& Elsevier]

Conclusion: A modified paraffin-embedding method could be applied to histopathological and immunohistochemical studies of the human cochlea. The complementary use of molecular and immunohistochemical techniques by means of this method is thus considered to be a valuable tool for the future study of the human inner ear. Objective: To propose a new paraffin-embedding method for the morphological and immunohistochemical study of the human cochlea. Methods: Five human temporal bones were harvested at autopsy. The temporal bone specimens were fixed in 20% buffered formalin, decalcified with EDTA, cropped to a cube of approximately 15 mm, embedded in paraffin, and then cut into 6 μm thick sections. The sections were stained with hematoxylin and eosin, and immunostained with anti-prestin and anti-neurofilament antibodies. Results: Although paraffin-embedded sections cannot show the excellent morphology of the delicate membranous labyrinth obtained with celloidin, this technique successfully preserved the morphology of the cochlea, especially the organ of Corti, thereby enabling us to obtain excellent immunostaining results. [ABSTRACT FROM AUTHOR]

Abstract: Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGen’s WaxFree method and our in-house phenol–chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambion’s RecoverAll method recovered the most amplifiable RNA. [Copyright &y& Elsevier]