Picrosirius red: a better polarizing stain

Collagen is a part of the extracellular matrix proteins that form an integral part of the connective tissue stroma. Collagen plays an important role in maintaining the structural and functional integrity of tissue. Due to the arrangement of its fibers, it illustrates the phenomenon of birefringence that is enhanced by special stains like van Gieson and Picrosirius Red. The aims of the present study were to compare the polarizing properties, shelf life and staining intensity of both these connective tissue stains to ascertain which gave better and most cost-effective results. A retrospective study was conducted in the Department of Oral Pathology where histopathologically diagnosed cases of fibroma were selected. Consecutive sections of formalin fixed and paraffin embedded tissues were stained with Picrosirius Red and van Gieson stains, respectively. This staining procedure was repeated on successive sections every 15th day for a period of 6 months. The polarizing property and staining intensity of the new and the previously stained sections along with the shelf life of the staining solutions were simultaneously evaluated and revaluated each time and tabulated. The data obtained was analyzed by using independent t-test and chi-square test. After a period of six months, the polarization intensity and stability of Picrosirius red was found to be better than van Gieson and this could also be appreciated more clearly in the stained sections. The p value was also found to be statistically more significant with Picrosirius Red as compared to van Gieson. Picrosirius Red Stain is not only more stable but also has better polarizing properties, staining intensity, and a longer shelf life as compared to van Gieson.     

Lack of vessel wall elastolysis in human invasive pulmonary aspergillosis.

In experimental studies, the apparent ability of Aspergillus fumigatus isolates to produce elastase in agar plates correlates with their ability to cause invasive pulmonary aspergillosis in mice pretreated with cortisone. Thus, elastase production may govern the pathogenicity of particular isolates. If this is so, then disruption of the elastic layers within blood vessel walls in invasive aspergillosis would be expected. To test this hypothesis, tissue blocks were prepared from nine patients with invasive pulmonary aspergillosis. Separate but immediately adjacent histological sections were stained by the Grocott and periodic acid-Schiff methods for fungal hyphae and by the elastic van Gieson technique for elastic tissue. Comparison of those segments of vessel walls infiltrated by hyphae with those not infiltrated by hyphae showed no overall loss of elastic tissue. Material from five of the cases was also stained with an unconventional combination of histochemical stains, allowing accurate identification of both fungal hyphae and elastic laminae in the same histological sections. The results showed no more disruption of elastic laminae than would be expected from simple physical displacement of elastic laminae. We conclude that if elastolysis contributes at all to invasion of vessel walls by aspergilli, then it seems to be very localized and/or transient.     

Relative Contributions of Collagen and Elastin to Elasticity of the Vocal Fold Under Tension

This study examined the contributions of collagen and elastin to the tensile elastic properties of the vocal fold lamina propria. Uniaxial stress–strain responses of vocal fold cover and vocal ligament specimens from 20 human larynges (12 males, 8 females) were quantified with sinusoidal stretch-release deformation in vitro. Mid-coronal sections of 12 specimens were examined histologically with Masson’s trichrome and elastin van Gieson stain to quantify the relative densities of collagen and elastin fibers. Results showed that significantly higher levels of collagen were found in the male vocal fold than female, for both the cover and the ligament. For male there was a significantly higher level of elastin in the cover than in the ligament. On average, the elastic modulus of the male cover was about twice that of the female at high-tensile strain (35–40%), whereas the male ligament was 3–5 times stiffer than the female in the same range. The ligament was stiffer than the cover for male, but the opposite was observed for female. These findings suggested that collagen and elastin could contribute differentially to elasticity of the cover and the ligament. The data may provide guidance for surgical reconstruction and tissue engineering of different lamina propria layers. A version of this paper was presented at the 2005 Annual Meeting of the American Academy of Otolaryngology – Head & Neck Surgery Foundation, Los Angeles, CA, September 25–28, 2005.     

Study on the Role of Histochemical Stains in Identifying Merkel Cells in Dogs

Merkel cells (MCs) are neuroendocrine cells involved with tactile sense, growth, differentiation, and homeostasis of the skin as well as in different cutaneous diseases. Specific staining techniques are required for their identification because they are not easily visible in paraffin sections stained with hematoxylin and eosin. The present study assess the histochemical features of the MCs in dogs comparing with those described for other mammals in the literature and with the use of immunohistochemistry. A systematic study of samples from MCs-rich areas from healthy dogs was carried out by use of several histologic stains, including metachromatic staining, silver stains, methylene blue, periodic acid–Schiff stain, and osmium-based staining method. MCs were detected by the Grimelius argyrophilic stain in 86.7% of the specimens. The staining was showed as dark-brown granular cytoplasmic and consistently polarized to the basal cell cytoplasm matching with the cellular distribution of the characteristic neurosecretory granules. Some modifications in the standard staining protocol, including rinsing, silver reimpregnation, and counterstain dye, enhanced the MCs identification in stratified squamous epithelium. When compared with Cytokeratin 20-immunolabeled serial sections several MCs appeared nonstained with the argyrophilic method. These differences in MC numbers between stains were statistically significant. Other histologic stains failed to identify MCs in the specimens. The results of this study indicate that Grimelius argyrophilic stain is a suitable method for demonstration of MCs in the stratified squamous epithelium of skin and mucosa. Discussion on its utility when compared with immunohistochemistry and a review of the scientific literature is also presented. Anat Rec, 302:1458–1464, 2019. © 2018 Wiley Periodicals, Inc.     

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)     

Routine elastic staining assists detection of vascular invasion in colorectal cancer

AIM: To determine the value of routine staining of colorectal cancer (CRC) specimens with elastic stains. METHODS AND RESULTS: Four hundred and ninety-eight cases of CRC were included. In 208 cases, vascular invasion (VI) was assessed using elastic stains [Elastic van Gieson or Elastin haematoxylin and eosin (H&E)] that were introduced as routine staining in CRC in our hospital. As a control, 290 cases in which VI was assessed solely on H&E staining were included. The site, stage and presence of vascular invasion were determined in both groups. The cost, time and workload resulting from the addition of these stains were also taken into account. The sensitivity of detection of VI in CRC was significantly improved after the introduction of these elastic stains in our routine practice (46.2% compared with 35.5% in the control group; P = 0.014). Particular improvement was noted in Dukes' stage A and B. We also noted that the net benefits gained from the introduction of these special stains outweighed the small extra cost and effort and, in addition, saved time for the reporting pathologists. CONCLUSION: Routine elastic stains are very useful and practical in evaluation of VI status in CRC and we recommend implementing these stains in routine pathological practice to improve patient care.     

The effects of multiple pregnancies and age on the elastic tissue of uterine arteries in the guinea pig

Mesometrial arteries and representative areas of the uterus were obtained from young and old postpartum guinea pigs. The tissues were fixed in Bouin's solution and then stained with Taenzer's Orcein and Verhoeff's elastic stains. Picro-indigo-carmine and Van Gieson were used as counterstains. It was found that the distribution of elastic tissue and the number of elastic and muscle layers in the arterial walls in general reflected the number of pregnancies the animal had had. This was found to be reliable only up to three pregnancies, after which the animals could be referred to only as being multiparous. There appeared to be a continuous increase in the amount of elastin in the blood vessels with each successive pregnancy. Further, aged animals showed more elastin in their vessels than younger ones of comparable parity. The formation of the new internal elastic membrane appeared to be delayed in older females. Our observations suggest that there might be a decrease in the postpartum resorption of elastin as a result of successive pregnancies and aging. They also suggest that the delay in the formation of the new internal elastic membrane in older postpartum females is probably due to aging.     

Diagnostic histochemistry in hepatic pathology

Histochemistry has an important, continuing role in the current assessment of hepatic biopsies and resection specimens. The evaluation of connective tissue elements in the liver can be accomplished with such methods as the Masson trichrome, Snook reticulin, Vierhoff van Gieson, orcein, and Victoria blue stains. The results contribute to the diagnosis of acute and chronic hepatitis, submassive necrosis, venous outflow obstruction, steatohepatitis, and cirrhosis. Fat stains done on frozen sections of liver tissue are routinely performed in the evaluation of donor liver allograft biopsies. Iron stains such as Perls’ method and the Prussian blue technique contribute to the recognition of hemochromatosis and hemosiderosis. The rhodanine, orcein, and Timm stains for copper are used in the characterization of chronic cholestatic liver disease and Wilson's disease. Labeling of carbohydrate-based moieties in various disorders is accomplished with the digested and undigested periodic acid-Schiff method, and Congo red or crystal violet stains can be employed to detect amyloid deposition. Lastly, evaluations of the thickness of the cell plates and continuity of the reticulin framework, as seen with the Snook reticulin stain, can contribute to the diagnostic separation of benign from malignant hepatocellular neoplasms.     

Routine argyrophil techniques detect Rickettsia rickettsii in tissues of patients with fatal Rocky Mountain spotted fever

Rickettsia rickettsii, a bacterial tickborne pathogen that causes Rocky Mountain spotted fever (RMSF), stains poorly or not at all with conventional tissue Gram techniques, and contemporary visualization of the pathogen in formalin-fixed, paraffin-embedded tissues has relied almost entirely on immunohistochemical staining methods that are generally limited to specialized research laboratories or national reference centers. To our knowledge, previously described argyrophil-based histochemical techniques have not successfully detected rickettsiae in formalin-fixed, paraffin-embedded tissues. To investigate the ability of standard silver impregnation techniques to demonstrate the occurrence and distribution of R. rickettsii in tissues of patients with RMSF confirmed by molecular and immunohistochemical methods, three widely recognized and commercially available silver impregnation methods (Warthin–Starry, Steiner, and Dieterle’s) were applied to various tissues obtained at autopsy from 10 patients with fatal RMSF. R. rickettsii bacteria were demonstrated in one or more tissues of all patients, using each of the argyrophil-based methods, and appeared as small, dark brown-to-black lanceolate rods, often in pairs and occasionally surrounded by a faint halo. Rickettsiae were identified most consistently in small arteries and arterioles of liver, kidney, and leptomeninges, and were localized predominantly to the cytoplasm of endothelial cells and less often within the internal elastic lamella and smooth muscle of the media. This validation of argyrophilic techniques to detect R. rickettsii demonstrates the utility of inexpensive core histochemical methods in the diagnosis of infectious agents in pathology specimens and may have utility in certain resource-limited settings where RMSF is endemic.     

Elastin and collagen IV double staining: A refined method to detect blood vessel invasion in breast cancer

Blood vessel invasion (BVI) is a prognostic indicator in various cancers. Elastic stain, which highlights blood vessel walls, is commonly used to detect BVI. In the breast, however, its diagnostic usefulness is limited because it also highlights some intraductal carcinoma components, which often mimic BVI. In this study, we aimed to improve BVI detection in breast cancer and developed a double staining: Victoria blue for elastin and immunohistochemistry for collagen IV. Collagen IV fibers were retained along the basement membranes of intraductal carcinoma components, whereas they were rearranged or lost in BVI. From these observations, we defined BVI as the presence of tumor cells inside an elastic ring with a rearrangement or loss of collagen IV fibers. Using these criteria, we found BVI in 148 cases (49%) among 304 cases of primary operable invasive breast carcinoma, and the presence of BVI correlated significantly with poor prognosis. By contrast, we detected BVI in 94 cases (31%) or 14 cases (5%) by elastic van Gieson or CD31 immunostaining among the same cases, respectively, with no statistically significant association with prognosis. Thus, elastin and collagen IV double staining facilitates the detection of BVI in breast cancer and is useful to predict prognosis.     

Value of elastin staining in the assessment of peritoneal implants associated with ovarian serous borderline tumours

AIMS: To determine whether elastin stains aid in classifying peritoneal implants associated with ovarian serous borderline tumours (SBT). METHODS AND RESULTS: The study group comprised 80 implants (nine invasive and 71 non-invasive) from 28 patients with ovarian SBT. Elastin stains were performed using histochemical and immunohistochemical methods to demonstrate the peritoneal elastic lamina (PEL), and evaluated with regard to assessment of the subtype of implant. The elastin stains demonstrated the PEL in most anatomical sites other than the omentum and the bladder and were considered helpful in 44/80 (55%) cases. The stains were most useful in the assessment of poorly oriented or traumatized biopsy specimens and in confirming the superficial distribution of non-invasive implants. The staining was non-contributory in most of the remaining biopsies, because the PEL was not identified. CONCLUSIONS: Demonstration of the PEL using elastin stains can be useful in the subclassification of implants associated with ovarian SBT and is of most value in confirming the superficial distribution of non-invasive lesions. However, evaluation is limited by the absence of a defined elastic layer in a proportion of biopsy specimens, possibly reflecting their superficial location, as well as absence of a distinct PEL in sites such as the omentum.     

Studies on elephant aortic elastic tissue. I. The histochemistry and fine structure of the fiber

Elephant arterial elastic laminae were shown to be refractory to staining by orcein or the resorcin dyes, both normally regarded as routine elastic tissue stains. To investigate this more thoroughly, elastin was isolated from arterial tissue by alkaline hydrolysis and studied in vitro. Compared to elastin from other species, elephant elastin was found to resist alkaline hydrolysis to a greater extent, to possess a greater UV absorption at 275 nm, and to show an unusual fluorescence at 415 nm. Electron micrographs of elastic fibres in situ demonstrated the presence of large amounts of a microfibrillar sheath surrounding the amorphous core.These results are interpreted to indicate the presence, in elephant arterial elastic tissue, of unusually large amounts of a nonelastin component which interferes with the normal staining reactions.     

Use of Heat to Improve Connective Tissue Stains: Modifications of Masson,Movat, and Fibrin Stains

Abstract

Three connective tissue methods are presented: modifications of Masson trichrome, Movat pentachrome and a fibrin method. A modified Verhoeff hematoxylin preheated and applied in the 60°C paraffin oven was used for all methods. The Movat pentachrome modification additionally included staining with alcian blue before application of Verhoeff hematoxylin, and the fibrin method was stained with lissamine fast yellow before application of the working red stain. All sections were stained with a working dilution of Biebrich scarlet and acid fuchsin, rinsed, and differentiated with phosphotungstic acid in a 60°C paraffin oven. Demonstration of collagen in the modifications of Masson and fibrin was done with either light green or aniline blue; saffron was used in the Movat pentachrome.

All 3 techniques were improved in quality and precision with the aid of heat. Although fibrin was demonstrated in all techniques, minute quantities were better seen in the fibrin stain because the red cells were stained in a different color. These modified stains demonstrated several entities in a single slide preparation in about 20 min. (The J Histotechnol 16:349, 1993)     

A modified silver methenamine Masson trichrome stain using methyl green for staining of renal biopsies

Renal pathology uses a battery of stains to allow proper assessment of all renal components. One of the most useful of these stains is the silver methenamine with a Masson trichrome counterstain (SMMT). The SMMT stain uses thin 1-um sections to identify immune complexes deposits on the basement membranes and in the mesangium of the glomerulus and can provide an excellent and inexpensive method. Problems can, however, occur when the stain is not performed by a technician or the stain needs to be performed in large numbers. A variation to the SMMT method has proved to provide a better, more robust and reproducible counterstain. The new method substitutes Light Green SF, used in the traditional Masson trichrome stain, with Methyl Green, a dye with much smaller molecule size, allowing better penetration and a stronger bonding to the tissue components.     

Pulmonary elastic fiber degradation in paraquat toxicity. An electron microscopic immunohistochemical study

To study the morphologic alterations of pulmonary elastic fibers in cynomolgus monkeys with paraquat toxicity, peroxidase- and ferritin-labeled antielastin antibodies were used for the light and electron microscopic localization of elastin. One week after paraquat, alveolitis, tissue damage and alveolar dilatation were present; elastic fibers were frayed and more diffusely and intensely stained than those of control animals. In the latter, staining was localized in peripheral regions of the amorphous components and, to a lesser extent, in some microfibrils of elastic fibers. At 3 to 4 weeks, diffuse staining was evident in damaged interstitial elastic fibers and in newly formed elastic fibers in areas of intraalveolar fibrosis. At 8 weeks, the interstitium contained many elastic fibers which showed staining only in peripheral regions of the amorphous components. These observations suggest that: 1) preembedding immunohistochemical staining for elastin is localized in peripheral regions of normal elastic fibers because the antielastin antibody can penetrate into mature and undamaged amorphous components only to a very limited extent; 2) in early stages of paraquat toxicity this staining is more diffuse and intense because elastase from inflammatory cells partially degrades the elastic fibers and permits greater penetration of the antibody into the amorphous materials; 3) in later stages the staining pattern returns to normal as inflammation subsides and elastic fibers are repaired; however, newly formed elastic fibers in areas of intraalveolar fibrosis stain diffusely, reflecting increased penetration of the antibody because of immaturity and incomplete cross-linking, and 4) degeneration of elastic fibers of alveolar walls in paraquat lung may lead to alveolar dilatation, which is associated with irregular fibrosis and constitutes one of the processes of pulmonary structural remodeling in paraquat lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

三种改良的阮森吡啶-硝酸银法显示大鼠脊髓组织的比较

目的 对3种改良的阮森吡啶-硝酸银法(Ranson)在显示大鼠脊髓组织中的应用进行比较. 方法 对照组采用传统的阮森吡啶-硝酸银法,即取大鼠的新鲜脊髓组织,在酒精中加纯氨水固定,经纯吡啶、硝酸银、还原液染色,常规石蜡切片并观察.实验组采用改良的阮森吡啶-硝酸银法,即分别采用NaOH溶液、Na2CO3溶液和95%酒精替换在对照组中使用的纯氨水,其他染色步骤同对照组. 结果 实验组的脊髓组织切片分别经3种改良的阮森吡啶-硝酸银法染色,神经元的胞体和突起均呈黄棕色,神经纤维呈棕褐色,切片染色结果与对照组比较无明显差异,尤以酒精替换组的染色效果为最佳. 结论 以改良的阮森吡啶-硝酸银法显示大鼠脊髓组织中神经元及神经纤维染色效果良好,方法更加简便易行,且避免了氨水的刺激气味,有利于改善教学和科研的局部工作环境.     

A novel mitochondria-targeting method using special staining for the detection of apoptotic hepatocytes

ABSTRACT

Early detection of apoptotic cells on histological slides is of major importance for both diagnostic and research areas. In the current study, the aim was to propose a convenient method to stain the mitochondria and establish whether hepatocytes undergoing apoptosis can be identified in tissue sections using the proposed method. Liver tissue from five adult chinchillas was fixed with 10% neutral buffered formalin for Goldner’s trichrome (GT) and Groat’s iron hematoxylin and eosin (HE) stains and with Kolster’s fixative for the Heidenhain’s iron hematoxylin procedure. The HE and GT-stained sections showed the morphological features consistent with apoptosis i.e., homogenous intensely acidophilic cytoplasm, cell shrinkage with an irregular outline, nuclear shrinkage with cloudy karyoplasm, and karyopyknosis in the late stage. Sections stained with Heidenhain’s iron hematoxylin method was used to pinpoint mitochondria and revealed cells which were undergoing the first stages of the apoptosis process i.e., disappearance of mitochondria from the cell, chromatin condensation and margination, paracentral localization of nucleoli, and vacuolated nuclei. In more advanced stages of apoptosis, cells presented significant nuclear and cytoplasmic changes. It was concluded that this is the first report targeting the mitochondria, by performing inexpensive histological staining techniques, in order to assess dead cells in situ.     

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.     

咽部特殊感染症病理特点及病原体检测的探讨

目的 探讨咽部特殊感染性疾病的病理特点,研究其病原体的检测方法,以提高特殊感染症的诊断水平.方法 分析北京同仁医院1998年1月至2008年1月咽部61例黏膜溃疡病患者的临床病理资料,以简易的组织芯片染色技术,对61例咽部黏膜溃疡组织,用PAS、Giemsa、革兰、美蓝、改良的Warthin-Starry(W-S)及抗酸染色进行集中染色,比较各种染色方法对病原体的染色效果,找出不同病原体的最佳染色方法.结果 组织切片内发现密螺旋体23例,短螺旋体10例,分枝杆菌4例,鼻硬结杆菌4例,霉菌侵袭1例,细菌和白色念珠菌混合感染1例,扁桃陷窝放线菌2例.非特异性细菌感染16例.结论 螺旋体、鼻硬结杆菌均以改良的W-S染色效果最佳,真菌染色应根据真菌的不同类型联合应用革兰及PAS、W-S染色进行鉴别,分枝杆菌以传统的抗酸染色效果最好.     

Benign vaginal polyp: a histological, histochemical and immunohistochemical study of 20 polyps with comparison to normal vaginal subepithelial layer

Twenty benign vaginal polyps from 18 patients, together with sections from normal vaginal epithelium, were studied histologically, histochemically using elastic van Gieson stain and immunohistochemically using monoclonal antibodies against vimentin, desmin and actin. The striking finding was the similarity, both histologically and immunohistochemically, of the stroma of vaginal polyps to that of the loose subepithelial layer found in normal vagina. The important difference was the marked degeneration of the elastic tissue, increased number of stellate and giant fibroblasts and subepithelial condensation of fibroblasts in the polyps. These findings support the hypothesis that vaginal polyps may represent a reactive hyperplasia of the loose subepithelial zone of the vaginal wall.