Paraffin Wax-embedded Tissue Sections Research Articles

Differential diagnosis of Crohn's disease and intestinal tuberculosis based on ATR-FTIR spectroscopy combined with machine learning.

Crohn's disease (CD) is often misdiagnosed as intestinal tuberculosis (ITB). However, the treatment and prognosis of these two diseases are dramatically different. Therefore, it is important to develop a method to identify CD and ITB with high accuracy, specificity, and speed. To develop a method to identify CD and ITB with high accuracy, specificity, and speed. A total of 72 paraffin wax-embedded tissue sections were pathologically and clinically diagnosed as CD or ITB. Paraffin wax-embedded tissue sections were attached to a metal coating and measured using attenuated total reflectance fourier transform infrared spectroscopy at mid-infrared wavelengths combined with XGBoost for differential diagnosis. The results showed that the paraffin wax-embedded specimens of CD and ITB were significantly different in their spectral signals at 1074 cm-1 and 1234 cm-1 bands, and the differential diagnosis model based on spectral characteristics combined with machine learning showed accuracy, specificity, and sensitivity of 91.84%, 92.59%, and 90.90%, respectively, for the differential diagnosis of CD and ITB. Information on the mid-infrared region can reveal the different histological components of CD and ITB at the molecular level, and spectral analysis combined with machine learning to establish a diagnostic model is expected to become a new method for the differential diagnosis of CD and ITB.

Primary Multicentric Pulmonary Low-grade Fibromyxoid Sarcoma and Chelonid Alphaherpesvirus 5 Detection in a Leatherback Sea Turtle (Dermochelys coriacea)

We describe the gross, microscopical, histochemical and immunohistochemical features of a sclerosing pneumopathic disease process resembling primary multicentric pulmonary low-grade fibromyxoid sarcoma in a juvenile female leatherback sea turtle (Dermochelys coriacea). The animal was fresh, presented in good body condition and stranded dead in Aracaju, Sergipe state, Brazil, in September, 2017. Grossly, the lungs were enlarged bilaterally and the parenchyma was replaced by large, coalescing, white, firm masses that extended into the bronchi and bronchioles and to the pleura. Microscopically, these masses consisted of paucicellular populations of well-differentiated, spindle-shaped fibroblasts with low pleomorphism and low mitotic count, but tissue invasion. Abundant collagen in compact areas merged with peripheral fibromyxoid foci and inflamed stroma. Antibodies specific for cytokeratins AE1/AE3 and smooth muscle actin (SMA) labelled pneumocytes lining the remaining distorted alveoli and the hypertrophied and hyperplastic bronchial muscles, respectively. Tumour cells were negative for SMA; neither neoplastic nor normal tissues cross-reacted with antibodies specific for vimentin or Ki67. Chelonid alphaherpesvirus 5 (ChHV5) polymerase chain reaction analysis from formalin-fixed, paraffin wax-embedded lung tissue sections amplified a 450 base pair fragment of DNA-polymerase (UL30 region) that had 100% homology to sequences previously detected in green sea turtles (Chelonia mydas) on the Brazilian coast. Enterocolitis was a concomitant condition that likely caused morbidity in this case. These findings contribute to the body of knowledge on sea turtle health and expand the known geographical range for ChHV5 in the southern hemisphere.

Validation of Antigen Retrieval is Important in Immunohistochemical Protocols for Detection of Prions in Paraffin Wax-embedded Tissue Sections

Validation of Antigen Retrieval is Important in Immunohistochemical Protocols for Detection of Prions in Paraffin Wax-embedded Tissue Sections

Immunohistochemical Validation of Spontaneously Arising Canine Osteosarcoma as a Model for Human Osteosarcoma

Osteosarcoma (OS) originates from bone-forming mesenchymal cells and represents one of the primary bone tumours. It is the most common primary bone tumour in dogs and man. The characterization of an appropriate natural disease animal model to study human OS is essential to elucidate the pathogenesis of the disease. This study aimed to validate canine OS as a model for the human disease by evaluating immunohistochemically the expression of markers known to be important in human OS. The immunohistochemical panel included vimentin, alkaline phosphatase (ALP), desmin, S100, neuron-specific enolase (NSE), runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 4 (BMP4). Immunohistochemistry was conducted on formalin-fixed, paraffin wax-embedded tissue sections from 59 dogs with confirmed primary OS. Vimentin, ALP, Runx2 and BMP4 were highly expressed by all tumours, while desmin, S100 and NSE were expressed variably. The findings were similar to those described previously for human OS and suggest that canine OS may represent a useful model for the study of the human disease.

Significance of apoptosis related proteins on malignant transformation of ovarian tumors: A comparison between Bcl-2/Bax ratio and p53 immunoreactivity

In this study, we compared the immunoreactivities of Bcl-2, Bax and p53 proteins in ovarian tumors and related the immunohistochemical findings to the histological type of the tumors. Formalin-fixed, paraffin wax-embedded tissue sections from 40 patients who had serous-mucinous borderline tumors and serous-mucinous adenocarcinoma of the ovary (n=10 each) were stained with hematoxylin–eosin (H&E). After histopathological examination, serial sections were stained immunohistochemically with primary antibodies to Bcl-2, Bax and p53 using an avidin–biotin-peroxidase method. A semi-quantitative grading system was used to compare the immunohistochemical staining intensities. The nuclear DNA fragmentation of apoptosis was determined using TUNEL method. As a result of immunohistochemical staining, increased immunoreactivity of Bcl-2 was observed in adenocarcinomas when compared to borderline tumors (P<0.001). Strong immunoreactivity of Bcl-2 and mild immunoreactivities of Bax and p53 were detected in ovarian adenocarcinomas. There were no significant statistical differences in the immunoreactivity of Bax among the histological type of ovarian tumors. Whereas a balance was observed between the immunoreactivities of Bcl-2 and Bax in the borderline cases, and this balance was strongly changed toward the anti-apoptotic Bcl-2 protein in patients with adenocarcinoma. TUNEL staining of sections indicated apoptotic cells in the serous borderline tumors were about 8-fold higher than in the serous adenocarcinoma. The results of this study on apoptosis-related factors might help to develop novel protective and therapeutic approaches, such as isoflavonoids and isothiocyanates, which were associated with decreased Bcl-2/Bax ratio, against the malignant epithelial ovarian tumors.

Generalized Encephalitozoonosis in a Young Kitten with Cerebellar Hypoplasia

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.

Antigen Retrieval Using Sodium Hydroxide for Prion Immunohistochemistry in Bovine Spongiform Encephalopathy and Scrapie

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 (PrP(Sc)) immunolabelling. To detect PrP(Sc) 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 PrP(Sc) 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 10 μg/ml PK in phosphate buffered saline containing 0.1% Triton-X at room temperature for 10 min and 150 mM NaOH at 60 °C for 10 min. By this simple procedure, PrP(Sc) was visualized in the brain of animals with BSE and scrapie using a range of anti-PrP primary antibodies.

Changes in chromatin structure during processing of wax-embedded tissue sections

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections.

C-Kit (CD117) expression in classic Kaposi’s sarcoma

Kaposi's sarcoma is a multicentric, low-grade, vascular neoplasia. Human herpesvirus 8 is associated with all epidemiological forms of KS and has been shown in vitro to induce the tyrosine receptor kinase c-Kit in infected cells. To investigate the expression of c-Kit in cases of classic KS and to clarify its association with clinicopathological parameters and HHV8 latency-associated nuclear antigen-1 expression. In total, 35 cases of classic KS at various histological stages were included in the study. Age and gender of the patients and location and histological stage of the tumours were recorded. Formalin-fixed, paraffin wax-embedded tissue sections were stained by immunohistochemistry with antibodies to c-Kit and HHV8. c-Kit immunoreactivity was found in 22 cases and HHV8 immunoreactivity was present in all cases. There was no correlation in c-Kit immunoreactivity between clinicopathological parameters and HHV8 immunoreactivity. The results of our study show that in cases of classic KS there is a high rate of c-Kit immunoreactivity, but c-Kit expression does not show any correlation with HHV8 immunoreactivity.

Significance of tyrosine kinase activity on malign transformation of ovarian tumors: A comparison between EGF-R and TGF- α

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF- α) are members of the polypeptide growth factor family. The epidermal growth factor-receptor (EGF-R) is a receptor tyrosine kinase of the ErbB family. Many types of cancer, including ovarian cancer, display enhanced EGF-R immunoreactivity on their cell surface membranes. Also, an increase in TGF- α synthesis and secretion usually occurs in human carcinoma cell lines. In this study, we compared the immunoreactivities of TGF- α and EGF-R in ovarian tumors and related immunohistochemical findings to the histological type of the tumors. Formalin-fixed, paraffin wax-embedded tissue sections from 40 patients who had serous-mucinous borderline tumor and serous-mucinous adenocarcinoma of the ovary ( n=10 each) were stained with hematoxylin–eosin and labeled for binding of primary antibodies against TGF- α and EGF-R using an avidin–biotin–peroxidase method. A semi-quantitative grading system was used to compare immunohistochemical labeling intensities. Increased immunoreactivity of EGF-R and moderate immunoreactivity of TGF- α was detected in adenocarcinomas. There was no significant difference in the immunoreactivity of TGF- α among the histologic types of ovarian tumors. The results of this study support the hypothesis that EGF-R may be a more useful marker than TGF- α in epithelial ovarian tumors.

Histopathological and ultrastructural effects of Losartan on embryonic rat kidney

The aim of our study was to investigate the histopathological, immunohistochemical and ultrastructural effects of Losartan (a selective angiotensin II type-1 receptor blocker) on renal development in rats. Twelve pregnant rats were divided into control and experimental groups. In the experimental group, Losartan (10 mg/kg/day) was given via nasogastric tube, between the sixth day of implantation and time of sacrifice on embryonic days 18 and 20. All formalin-fixed, paraffin wax-embedded renal tissue sections were stained with hematoxylin and eosin or labelled for binding of primary antibodies against transforming growth factor-beta (TGF-beta 1,-2,-3) using an avidin-biotin-peroxidase method. For electron microscopic examination, samples were fixed with glutaraldehyde and osmium tetroxide and embedded in araldite. Glomerular basement membrane (GBM) thickness was measured and compared using an unpaired t-test. Angiotensin II type-1 receptor antagonism by Losartan inhibited renal growth and delayed nephron maturation. Increased immunoreactivity of TGF-beta's was observed in developing nephron precursors and interstitial cells in the experimental group. Electron microscopical examination showed that thickening of the GBM was normal in the control group but an irregular thickening was seen in the experimental group (p < 0.001). It was also seen that epithelial cells of developing tubules underwent apoptosis in the experimental group. Thus, renal development in rats seems to depend on an intact renin-angiotensin system.

Differential expression of activation‐induced cytidine deaminase (AID) in nodular lymphocyte‐predominant and classical Hodgkin lymphoma

Activation-induced cytidine deaminase (AID) is indispensable for class switch recombination and somatic hypermutation of immunoglobulin genes. Expression of AID has been detected in germinal centre centroblasts and in lymphomas derived from germinal centre cells. However, in situ studies of AID expression have until now been hampered by a lack of antibodies suitable for immunohistochemistry. To overcome this problem, an AID-specific monoclonal antibody suitable for immunohistochemical staining of formalin-fixed, paraffin wax-embedded tissue sections has been generated. This antibody was shown to detect AID expression in normal germinal centre B-cells as well as in non-Hodgkin lymphomas with a putative germinal centre origin. Using this antibody, a virtually exclusive cytoplasmic localization of AID in normal and neoplastic B-cells is shown. Employing a combination of immunohistochemistry and AID-specific in situ hybridization, it is demonstrated that AID is consistently expressed in the neoplastic cells of nodular lymphocyte-predominant Hodgkin lymphoma (HLnlp) but only infrequently in classical HL (cHL). This is in keeping with the notion that tumour cells of HLnlp represent transformed germinal centre B-cells showing evidence of somatic hypermutation. AID represents an additional marker useful in the differential diagnosis of HLnlp and cHL.

Nm23-H1 expression and loss of heterozygosity in colon adenocarcinoma

Background: The discovery that genetic alterations in oncogenes and tumour suppressor genes accompany tumour formation in many human tumours has encouraged the search for genes that promote or suppress tumour...

Canine Lymphomas: a Morphological and Immunohistochemical Study of 55 Cases, with Observations on p53 Immunoexpression

The purpose was to determine the immunophenotype of canine lymphomas (CLs) classified according to the WHO nomenclature for domestic animals, and to relate these findings to the immunoexpression of p53 protein. Lymphomas were immunophenotyped with antibodies to CD79a, CD3, and p53 protein, suitable for paraffin wax-embedded tissue sections. Of 55 cases, 40 (72.7%) were of the B-cell phenotype, 12 (21.8%) of the T-cell phenotype, and three (5.4%) were non-B-non-T lymphomas. Of the 40 B-cell lymphomas, 31 were of the large B-cell type, six were lymphoplasmacytic, one lymphocytic, one follicular (grade II) and one unclassified low-grade. One of the peripheral T-cell lymphomas was morphologically consistent with anaplastic large cell lymphoma. Immunolabelling for p53 occurred in 24 B-cell and seven T-cell lymphomas. Lymphomas with high grade histology showed a significantly increased frequency of p53 positivity ( P=0.01). Positivity for p53 (more than 10% positive cells) tended to be associated with the T-cell phenotype ( P=0.06). Mean patient age was significantly higher in p53-positive cases ( P=0.02). These data are comparable with findings in human lymphomas.

The role of angiogenesis in vulvar cancer, vulvar intraepithelial neoplasia, and vulvar lichen sclerosus as determined by microvessel density analysis

Objectives We compared microvessel density (MVD) in normal, benign, preneoplastic, and neoplastic (squamous cell carcinoma (SCC)) vulvar disease to ascertain if this parameter could identify cases with lichen sclerosus (LS) and high-grade vulvar intraepithelial neoplasia (VIN3) at risk of developing malignancy. Methods Microvessels were immunohistochemically stained in paraffin wax-embedded vulvar tissue sections with anti-von Willebrand factor (vWF) antibody using the streptavidin–biotin–horseradish peroxidase complex technique. Three “hot spots” with the greatest MVD were identified within 200 μm of the subepithelial dermis under low magnification (×40 and ×100). The highest (HVD) and average (AVD) MVDs were quantified for each sample under high magnification (×200) using an image analysis system. Results HVD and AVD showed similar significant differences. SCC had significantly the highest MVD followed by VIN3, normal vulva, and LS. LS had significantly the lowest MVD, even lower than that of normal vulva. Two cases of VIN3 had much higher HVD (9.16 and 9.61) and AVD (6.89 and 7.71) compared with the main cluster of cases. Conclusion In vulvar LS, MVD, as assessed by HVD/AVD, is not a useful parameter in determining potential malignant progression, while in VIN3 this parameter could be valuable in identifying cases at greatest risk of progression to invasive disease.

Human papillomavirus associated with oesophageal cancer

Aim: To study the prevalence and the different types of human papillomavirus (HPV) in patients with oesophageal cancer from a high risk area of South Africa (Transkei). Methods: DNA samples...

Progesterone receptors in normal, dysplastic and tumourous feline mammary glands. Comparison with oestrogen receptors status

The aims of the study were to standardise an immunohistochemical (ihc) method for the detection of progesterone receptors (pr) on formalin-fixed, paraffin wax-embedded tissue sections of feline mammary gland tumours and dysplasias, comparing the results with those obtained using the radiolabelled ligand dextran coated charcoal (dcc) assay applied to frozen tissue samples from the same cases. Also, to define the immunohistochemical distribution of pr in the different cellular compartments of the lesions and to compare the oestrogen receptor (er) and pr status of the feline mammary lesions. Proliferative mammary lesions collected from 34 cats were studied; 25 malignant tumours and 9 benign tumours and dysplasias. pr protein was present at a concentration of 5fmolmg−1 (positivity threshold) in 37·5 per cent of malignant tumours and 66·7 per cent of benign tumours and dysplasias while immunoreative products to pr antibody were found in the nuclei of tumour cells in 38·5 per cent and 66·7 per cent of the cases, respectively. Concordance between dcc-pr and ihc-pr was 88·5 per cent (P<0·001). The specificity (true negatives) and sensitivity (true positives) of the ihc method were 89·4 per cent and 87·5 per cent respectively. The presence of pr was linked to the absence of ovariectomy (P<0·02). Oestrogen receptors, detected by either method, were also detected in half the cases in which pr had been detected. In malignant tumours, the most prevalent groups were the er + pr + ander −pr + groups.

Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the symbiotic sea anemone Aiptasia sp.

Different cell death pathways were investigated during bleaching in the sea anemone Aiptasia sp. in response to hyperthermic treatment. Using a suite of techniques, (haematoxylin and eosin staining of paraffin wax-embedded tissue sections, in-situ end labelling (ISEL) of fragmented DNA, agarose gel electrophoresis electron microscopy) both necrotic and programmed cell death (PCD) activity were indicated. After a treatment period of 4 days, the host endoderm tissues underwent necrotic cell death. This was indicated by widespread cellular degradation, dilation of cell cytoplasm and organelles, cell swelling and rupture, irregular pyknotic condensation of nuclear chromatin, and abundant cell debris. Host cell necrosis was associated with the release of zooxanthellae with a normal, healthy appearance into the coelenteron. Longer periods of hyperthermic treatment (7 days) were correlated with further animal cell degradation and the in-situ degradation of zooxanthellae remaining within the degraded endoderm. Within the same degraded endoderm tissue, the degradation of zooxanthellae resulted from two forms of cell death occurring simultaneously, which were identified as programmed cell death and cell necrosis. Programmed cell death of zooxanthellae was characterised by condensation of the cytoplasm and organelles, cell shrinkage, formation of accumulation bodies at the periphery of the cell wall, and DNA fragmentation. Cell necrosis of zooxanthellae was characterised by dilation of the cytoplasm and organelles, cell swelling and lysis, dispersion of cell component debris, and DNA fragmentation. The existence of a programmed cell death pathway within zooxanthellae is important to the understanding of coral bleaching events, raising interesting questions regarding the evolution of this process and the activation of the cellular trigger mechanisms involved.

A new monoclonal antibody, P2A8(6), that specifically recognizes a novel epitope on the multidrug resistance-associated protein 1 (MRP1), but not on MRP2 nor MRP3.

Multidrug resistance (MDR) is a major problem in the chemotherapeutic treatment of cancer. Overexpression of the multidrug resistance-associated protein 1 (MRP1), is associated with MDR in certain tumors. A number of MRP1-specific MAbs, which facilitate both clinical and experimental investigations of this protein, are available. To add to this panel of existing antibodies, we have now generated an additional MRP1-specific monoclonal antibody (MAb), P2A8(6), which detects a unique heat stable epitope on the MRP1 molecule. Female Wistar rats were immunized via footpad injections with a combination of two short synthetic peptides corresponding to amino acids 235-246 (peptide A) and 246-260 (peptide B) of the MRP1 protein. Immune reactive B cells were then isolated from the popliteal lymph nodes for fusion with SP2/O-Ag14 myeloma cells. Resultant hybridoma supernatants were screened for MRP1-specific antibody production. Antibody P2A8(6) was characterized by Western blotting and immunocytochemistry on paired multidrug resistant (MRP1 overexpressing) and sensitive parental cell lines. The antibody detects a protein of 190 kDa in MRP1-expressing cell lines but not in MRP2- or MRP3-transfected cell lines. P2A8(6) stains drug-selected and MRP1-transfected cell lines homogeneously by immunocytochemistry and recognizes MRP1 by immunohistochemistry on formalin-fixed paraffin wax-embedded tissue sections. Peptide inhibition studies confirm that P2A8(6) reacts with peptide B (amino acids 246-260), therefore recognizing a different epitope from that of all currently available MRP1 MAbs. This new MAb, chosen for its specificity to the MRP1 protein, may be a useful addition to the currently available range of MRP1-specific MAbs.

Expression of sodium iodide symporter in benign and malignant human thyroid tissues.

The extent of human sodium iodide symporter (hNIS) expression in different kinds of human thyroid cancer tissues and cell lines remains controversial. In this study, polyclonal antibodies to hNIS were used to analyze the expression of symporter protein in benign and malignant human thyroid tissues. Formalin-fixed, paraffin wax-embedded tissue sections were used. Staining was performed using primary polyclonal antibody of rabbit anti-human hNIS diluted in PBS (1:500). Results showed that 2 of 3 normal tissue, 3 of 6 nodular hyperplasia, one follicular adenoma, 3 of 11 papillary thyroid carcinoma, 1 of 5 follicular carcinoma and none of 3 metastatic thyroid epithelial tissue specimens stained positively for hNIS. A higher percentage of positive staining for symporter protein was found in benign thyroid tissues including normal thyroid tissue, nodular hyperplasia, and adenoma (60%). In contrast, papillary and follicular thyroid carcinomas demonstrated lower symporter protein expression (20%). In conclusion, although the number of tissue samples examined in this study was small, hNIS staining found a higher ratio of symporter protein expression in normal and benign thyroid tissues compared with malignant tissues. Determination of the reason for discrepancies in the expression of hNIS in in vivo and in vitro studies will require further investigation.