A combination Prussian blue – hematoxylin and eosin staining technique for identification of iron and other histological features

The Prussian blue reaction (PB) detects ferric iron in histological sections but the nuclear fast red (NFR) counterstain does not selectively stain the surrounding tissue and cellular features very well. The PB/NFR stain has the advantage of detecting iron located in tissue sections, but a significant disadvantage of having poorly differentiated tissue components, as compared to a routine hematoxylin and eosin (H&E). We developed a combination of Gomori’s Prussian blue/H&E staining method (PB/H&E), and modified the technique for best performance and clarity, then assessed the ability of this new combination stain to differentiate histological features of the tissue and identify iron. Serial sections from seven formalin fixed paraffin-embedded liver samples previously diagnosed with the presence of ferric iron were subjected to our routine H&E, routine PB/NFR and three trials of the new Prussian blue/H&E combination (PB/H&E). The technique that best differentiated the histological components of tissues containing iron was further tested on liver sections from a variety of species to verify consistency i.e. equivalence in staining intensity, concentration, brightness between sections of the same sample and quality i.e. coloration, vividness, recognizable differentiation of tissue components, improved staining.     

同时显示肾微血管和细胞构筑的实验研究

目的：在媒染微血管的同时显示肾内细胞构筑，以了解肾微血管与肾小管之间的相互关系。方法：用单宁酸一氯化铁法(TA—be)媒染肾微血管，再以苏木精、H—E法复染来观察肾小管细胞结构。结果：经复染的2组切片各级血管和细胞均染色良好，血管壁平滑肌纤维、内皮细胞界限清晰，细胞核蓝染，H—E染色的细胞质呈深浅不同的红色。因切片较厚，保证了微血管的立体感，细胞的染色效果却不及薄切片的鲜艳。结论：TA—Fe法配合苏木精或HE复染，可同时显示肾微血管和细胞构筑。     

DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis.

Myocardial tissue taken from 19 autopsy cases of myocardial infarction were examined both by the nick and labeling method (NELM) and by DNA agarose gel electrophoresis in order to demonstrate the localization of cells with fragmented DNA and to confirm the internucleosomal cleavage of DNA biochemically. The nuclei corresponding to those with the histological features of acute myocardial infarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positive with the nick end labeling method. Myocardial cells corresponding to those with nick end labeling method-stained nuclei, on the other hand, had mostly pyknotic and karyolytic nuclei and some unremarkable nuclei, even nuclear ghosts, and showed degenerated cytoplasm, including contraction band necrosis in H&E-stained preparations. The agarose gel electrophoresis of DNA extracted from the corresponding areas mentioned above showed the ladder pattern of internucleosomal cleavage characteristic of apoptosis. The present study revealed that infarcted myocardial cells with nuclear outlines, even nuclear ghosts, showed a distinct DNA fragmentation with the ladder pattern of internucleosomal cleavage. It is concluded from this study that the damaged myocardial cells of acute myocardial infarction represent a coagulation necrosis having the biochemical nature of apoptosis.     

Modification of Brain Processing Techniques: Tissue Embedding in Glycol Methacrylate and Stain Modifications

Abstract

Increased interest in quantitation of the histopathological changes in a variety of neurological disorders (including neurodegenerative disease such as Alzheimer's disease) continues in an attempt to develop specific clinical-histopathological correlations. Most previous efforts at quantitation have used paraffin embedded sections of brain tissue, although plastic embedded sections have recently become a preferable alternative because they provide greatly reduced tissue shrinkage and distortion during processing, and greater clarity and improved resolution to the tissue sections. We have developed techniques for glycol methacrylate embedding and sectioning of brain tissue blocks on a standard histology laboratory microtome. In addition, we have modified routine diagnostic and investigational neurohistological stains for use in glycol methacrylate embedded brain sections, including hematoxylin and eosin, modified Bielschowsky stain, Jamarri silver technique, Einarson's Nissl stain, gallocyanin-Darrow red myelin stain, and the thioflavine-S-hematoxylin stain. The use of plastic embedded sections with appropriate stains will permit critical histopathological evaluation of nervous system tissue from patients with a variety of neurological disorders. (J Histotechnol 12:201, 1989)     

缺氧导致体外培养乳鼠心肌细胞凋亡的研究

目的:通过体外培养乳鼠心肌细胞模拟缺氧模型,探讨心肌细胞缺氧损伤时是否发生心肌细胞凋亡。方法:取(1~3)d新生SD乳鼠心肌进行心肌细胞培养,采用模拟缺氧模型,通过HE染色、流式细胞仪等手段观察检测细胞凋亡的改变。结果:在本实验心肌细胞缺氧模型中,HE染色可见凋亡细胞体积变小,胞核浓缩,胞质嗜酸性增强。流式细胞仪检测出明显的凋亡峰。结论:本实验培养乳鼠心肌细胞缺氧模型可导致心肌细胞凋亡,可通过HE染色观察凋亡细胞形态学的变化,流式细胞术检测细胞DNA含量而对凋亡细胞进行定量分析。     

Morphometric quantification of apoptotic stages in cell culture

Apoptosis is an active process of self-destruction, whereby cells undergo physiological cell death. It occurs during development and regulation of tissue homeostasis or as a result of changes in environmental stimuli. Chromatin condensation and nuclear fragmentation, which are typical features of apoptotic nuclei, are usually quantified by fluorescent DNA dyes. The present study reports a reliable method to analyze morphological apoptotic stages in cultured cells, using light microscopy. We used the human neuroblastoma cell line SK-N-BE as a model to study apoptosis induced by inadequate cell-matrix interactions. Apoptosis was detected on cells cultured for different time intervals on polyHEMA, poly-L-lysine or collagen I. Quantitative morphometric and densitometric analysis after hematoxylin nuclear staining and caspase-3 immunocytochemistry, as markers of occurring apoptosis, were performed. Our method identifies different stages of caspase-3 activation and the subsequent DNA fragmentation and condensation. This experimental procedure enables us to detect slight differences in apoptosis progression by morphological analysis.     

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.     

Detection of the M30 neoepitope as a new tool to quantify liver apoptosis: timing and patterns of positivity on frozen and paraffin-embedded sections

One of the first stages of apoptosis is cytokeratin cleavage mediated by caspases, which is associated with the expression of a neoepitope, the cleavage site of cytokeratin 18, identifiable by the M30 monoclonal antibody. The aim of this study was to evaluate the timing of neoantigen expression and its modifications in the various morphologic stages of apoptosis on frozen and paraffin-embedded sections from liver biopsies of patients with chronic hepatitis or transplanted liver. The appearance of this neoepitope coincides with the gradual disappearance of cytokeratins, with the appearance of nuclear DNA fragmentation, and with the presence of Councilman bodies. The staining patterns on paraffin-embedded sections of liver specimens were similar to those found in frozen sections, with a reduced sensitivity. The M30 antibody is correlated with apoptosis, and its specificity for epithelial cells makes this method the first choice for routine evaluation of apoptosis in liver epithelial cells.     

Apoptotic bodies as a morphological feature in serous ovarian carcinoma: correlation with nuclear grade, Ki-67 and mitotic indices

This study evaluated the relation of apoptosis with mitotic activity, Ki-67 indices, and nuclear and architectural grades in serous ovarian carcinoma. Apoptotic body (ABI) and mitotic indices (MI) were obtained by examining hematoxylin and eosin-stained sections from 35 serous ovarian carcinomas for apoptotic bodies and mitotic figures. ABI and MI were reported as the number of apoptotic bodies, and mitotic figures and immunostaining for Ki-67, respectively, as positive cells in 1000 cells. Nuclear grade was determined as grade 1 [n = 11], grade 2 [n = 13], and grade 3 [n = 11] according to recently defined criteria. There was a significant correlation between Ki-67 indices and ABI (p < 0.0001), Ki-67 and MI (p < 0.0001), as well as between MI and ABI (p < 0.0001). ABI increased with nuclear grade (p < 0.0001) and the type of the histological differentiation pattern (from glandular to papillary and solid architectural patterns) (p < 0.0001). Apoptosis, quantitated by ABI, is positively correlated with proliferation, thereby constituting a factor in the regulation of tumor growth of serous ovarian carcinomas. The positive correlation between ABI and increasing nuclear grade, mitotic activity, and architectural growth pattern may indicate that apoptotic bodies are another variable for grading serous ovarian carcinomas.     

Paneth Cell Identification in the Small Intestine of Guinea Pig Offsprings (Cavia porcellus)

The aim of this study was to determine the presence, number, and morphometrical characteristics of Paneth cells (PC) in the small intestine of guinea pigs during lactation. We used 48 pups from 0 to 15 days old. Samples from small intestine were fixed in 10% buffered formaldehyde (pH 7.4) and processed for histological and morphometrical studies using hematoxylin and eosin (HE), Phloxine tartrazine or Masson's Trichome staining, or immunohistochemistry for lysozyme. PC were morphologically identified at day 2 using Masson's Trichome or Phloxine tartrazine stainings, and at day 4 using HE, whereas using immunohistochemistry they were recognized from birth. Morphometrical differences were found between the intestinal sections at each age studied, and within each section during the first weeks of life. In all developmental stage, the highest number of PC was observed in the duodenum of 13 days old guinea pigs. Our results confirm the presence of PC in the small intestine of guinea pigs from birth. Anat Rec, 297:856–863, 2014. © 2014 Wiley Periodicals, Inc.     

Eastwood Congo Red Method For Demonstrating Amyloid

Abstract

A modification of Eastwood's Congo red method is described. The method, which substitutes iron hematoxylin in place of alum hematoxylin, appears to enhance the staining of amyloid when viewing with light microscopy, at the same time quenching background fluorescence when viewing with fluorescence microscopy. The method gives consistent and reliable staining results, and the Congo red staining solution has excellent stability. (The J Histotechnol11:105, 1988.)     

Comparison between pH 2·5 Alcian blue stain and Hale’s dialyzed iron technique for demonstrating acid mucin in colorectal carcinoma

Abstract

This study compared the value of pH 2·5 Alcian blue and Hale’s dialyzed iron methods for demonstrating acid mucin in colorectal carcinoma. A total of 40 colorectal biopsies from known positive cases were examined. Sections of the biopsies were stained using pH 2·5 Alcian blue method and Hale’s dialyzed iron technique, and classified as ‘good’ or ‘excellent’ according to staining intensity and efficiency, using a semi-quantitative scoring system. The pH 2·5 Alcian blue method was found to provide a higher frequency of ‘excellent’ scores compared to Hale’s dialyzed iron technique, 32 (80%) versus 21 (52.5%) respectively (P = 0·018). Both methods provide at least a minimal level of satisfactory staining results and seem to demonstrate the same amounts of acid mucins; however, if high quality Alcian blue dyes are available, pH 2·5 Alcian blue method is especially recommended to stain colorectal carcinoma acid mucins because it provides a stronger staining intensity and coloration compared to Hale’s dialyzed iron technique.     

Ultrastructural Analysis of Apoptosis Induced by the Monoclonal Antibody Anti-APO-1 on a Lymphoblastoid B Cell Line

Apoptosis (programmed cell death) is the most common form of death in eukaryotic cells. We recently described a monoclonal antibody, anti-APO-1, which induces apoptosis of cells from the human B-lymphoblastoid line SKW 6.4 and of cells from a variety of other human lymphoid cell lines. This model of apoptosis was now studied ultrastructurally.

SKW 6.4 cells undergoing apoptosis showed the following morphological changes: condensation of the cytoplasm and karyoplasm, formation of large electron-opaque aggregates of the chromatin lining the nuclear membrane, “blebbing” of the cell membrane at an early stage of apoptosis, and dilatation of the mitochondria. Two hours after adding anti-APO-1, the nuclear membrane was ruptured. Occasionally, large vesicular enlargement of endoplasmic reticulum or Golgi appeared in the cytoplasm. Finally, total breakdown of all cell membranes and cellular disintegration was observed.     

Apoptosis and Accidental Cell Death in Cultured Human Keratinocytes after Thermal Injury

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72°C for 1 second. After exposure to temperatures of 58 to 59°C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66°C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72°C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59°C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.     

CK19表达及其在喉鳞癌淋巴结微转移诊断中的应用

目的:研究用免疫组化方法检测CK19及其在喉鳞癌淋巴结微转移诊断中应用与临床病理意义。方法:对40例喉鳞癌患者及清扫淋巴结163个(常规临床检查颈部淋巴结阴性pN₀)进行常规病理检查(HE染色)和抗角蛋白19抗体的免疫组化检测。结果:40例喉鳞癌组织中CK19均表达阳性,40例颈淋巴结常规病理检查(HE染色)发现5例有转移者(共23枚淋巴结),CK19检测也为阳性。19个淋巴结常规病理检查未发现转移者,CK19也表达阳性。随着肿瘤T分期的增加,淋巴结CK19表达阳性率亦增加。CK19表达阳性者预后较阴性者差。结论:CK19免疫组化法是检测喉鳞癌淋巴结微转移的敏感而便捷的方法,而检测喉鳞癌微转移有助于判断肿瘤进展程度与预后,特别对筛选组织学检查淋巴结阴性但存在微转移的病人有实用价值。     

Establishment of digital cutoff values for intraepithelial lymphocytes in biopsies from colonic mucosa with lymphocytic colitis

Background/IntroductionLymphocytic colitis (LC) and the incomplete form (LCi) are common causes of chronic watery diarrhea. Endoscopy is often inconspicuous, and the diagnosis relies on histopathological assessment of colonic biopsies. Digital Image Analysis (DIA) eliminates interobserver variation. The aim of this study was to establish digital cutoff values for LC and LCi on CD3 stained slides.Material and methodsOne hundred and six patients with a hematoxylin and eosin (HE) diagnosis of normal colonic mucosa (N = 19), non-specific reactive changes (N = 24), LCi (N = 23) and LC (N = 40) were eligible for analysis. The number of intraepithelial lymphocytes (IELs) reached by DIA in the total surface epithelium and in hot spots of the biopsies was compared with the diagnostic category assigned by the pathologists based on HE stained slides. The digitalized slides were analyzed for number of IELs using Visiopharm Quantitative Digital Pathology software. All digitalized slides were examined manually to identify differences in the approach to the evaluation of the biopsies by the pathologists and DIA.ResultsThe median IEL counts and interquartile range in the total surface epithelium were 3.6 (3.2–4.3), 4.4 (3.4–5.3), 19.8 (16.6–30.0) and 41.3 (37.0–47.8) in normal colon mucosa, mucosa with non-specific reactive changes, LCi and LC, respectively. Discrimination between normal mucosa and non-specific reactive changes was not possible. Digital cutoff values with the best separation between non-LC, LCi and LC were > 13 IELs/100 epithelial cells for LCi and > 36 IELs/100 epithelial cells for LC. These cutoff values resulted in an agreement between the pathologist’s and DIA that was very good with a kappa value of 0.90.ConclusionDespite differences among the approach of DIA and the pathologist’s assessment of IELs in colonic mucosa DIA is able discriminate between the HE based diagnoses of the three subgroups non-LC, LCi and LC with high accuracy.     

A Nuclear Artifact of Lymphoid Tissue

Abstract

Several tissue sections of a cervical lymph node diagnosed as lymphoma presented on light microscopy with an unusual nuclear artifact of lymphoid cells after fixation in 10% neutral buffered formalin and staining with hematoxylin and eosin. Nuclei appeared shrunken with homogeneous dispersion of chromatin and spiked at their periphery. Inadequate irnrnersion of tissue sections in one paraffin bath, leaving blocks uncovered, was found to have caused the artifact. When routine care was exercised in replacing, or at least rotating, paraffin baths each time the tissue processor was changed to fresh fixative, dehydrants, and clearing agents, the problem was resolved. (The J Histotechnol 16:365, 1993)     

Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts

The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) technique has been extensively used for the detection and quantification of apoptosis in histological tissue sections. However, the interpretation and specificity of this assay have been controversial. With accumulating knowledge of the molecular mechanisms of cell death and the discovery of the caspases as key mediators of apoptosis, more direct and earlier measurements of apoptosis in tissue sections have emerged. This study, using antibodies that specifically recognize activated caspase-3 and caspase-cleaved cytokeratin (CK) 18, evaluated whether immunohistochemical stains would improve the detection and quantification of apoptosis in tissue sections, compared with the TUNEL assay. Tumour xenografts of the prostate cancer cell line PC-3 were used as an example, since these tissues contain large numbers of cells undergoing apoptosis. Apoptotic cells were quantified and apoptotic indices were calculated by computer-assisted image analysis following identification of apoptotic cells by morphological analysis, the TUNEL assay, activated caspase-3 and cleaved CK18 immunohistochemistry. The results indicated that activated caspase-3 immunohistochemistry was an easy, sensitive, and reliable method for detecting and quantifying apoptosis in this model. An excellent correlation (R = 0.89) between the apoptotic indices obtained using activated caspase-3 and cleaved CK18 immunostaining was observed. A good correlation (R = 0.75) between the apoptotic indices obtained using activated caspase-3 immunostaining and the TUNEL assay was also found. Activated caspase-3 immunohistochemistry is therefore recommended for the detection and quantification of apoptosis in tissue sections.     

Histogenesis of dieldrin and DDT-induced hepatocellular carcinoma in Balb/c mice

Male, Balb/c mice were fed diets containing dieldrin (10 ppm) and DDT (100-175 ppm) for 75 weeks. Control and treated mice were serially killed and their livers analyzed by histological and histochemical procedures after 2, 4, 8, 16, 36, 52 and 75 weeks of exposure. Mice administered both chlorinated hydrocarbons initially responded with centrolobular hepatocytomegaly. The cells were characterized by decreased glucose-6-phosphatase and succinate dehydrogenase activity. At later periods 52 through 75 weeks, foci of phenotypically-altered hepatocytes were noted. The cells of these lesions were basophilic or clear-staining in hematoxylin and eosin-stained sections and displayed increased gamma glutamyl transpeptidase activity. In mice preloaded with iron dextran, cells of foci were negative for iron when the surrounding parenchyma was siderotic. Hepatocellular adenomas (HA) and carcinomas (HPC) were composed of cells with increased gamma glutamyl transpeptidase and glucose-6-phosphate dehydrogenase and decreased glucose-6-phosphatase and succinate dehydrogenase activity. In iron loaded mice, the cells of HA and HPC did not stain for iron in otherwise siderotic surroundings. Both hepatocellular foci and adenomas may be potential precursors of mouse hepatocellular carcinomas.