Staining efficacy assessment of a differential routine and special stains for pathological stromal calcifications in maxillofacial lesions

ABSTRACT

Head and neck connective tissue lesions may have diverse calcifications within the fibrous connective tissue stroma. The perplexity involved in the identification and determination of the nature or degree of calcification through routine hematoxylin and eosin (H&E) stains necessitates the usage of a specific, simple, and cost- and time-effective differential staining techniques. The aim of the present study was to develop criteria to distinguish bone formation from bone resorption using methylene blue-acid fuchsin (MB/AF) stain and the role of collagen fibers in the identification of stromal calcifications using polarizing microscopy with picrosirius red stain. Twenty cases with pathological diagnoses for various stromal calcifications in maxillofacial lesions were retrieved from the departmental archives. Decalcified formalin fixed paraffin embedded tissue sections were stained with hematoxylin and eosin, Masson’s trichrome (MT), methylene blue-acid fuchsin (MB/AF), and picrosirius red. The stained sections were assessed to identify the calcifications found in the surrounding connective tissue stroma. It was observed that most cases showed maximum staining intensity with MB/AF stain as compared to the other staining methods. Moreover, the results suggested that contrast between calcification and stromal soft tissue was best distinguished with the MB/AF stain except in the case of dystrophic calcifications. Along with this, polarizing microscopy with picrosirius red enables better characterization of stromal components. Although the H&E stain and a connective tissue stain i.e. Masson’s trichrome, are employed routinely in histopathology; the use of special stains such as MB-AF and picrosirius red facilitates the identification of calcifications from the stromal tissues.     

Embolized crospovidone (poly[N-vinyl-2-pyrrolidone]) in the lungs of intravenous drug users.

Crospovidone is an insoluble polymer of N-vinyl-2-pyrrolidone that is used as a disintegrant in pharmaceutical tablets. It can potentially embolize to the lung when aqueous tablet suspensions are injected intravenously. In this report, we identified embolized crospovidone in autopsy-derived lung tissue from three adult IV drug users, 1 man and 2 women, whose ages respectively were 27, 38, and 40 years. Suspected crospovidone was compared with pharmaceutical-grade crospovidone by means of histochemical stains, transmission electron microscopy, and infrared spectroscopy. Similar particles were also observed by light microscopy in a 4-mg tablet of hydromorphone, a preparation prescribed to two of the patients. Two patients had sickle cell disease and were taking methadone and/or hydromorphone for pain management; the third was receiving parenteral hyperalimentation after small bowel resection. Crospovidone appeared as deeply basophilic, coral-like particles within pulmonary arteries and in extravascular foreign-body granulomas. Intrapulmonary crospovidone stained similarly to the pure substance, including intense staining with mucicarmine, Congo red, and Masson trichrome. With Movat pentachrome stain, both intravascular and purified crospovidone appeared orange-yellow, whereas most interstitial particles associated with giant cells stained blue-green. Alcian blue failed to stain intravascular or purified crospovidone but strongly decorated some phagocytized particles. Ultrastructurally, both purified powder and tissue deposits of crospovidone appeared as irregular, electron dense, laminated, and finely granular material. Intrapulmonary crospovidone was associated with inflammatory cells and exhibited degenerative changes. By infrared spectroscopy, crospovidone in tissue had the same spectral characteristics as pharmaceutical grade crospovidone and the library reference, polyvinylpyrrolidone (PVP). We conclude that crospovidone contributes to pulmonary vascular injury in some persons who illicitly inject pharmaceutical tablets. It is readily identifiable histologically and distinguishable from other tablet constituents, such as cornstarch, talc, and microcrystalline cellulose. The variable staining with Alcian blue and Movat suggests that crospovidone is altered in vivo by the inflammatory response.     

组织化学染色技术在超薄生物塑化标本中的应用

目的 探讨组织化学染色技术是否可以应用于塑化标本并验证染色塑化标本是否具有自发荧光。 方法 选取1个手掌标本经超薄生物塑化技术做成组织块,进行连续切片,切片数量56张,并按切片顺序进行染色处理：原始切片,苏木素-伊红染色,凡尔霍夫- 酸性品红染色,亚甲蓝-天青Ⅱ号染色,在扫描仪、光学显微镜和激光扫描共焦显微镜下观察染色效果和组织结构特点并进行比较。 结果 3种染色技术都可应用于塑化切片染色。苏木素-伊红染色显示肌肉、结缔组织呈红色或紫红色,骨质呈紫蓝色或蓝色。凡尔霍夫-酸性品红染色显示弹力纤维呈黑色,胶原呈红色,其他组织为黄色或棕黄色。亚甲蓝-天青Ⅱ号染色显示肌腱呈紫红色,骨质呈粉红色,软骨呈紫罗兰,其他组织呈紫色。但细胞内结构的染色效果并不理想。在激光扫描共焦显微镜下,胶原纤维、弹力纤维和肌肉纤维具有自发荧光,结构清晰可辨。结论 常用的组织化学染色技术适合于超薄塑化切片染色,染色切片的各种组织结构比未染色的观察效果好。染色后,塑化切片中具有自发荧光的结构在激光扫描共焦显微镜下亦可清晰显影。     

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.     

三色染色法和免疫细胞化学法显示培养的神经轴索及雪旺细胞

本研究目的在于用三色染色法和免疫细胞化学反应显示培养的周围神经中的轴索及雪旺细胞。将大鼠背根神经节体外培养于聚吡咯膜上 2周 ;用苏木精、固绿 FCF、变色素 2 R及磷钨酸等配制的染色液染色 ;或用抗 S-10 0蛋白和抗神经微丝蛋白抗体进行免疫细胞化学法反应。结果证明 ,在三色染色法中神经节神经元发出的长突起呈蓝绿色 ,细胞核呈红色或紫红色 ,胞质呈浅灰色。免疫细胞化学反应证明神经节发出的长突起为轴索 ,紫红色核和浅灰色胞质的细胞为雪旺细胞。本文结果提示 ,三色染色法能区别显示培养的周围神经组织中的轴索及雪旺细胞。     

Novel Histochemical Stain for Tinctorial Detection of Cancer and Neoplastic Cells

Abstract

Zetiq has introduced a histochemical stain that claims to tinctorially identify cancer and neoplastic cells. Because of the potential importance of such a capability, we undertook to investigate Zetiq's CellDetect® staining technology as applied to cultured cell lines as well as an initial sample of clinical cases. This goal was pursued by investigating four types of comparisons: 1) cancer cell lines before and after differentiation; 2) cervical squamous-cell carcinoma (SCC) biopsies to non-neoplastic squamous epithelium; 3) SCCs to neoplastic, nonmalignant squamous epithelium; and 4) neo-plastic squamous cells to non-neoplastic squamous cells in cytological preparations. The clinical material was also stained with hematoxylin and eosin (biopsies) or Pap (cytologies) for diagnostic purposes. We found that all CellDetect®-stained cells exhibited one of the two tinctorial outcomes. Cell lines with malignant phenotype uniformly had red/purple cytoplasm, whereas the differentiated phenotype changed the color to blue/green. Moreover, these two color values are sufficiently distinct that they can be readily distinguished quantitatively with an ELISA reader when applied to cells in tissue culture. Biopsies of SCC and non-neoplastic tissues exhibit the same two color values: cancers had red/purple cytoplasm, whereas non-neoplastic tissues were green/blue stained. In contrast, neoplastic, nonmalignant tissues (dysplasias) stained red/purple, similar to SCCs. Cytological preparations gave similar results: neoplastic cells stained red/purple, whereas non-neoplastic cells exhibited green/blue cytoplasm. The study demonstrated that the staining was rapid and reproducible. Conclusion: The CellDetect® stain allows detecting cancer and neoplastic cells tinctorially based on a rapid, reproducible histochemical process.     

Histological analysis of clipped human intracranial aneurysms and parent arteries with short-term follow-up

BackgroundSurgical clipping of intracranial aneurysms is the gold standard for the prevention of rupture. However, the biological processes that occur following clipping are poorly understood. To better understand these effects, retrieved and clipped human intracranial aneurysms were examined histologically.MethodsAt autopsy, 17 aneurysms from 10 patients were retrieved 3–21 days after clipping. The tissues were embedded in paraffin, and microtome sections were stained using hematoxylin–eosin and Movat pentachrome. Using light microscopy, clip placement relative to the internal elastic lamina of the parent artery, endothelialization of the aneurysm neck, thrombus organization inside the aneurysm sac, inflammation in the sac, wall, and parent artery, and atherosclerotic changes were determined.ResultsDespite complete reconstruction of the artery with the clip, diseased vessel wall was frequently observed outside the clip. By 10 days postsurgery, the beginnings of endothelialization and neointima formation were observed at the neck. However, the neck coverage was variable and incomplete at these early time points. Thrombus organization inside the aneurysm sac was rarely observed, and inflammatory cells were not present inside the aneurysm sac. Inflammatory cells were commonly observed in the aneurysm wall, and atherosclerotic change was present in each sample.ConclusionsComplete aneurysm exclusion and apposition of healthy arterial wall occurred infrequently in our series. Endothelialization and neointima formation at the aneurysm neck take some time to complete and are often incomplete. The effectiveness of aneurysm clipping is related to the mechanics of aneurysm exclusion rather than the processes of endothelialization and neointima formation.SummaryComplete aneurysm exclusion and apposition of healthy arterial wall occurred infrequently in our series. Endothelialization and neointima formation at the aneurysm neck take some time to complete and are often incomplete. The effectiveness of aneurysm clipping is related to the mechanics of aneurysm exclusion rather than the processes of endothelialization and neointima formation.     

Collagen-rich crystalloids in a scarred vascularized cornea

Collagen-rich crystalloids, also referred to as collagenous crystalloids, are uncommon findings in benign salivary gland tumors with myoepithelial differentiation and in cutaneous neoplasms. Herein, we report the presence of collagen-rich crystalloids in the scarred, vascularized cornea of a 56-year-old woman. The patient underwent penetrating keratoplasty, and microscopic examination of hematoxylin-eosin-stained sections disclosed large aggregates of refractile material within the corneal stroma. The deposits were partially birefringent when viewed with polarized light and composed of radially arranged columns (long rectangles) with rounded to pointed tips. The deposits had tinctorial properties of collagen using Masson trichrome and the van Gieson method for collagen, and they stained with Alcian blue, pH 2.5, and Verhoeff elastic stain. They did not stain with Gomori methenamine silver, Snook reticulin stain, or tyrosine using the Baker modification of the Millon reaction. This is the first report, to our knowledge, of collagen-rich crystalloids in the cornea. Their presence in the cornea indicates that these structures may occur in the absence of neoplasia.     

A small reduction in control tissue size yields a significant reduction in digital image storage cost

As digital pathology is becoming more prevalent, the cost of image storage becomes factor to consider. The purpose of this study was to test the effect of reducing the tissue size of frequently scanned slides on digital whole slide image file size and file storage cost. We investigated the file sizes of our most commonly scanned slides – the daily special stain controls. The average file size of these stains ranges from 109 to 476 MB. All of the Masson trichrome slides, Movat pentachrome slides, and PAS slides were reviewed from 2015, and the tissue measurements and file sizes were recorded. New, smaller control tissue blocks were created. Once scanned, the tissue measurements and file sizes were also recorded. Overall, we reduced the size of the tissue on the slide by an average of 74%. We calculate that for these three control tissues alone, $3465 in storage costs could be saved every year by reducing the size of the tissue on the slide.     

Characterization of acrylic polyamide plastic embolization particles in vitro and in human tissue sections by light microscopy, infrared microspectroscopy and scanning electron microscopy with energy dispersive X-ray analysis.

Vascular embolization is a well-established practice for the treatment of tumors and vascular lesions. Rounded beads (microspheres) of various materials (collagen, dextran and trisacryl-polymer-gelatin) were developed to solve problems encountered with earlier versions of embolic material. We performed histochemistry, Fourier transform infrared microspectroscopy and scanning electron microscopy with energy dispersive X-ray analysis on two uterine and one hepatic specimen with unidentified intravascular foreign material, and examined a reference embolization product for comparison. The hematoxylin and eosin stained tissue sections showed multiple foci with unidentified intravascular foreign material and fibrous obliteration of vessel lumens. Only one case had a clinical history of previous embolization but without specifying the material used. One case was submitted for identification of a 'parasite'. The material stained positively with Sirius red and mucicarmine, variably with Masson's trichrome stain and Movat pentachrome, and did not stain centrally with periodic acid Schiff with diastase. Infrared spectrophotometric analysis of the material from all three cases demonstrated the spectrum of acrylic polyamide plastic. A control sample of EmboGold exhibited infrared microspectroscopic spectra similar to the three tissue specimens. Analysis by scanning electron microscopy with energy dispersive X-ray analysis demonstrated some differences in elemental composition between the tissue sections and the selected reference material. To our knowledge, this is the first report of infrared spectrophotometric analysis with scanning electron microscopy with energy dispersive X-ray analysis of an acrylic polyamide plastic embolization product both in vitro and in human histologic tissue sections. In cases lacking appropriate clinical information, identification by these methods and/or a panel of special stains may assist pathologists unfamiliar with this material's light microscopic appearance.     

CD117-positive cells and mast cells in adult human cardiac valves--observations and implications for the creation of bioengineered grafts.

BACKGROUND: There is no report to date of stem cells in human cardiac valves. We examined their possible presence, number, and distribution in valves removed at cardiac surgery from patients with a variety of underlying valve pathologies. METHODS: Grossly normal aortic and mitral valves were obtained from live heart transplant patients. Surgically excised valves with rheumatic mitral stenosis, aortic valve age-related degeneration, aortic valve changes of aortoannular ectasia, and mitral valves with myxomatous degeneration were studied. Immunohistochemical and histochemical studies were performed on sequential valve sections, including hematoxylin and eosin, hematoxylin phloxine saffron, Movat pentachrome, toluidine blue, CD31, CD34, and CD117. RESULTS: There were small clusters of CD117-positive cells in the fibrosa and spongiosa of mitral and aortic valves from all groups of valves. Sequential sectioning and staining showed that almost all of these cells were mast cells. However, in the mitral myxomatous valves and the mitral rheumatic valves, there were rare CD117-positive cells that did not have corresponding toluidine blue staining and thus could be valve mesenchymal stem cells. CONCLUSIONS: Most of the CD117-positive cells in normal and diseased adult heart valves are mast cells. These valve cells could play a role in valve pathology and injury. A very small number of possible valve stem cells were also identified. It is unlikely that these valve stem cells are sufficient in number to allow isolation and expansion for tissue engineering purposes.     

Special Stains in the Microwave Oven

Abstract

We describe the modification of a number of stains for the microwave oven: Grocott's methenamine silver, acid fast bacteria, Giemsa, Lillie's melanin, methyl green pyronin, Masson's trichrome, and glycogen double digestion. Their adaptation has markedly reduced the time needed for staining. The staining procedures described here can be used on paraffin sections, frozen sections on coated slides, touch preparations, and cytology specimens.     

Counterstains for the Uzman Rubeanic Acid Technique for Copper in Microscopy,Color and Monochrome Photomicrography

Abstract

For maximal color contrast with the dark green copper salt formed in the Uzman reaction, a light red counterstain is required. Nuclear fast red, which stains nuclei light red and cytoplasm a faint pink, gives excellent color and tonal contrasts. This results in high visibility of copper salts and delicately stained tissues, allowing the viewer both to identify tissues and to appreciate the location of the copper deposits. Nuclear fast red, eosin, methylene blue, metanil yellow, picric acid and Harris' hematoxylin were evaluated as counterstains. Nuclear fast red was the counterstain of choice for visual observation, color photomicrography and monochrome photomicrography. Counterstains of eosin and methylene blue were adequate; metanil yellow and picric acid were barely adequate. Hematoxylin and no counterstain were unacceptable for monochrome photomicrography. Nuclear fast red counterstained Uzman sections required a Wratten #11, #59 or #58 filter in monochrome photomicrography, the exact filter depending on the density of the counterstain. Lightly nuclear fast red counterstained tissues required the use of a #58 filter, the more densely counterstained sections, a #59 or #11 filter.     

Microwaveable Toluidine Blue Stain for Surface Staining of Undecalcified Bone Sections

Abstract

Toluidine blue stain has been widely used for staining undecalcified bone sections, but previous techniques required removal of embedding plastic or surface etching with both alcohol and acid solutions. In this study, solutions of toluidine blue at different pHs were used to stain unetched and acidetched sections. A group of the test staining solutions were heated in an effort to enhance section staining. Acid-etching prior to staining was found to be necessary to achieve bone matrix staining, but methanol etching could be eliminated if staining solutions were heated to 55–60°C. Sections stained with the heated solutions buffered to pH 7.0 or 8.0 had optimum morphology. (The J Histotechnol 17:357, 1994)     

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)     

Effects of isopropanol storage time on histochemical and immunohistochemical stains in lung tissue

Abstract

Objectives: Prolonged storage of tissues in formalin is known to result in progressively increasing cross-linking of proteins and subsequent difficulties in histochemical and immunohistochemical staining. One solution to this problem is to fix the tissues in formalin and then store them in alcohol. Our institution uses isopropanol for this purpose. However, little is known on how isopropanol storage may affect the quality of subsequent staining of sections taken from these tissues.

Methods: To test this hypothesis, lung tissue samples from a transplant donor lung were fixed for 24 hours in 10% neutral buffered formalin and then stored in 70% isopropanol at 4°C for 0 days, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, and 24 weeks. All lung tissue samples were stained for hematoxylin and eosin, Masson’s trichrome, periodic acid Schiff, CD31, CD68, and smooth muscle actin. Staining intensities were scored (1?=?weak; 2?=?moderate; and 3?=?intense) and the inter-observer variability was evaluated.

Results and discussion: There were no significant differences in the staining intensities of hematoxylin and eosin, Masson’s trichrome, periodic acid Schiff, CD31, CD68, and smooth muscle actin in the lung tissue samples with increasing storage time in isopropanol. Thus, the tissues that are stored in isopropanol at 4°C can yield good quality staining for research and clinical investigations.     

Small Intestinal Stromal Tumors with Skeinoid Fibers in Neurofibromatosis: Report of Four Cases with Ultrastructural Study of Skeinoid Fibers from Paraffin Blocks

Small intestinal stromal tumors (SIST) with skeinoid fibers (SF) are reported in four patients with neurofibromatosis. SF appeared as intercellular eosinophilic globules that stained positively on periodic acid-Schiff stain and bluish on Masson trichrome stain, like those reported previously. Electron microscopic confirmation of SF was carried out on tissue retrieved from paraffin blocks, which revealed diagnostic ultrastructural features of SF. The association of SIST with SF and neurofibromatosis enhances the diagnostic value of SF as a marker of neurogenic spindle cell tumors. This study also underscores the importance of electron microscopy for differentiation of SF from pseudoskeinoid structures, which may mimic SF on light microscopic examination.     

Small Intestinal Stromal Tumors with Skeinoid Fibers in Neurofibromatosis: Report of Four Cases with Ultrastructural Study of Skeinoid Fibers from Paraffin Blocks

Small intestinal stromal tumors (SIST) with skeinoid fibers (SF) are reported in four patients with neurofibromatosis. SF appeared as intercellular eosinophilic globules that stained positively on periodic acid-Schiff stain and bluish on Masson trichrome stain, like those reported previously. Electron microscopic confirmation of SF was carried out on tissue retrieved from paraffin blocks, which revealed diagnostic ultrastructural features of SF. The association of SIST with SF and neurofibromatosis enhances the diagnostic value of SF as a marker of neurogenic spindle cell tumors. This study also underscores the importance of electron microscopy for differentiation of SF from pseudoskeinoid structures, which may mimic SF on light microscopic examination.     

Citrate Buffer Alternative to Picric Acid for Masson Trichrome Stain

Abstract

An alternative to picric acid as a mordant in the staining of cytoplasm, keratin, muscle fibers, and intercellular fibers in the Masson trichrome stain is a citrate acid-sodium citrate buffer used as the microwave pretreatment of the tissue mounted on a slide. The Masson trichrome stain, a combination of Biebrich scarlet-acid fuchsin and aniline blue dye can be done within 1 hr and avoids the hazards associated with picric acid, a highly explosive compound in the dry state. (The J Histotechnol 19:341–342, 1996)     

Histopathological study on fat necrosis with comparison between agonal stage and acute pancreatitis

A histopathological study on fat necrosis with a comparison between patients in agonal stage and those with acute pancreatitis was presented. Fat necrosis was observed in 15 out of 56 autopsied cases (26.8%) which were selected randomly from the autopsy file. Fat necrosis was frequently stained blue or purple with Nile blue stain, suggestive of acid lipid. The necrosis was usually accompanied by fibrin thrombi in the capillaries and reparative foam cells, nor hemorrhage and hemosiderin deposition. In acute pancreatitis fat necrosis also reacted blue or purple with Nile blue stain in 8 out of 9 patients. Reparative reaction was composed of granulation tissue in 6 patients, frequently with organized thrombosis in the veins. Fat necrosis was accompanied by fibrin thrombi and either hemorrhage or hemosiderin deposition. Therefore, differences in both hemorrhage or hemosiderin deposition and reparative change were observed in fat necrosis between patients in agonal stage and those with acute pancreatitis.