Alkaline Congo Red Staining for Amyloid

Abstract

An investigation, primarily of the stock Congo red solution for the staining of amyloid, was made. It was found that some samples of Congo red contained a second component which was only slightly soluble in NaCl ethanol. This made definition of- a saturated solution impossible. However, 50 mg Congo red in 50 ml of NaCl ethanol mechanically stirred for 15 minutes, then adding 0.5 ml 1% NaOH and filtering and staining in this solution for 20 minutes, gave an adequate stain for amyloid.     

Diagnosis of renal amyloidosis using Congo red fluorescence

Early diagnosis and classification of amyloid deposition and differentiation from other glomerular fibrillar deposits relies on routine Congo red (CR) histochemistry. Congo red fluorescence (CRF) is an alternative method based on examination of the CR-stained section by ultraviolet (UV) light. The aim of this study is to investigate the usefulness of CRF, especially when applied to frozen kidney sections. Congo red fluorescence was applied to sections of frozen kidney biopsies prospectively and to paraffin sections retrospectively. The findings of CRF were compared to CR staining in bright light. Prospectively, 15 cases of amyloidosis were diagnosed on frozen sections and identical CR staining was found in all of the paraffin-stained sections. There were no false positives or negatives. Retrospectively, 146 renal biopsies previously stained with CR were re-evaluated with CRF. Eighty-seven CR positive cases were confirmed by CRF, and one new case was identified. Congo red fluorescence is simple to perform and more pronounced, therefore easier to evaluate than CR in bright light. Congo red, when combined with immunohistochemistry, is still visible under UV whereas CR is masked in bright light. Although not widely used, the CRF method for detecting amyloid is simple to use with a high specificity and sensitivity, and may be applied successfully to frozen sections.     

形态学检验方法在心肌组织样本中的应用

目的探讨形态学检验方法在心肌疾病中的应用,评价其优缺点。方法运用磷钨酸苏木素(PTAH)染色法、Masson 3色染色法、弹力纤维组织(ET)+Van Gieson(VG)染色法、刚果红染色法、过碘酸雪夫氏(PAS)染色法、革兰氏染色法、以及免疫组织化学法及透射电子显微镜对心肌组织进行检测。结果 HE染色对心肌组织的区分度不好;Masson 3色染色法对心肌细胞和胶原纤维有明确的区分作用;ET+VG染色法可以很好地区分弹力纤维、胶原纤维、肌纤维和淀粉样物质;刚果红染色主要用于淀粉样物质的检测;PAS反应可辅助诊断糖原累积症;免疫组织化学(IHC)技术在疾病模型的研究及对疾病的观察中具有重要辅助作用;电子显微镜对超微结构的观察是病因学诊断的重要手段。结论运用组织水平、亚细胞水平以及分子水平的形态检验方法,对心肌组织样本进行观察与评估,可以得到较好的效果。     

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)     

Environmentally Friendly Mercury-Free Hematoxylin

Abstract

A modified rapid method for preparing hematoxylin is described. The technique is based on avoidance of the heavy metals generally used in ripening procedures or other toxic and environmentally hazardous compounds. This formula results in a ready to use, high quality product that keeps well indefinitely. It can be used in almost every situation requiring hematoxylin staining, is equally good for staining plastic and paraffin embedded material as well as fresh frozen cryostat tissue sections, and can be used for cytological staining procedures. (The J Histotechnol 16:371, 1993)     

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.     

NSH in Action

Abstract

In his phosphotungstic acid-hematein (PTAH) formula, Mallory combined hematoxylin with phosphotungstic acid (PTA) and then added the oxidant potassium permanganate. As hematoxylin is oxidized, protons of the strongly acid PTA compete with the metal for binding sites and much hematein is coverted into hematoxylin-like compounds. Owing to this chemically unsound procedure, PTAH solutions require “ripening” for several months to yield optimal coloration of tissues. Therefore, we modified the method to facilitate chelate formation. Hematoxylin was oxidized with potassium permanganate for 30 minutes, PTA was added, and the solution was allowed to stand for one hour. In a simplified technic, the staining properties of this PTAH solution were similar to those of traditional formulas aged for six months. Owing to steric conditions in PTA, red and blue PTAH compounds are formed. These two fractions are bound to tissues via different chemical mechanisms. The blue component is sensitive to heat and is partly converted into red compounds during staining at 60°C. The modified PTAH solution proved suitable for tissues fixed in methacarn, 10% unbuffered or buffered neutral formalin, or Zenker-formol. However, overfixation with the latter three fixatives impaired binding of the blue component of PTAH in peripheral areas of blocks. (The J Histotechnol 11:153,1988.)     

The congo red stain revisited

The Congo red stain has undergone several modifications since it was first used by Bennhold in 1922 in order to increase the specificity for staining amyloid. Most of the laboratories in the United States use the method of Puchtler which uses alkaline Congo red solution. Some of the variables associated with the procedure were investigated by us. Our results showed the following: (1) amyloid showed green birefringence at all levels between 4 to 12 mu thick sections with better visualization of small deposits with increased thickness. Best results were obtained with 8 mu thick sections; (2) omission of the pretreatment with alkaline alcoholic solution of sodium chloride (NaCl) did not affect the sensitivity of the method; (3) the use of polar mounting media had no effect on amyloid and collagen birefringence; (4) 50 percent saturation of the Congo red staining solution with NaCl caused strong staining of collagen, elastic fibers and eosinophilic granules. In addition, collagen showed green birefringence and dichroism and its differentiation from amyloid became difficult; and (5) using the staining solution fully saturated with NaCl, no positive staining was seen with tissues other than amyloid. Collagen and elastic fibers showed red fluorescence which was of less intensity than amyloid. It is our conclusion that the method of Puchtler for detecting amyloid gives better results if the staining solution is fully saturated with NaCl. The pretreatment step may be deleted without compromising the quality of staining. Improved staining of amyloid enhances the specificity of green birefringence, dichroism, and red fluorescence.     

Abdominal fat tissue aspirate in human amyloidosis: light, electron, and immunofluorescence microscopic studies

Abdominal fat tissue aspirates from 12 patients with biopsy-proved amyloidosis were investigated by different morphologic techniques. By light microscopy, after staining of the fat tissue aspirates with Congo red and examination with a polarizing microscope, positive results were obtained in nine patients with amyloidosis, two of the three with primary (AL) amyloidosis and seven of the nine with secondary (AA) amyloidosis. By indirect immunofluorescence, using AA antiserum, positive results were obtained in five of the nine cases of AA amyloidosis (aspirates from these five patients were positive on Congo red staining). By electron microscopy, amyloid fibrils were observed in five cases of amyloidosis (two of the AL and three of the AA type, all positive on Congo red staining). Although amyloid was demonstrated less frequently by immunofluorescence and electron microscopy, perhaps because of the small numbers of fat particles examined, it seems that, with Congo red staining, abdominal fat tissue aspiration is a simple and sensitive method for the diagnosis of amyloidosis. Immunofluorescence studies allow discrimination between the different types of amyloidosis. The method could be used in patients in whom other types of tissue biopsy are not recommended because of risks of bleeding or other problems.     

Sensitivity and specificity of Congo red staining according to Romhányi. Comparison with Puchtler's or Bennhold's methods

The sensitivity and specificity of various Congo red staining methods is very important in the diagnosis of amyloidosis. When using a less sensitive staining method, some true positive cases of amyloidosis remain undetected. A more highly specific method potentially detects more cases and reveals amyloidosis in an earlier stage of deposition. In this paper, the Congo red staining method according to Romhányi is discussed in comparison with Puchtler's and Bennhold's methods. Using Romhányi's technique, there is no alcoholic differentiation, and thus no dye molecules are washed off the amyloid filaments. The binding of the oriented dye molecules is optimal for polarization microscopy. With this method, the polar hydrophilic mounting medium, gum Arabic is used. Mounted in this carbohydrate-containing, hydrophilic medium, the Congo red molecules are oriented parallel to the surface of the amyloid filaments and the sign is linear positive, corresponding to an additive character of topo-optical staining reactions. Otherwise, the Congo red molecules are oriented perpendicular to the surface of collagen, reducing the intensity of birefringence and even inducing an inversion of the original sign of the collagen birefringence. With alcoholic differentiation, Congo red dye molecules are extracted and this decreases the birefringence of amyloid deposits, i.e. minimal amyloid deposits may be missed. Using the apolar hydrophobic mounting medium, Canada balsam, an axis-parallel arrangement of Congo red dye molecules on the surface of collagen fibers and amyloid will occur, resulting in an additive topo-optical reaction with a green polarization color and a false positive diagnosis of amyloidosis (“phantom amyloidosis”).     

Sensitivity and specificity of Congo red staining according to Romhányi. Comparison with Puchtler's or Bennhold's methods

The sensitivity and specificity of various Congo red staining methods is very important in the diagnosis of amyloidosis. When using a less sensitive staining method, some true positive cases of amyloidosis remain undetected. A more highly specific method potentially detects more cases and reveals amyloidosis in an earlier stage of deposition. In this paper, the Congo red staining method according to Romhányi is discussed in comparison with Puchtler's and Bennhold's methods. Using Romhányi's technique, there is no alcoholic differentiation, and thus no dye molecules are washed off the amyloid filaments. The binding of the oriented dye molecules is optimal for polarization microscopy. With this method, the polar hydrophilic mounting medium, gum Arabic is used. Mounted in this carbohydrate-containing, hydrophilic medium, the Congo red molecules are oriented parallel to the surface of the amyloid filaments and the sign is linear positive, corresponding to an additive character of topo-optical staining reactions. Otherwise, the Congo red molecules are oriented perpendicular to the surface of collagen, reducing the intensity of birefringence and even inducing an inversion of the original sign of the collagen birefringence. With alcoholic differentiation, Congo red dye molecules are extracted and this decreases the birefringence of amyloid deposits, i.e. minimal amyloid deposits may be missed. Using the apolar hydrophobic mounting medium, Canada balsam, an axis-parallel arrangement of Congo red dye molecules on the surface of collagen fibers and amyloid will occur, resulting in an additive topo-optical reaction with a green polarization color and a false positive diagnosis of amyloidosis ("phantom amyloidosis").     

Development of Staining Controls for Campylobacter pylori

Abstract

Recent association of Carnpylobacterpylori with chronic gastritis and duodenal disease has increased the need for Carnpylobacter staining controls in paraffin embedded tissue. We used pure culture of C. pylori mixed with human tissue to prepare a positive staining control in the laboratory. The staining characteristics of C.pylori were evaluated by the following histopathologic staining methods: hematoxylin and eosin, Brown and Brenn, Brown-Hopps, modified Steiner, modified Giemsa, Gimenez, periodic acid-Schiff, and half-Gram. We found that the modified Steiner method, which takes 1-1½ hours and uses the microwave technique, demonstrated C. pylori with better contrast and morphological detail than the other methods. (The J Histotechnol 12:299, 1989)     

“Harris Hematoxylin,” What Harris Really Wrote and the Mechanism of Hemalum Stains

Abstract

In current textbooks, Harris “hematoxylin” stain includes differentiation in acid alcohol, followed by bluing of sections. However, Harris (1900) did not mention differentiation, but strongly recommended progressive staining with an acidified or diluted solution of his formula or with Mayer's acid hemalum. A review of the chemistry of hematoxylin and hemalum stains showed binding of unoxidized hematoxylin by tissues. Oxidation of hematoxylin yields anionic hematein, which does not contain a quinonoid ring. Two hematein ions form a chelate with an Al⁺⁺⁺ cation. Hemalum is bound to tissues by coordinate and hydrogen bonds. Dye binding by nuclei occurs mainly via non-ionic bonding. Protons of acids added to the hemalum solution or used in differentiation compete with the metal for binding sites. When appropriate amounts of acid are added to the hemalum solution, nuclei are stained selectively. Acidified hemalum solutions require only two steps, staining and washing, and yield perfectly reproducible staining patterns, even in the hands of trainees. (The J Histotechnol 10:257, 1987.)     

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)     

Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy

Amyloid fat pad aspiration specimens for cases with a clinical suspicion of amyloid typically are stained with Congo red and examined by brightfield microscopy. Congophilia with apple-green birefringence by polarization microscopy (PM) is considered diagnostic for amyloid. Examination of Congo red-stained slides by fluorescent microscopy (FM) is considered by some to be a more sensitive detection method. In this study, we assessed the utility of this technique in cytopathology archival slides from abdominal fat pad aspirations previously stained with Congo red dye. Seventy-eight cases of abdominal fat pad aspirations collected during the last 5 yr and stained with the Congo red procedure were obtained from archival files. Additionally, 20 adipose tissue material slides prepared from the surgical pathology specimens were examined as controls. One representative smear was examined in each case using FM equipped with rhodamine excitation/absorption (540/570 nm) filters. Relevant clinical information was obtained in all cases. Twelve cases (15.4%) of the 78 fat pad aspiration cases were reported originally as positive by Congo red stain using polarization and apple-green birefringence as diagnostic criteria. On review, four cases were deemed unsatisfactory. By FM examination 29 of the 74 (39.2%) cases were reclassified as positive for amyloid. The results were confirmed by immunohistochemical stain for amyloid P protein and electron microscopy. A number of similar distinct fluorescence and immunohistochemical patterns were recognized in the positive cases. Minimally weak fluorescence in the adipose tissue was observed in the control cases. The use of FM in Congo red-stained fat pad smears can improve the detection of amyloid in cytology preparations.     

Idiopathic Amyloidosis Due to AA Protein: A Report of 2 Cases

Abstract

Two cases of idiopathic amyloidosis are described. AA protein was found to be the major constituent of tissue amyloid in both, based on sensitivity of Congo red birefringence to pretreatment of slides with potassium permanganate and immunoperoxidase staining with a monospecific antiserum. However, an underlying inflammatory, infectious or neoplastic disorder was not identified. The occurrence of AA amyloidosis without clearcut underlying disease is rare, having been described in only thirteen previous instances. Recent clinical series suggest that such cases may have a different course and response to therapy from “primary” amyloidosis due to systemic light chain deposition. Such cases may also underscore the importance of genetic factors in the pathogenesis of AA amyloid and the importance of characterizing the amyloidoses biochemically, as well as clinically. (The J Histotechnol 12:137, 1989.)     

Anti-asthma potential of crocin and its effect on MAPK signaling pathway in a murine model of allergic airway disease

Abstract

Context: Crocin, a diterpenoid glucoside, has multitudinous activities such as anti-inflammation, anti-allergy, anti-oxidation and relaxing smooth muscles.

Objective: In this study, the potential of crocin as an anti-asthma agent was investigated in a murine model.

Materials and methods: BALB/c mice were sensitized and challenged by ovalbumin (OVA) to induce allergic airway inflammation, with crocin administered one hour before every OVA challenge. Airway hyper-reactivity was evaluated by lung function analysis systems. Leukocyte counts in bronchoalveolar lavage fluid (BALF) were measured by a hemocytometer and Diff-Quick-stained smears. Lung tissues were stained with hematoxylin–eosin, Congo red and methylene blue for histopathological inspection. Inflammatory mediators in serum, BALF and lung were measured by ELISA or RT-PCR. Effects of crocin on MAPK signaling pathways were investigated by western blot analysis.

Results: Crocin significantly suppressed airway inflammation and hyper-reactivity, reduced levels of BALF interleukin (IL-4), IL-5, IL-13 and tryptase, lung eosinophil peroxidase and serum OVA-specific IgE, and inhibited the expression of lung eotaxin, p-ERK, p-JNK and p-p38 in the OVA-challenged mice.

Conclusions: These results demonstrated that the suppression of crocin on airway inflammation and hyper-reactivity in a murine model, thus crocin might have a great potential to be a candidate for the treatment of asthma.     

Power and challenges of using zebrafish as a model for skeletal tissue imaging

Abstract

The zebrafish (Danio rerio) is now a widely used model organism in biomedical research. The species is also increasingly used for studying skeletal development and regeneration and for understanding human skeletal diseases. The small size of this model organism is an advantage and an extreme challenge for visualizing and diagnosing the animals’ skeleton. This applies especially to early stages of skeletal development. Similar challenges arise for the analysis of the skeleton of other small fish species, such as medaka (Oryzias latipes). High quality histological preparations and knowledge about the special quality of the zebrafish skeleton remain prerequisites for a correct analysis. In addition, new methods for fast and high-resolution 2D and 3D skeletal tissue screening are required for a maximal understanding of skeletal development. We, in this study, review advantages and limitations of adapting current visualization techniques for zebrafish skeletal research. We discuss the methods for in toto visualization, such as X-raying, micro-CT, Alizarin red staining and optical projection tomography. Techniques for in vivo imaging, such as second harmonic generation microscopy and two-photon excitation fluorescence, are also discussed. Finally, we explore the possibilities of light-sheet microscopy for the analysis of the zebrafish skeleton.     

Prevalence of amyloid and positive Congo red frequency among routine surgical specimens

Amyloidosis is characterized by an extracellular tissue deposition of one of a family of biochemical proteins that are abnormally folded. The deposits are often subtle, and can be missed on routine hematoxylin and eosin (H&E)-stained slides. Current literature does not offer an expected prevalence rate of amyloid or frequency of Congo red positivity among routine surgical pathology specimens in a referral bias-free setting. The objective of this study was to determine these parameters at a large community hospital. The pathology database was searched for all surgical pathology and autopsy cases diagnosed with amyloidosis from 2001–2013. All cases were reviewed and clinical parameters were recorded. Based on H&E interpretation, Congo red was performed on 218 cases. Of these, 36% confirmed positive birefringence. The prevalence of amyloid among routinely submitted pathology specimens was calculated as 0.027% over the 13-year interval. Amyloid was detected in less than 1% of routine surgical specimens. When suspicious H&E findings prompted Congo red staining, amyloidosis was confirmed about a third of the time over the 13-year period. Establishing an optimal rate of Congo red utilization may provide a standard measurement needed to ensure high amyloid detection rates among pathologists at the community level.     

An Artifact of H&E Staining: The Problem and Its Solution

Abstract

The problem of uneven hematoxylin and eosin (H&E) staining is a widespread phenomenon. When water due to incomplete clearing, as in a humid environment, was found to be the problem, substitution of toluene for xylene in the tissue processing procedure eliminated the artifact. The importance of an effective quality assurance in identifying equipment malfunction or contamination of reagents as a cause of uneven staining is stressed. (The J Histotechnol 13:193, 1990)