Comparison of histochemical staining techniques for detecting mast cells in oral lesions

ABSTRACT

Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.     

宫颈特殊染色技术对子宫颈癌及宫颈上皮内瘤变初筛的临床研究

目的:探讨宫颈特殊染色技术筛查方法对宫颈癌及癌前病变的筛查意义。方法:本研究通过对1963例就诊我院妇科门诊的患者进行宫颈特殊染色检查(FRD),以组织病理学检查结果为标准,分析FRD宫颈特殊染色的临床意义。结果:1963例患者行宫颈特殊染色检查及对初筛阳性患者行阴道镜下活检,根据活检病理结果进行分析,CINI阳性率80.77%℅,CINII81.25%,CINIII100%℅,侵润癌100%,总阳性率90.50%。结论:利用亚甲蓝显色和醋酸白化反应双重定位及指示,不仅可提高宫颈癌及癌前病变的检出率,而且操作简便,判读容易,结果快速,成本低廉。     

A method for the stabilization of toluidine blue vital staining for histological and ultrastructural studies

A method previously developed to stabilize methylene blue vital staining has been applied to toluidine blue, a basic dye with a closely related chemical structure commonly used as a vital stain for the screening of oral and esophageal lesions.The stain was made stable by adding 1% sodium silicotungstate to an aldehyde fixative (formaldehyde or glutaraldehyde). Sections were prepared for light and electron microscopy.     

CDX2在胃癌及癌前病变组织中的表达及其临床意义

目的研究CDX2在胃癌及癌前病变组织中的表达及其与临床病理因素之间的关系。方法采用免疫组织化学S-P法检测CDX2在胃癌及癌前病变组织中的表达,采用阿新兰/过碘酸雪夫氏染色(AB/PAS)和高铁二铵/阿新兰(HID/AB)染色对胃黏膜肠化生进行分型。结果 (1)CDX2在正常胃黏膜组织、慢性浅表性胃炎中不表达,CDX2在肠化、异型增生及胃癌中的阳性率(81.7%、50%、48.2%)均明显高于正常胃黏膜组织(P0.05),在肠化组织中的表达明显高于异型增生和胃癌(P0.05),在异型增生和胃癌中的阳性率差异无显著性(P0.05)。CDX2的表达与肠化的分型和异型增生的分级之间均无关系。(2)CDX2在肠型胃癌中的阳性率(67.4%)明显高于弥漫型和混合型胃癌(25.7%、42.9%);CDX2的表达与胃癌分化程度呈正相关,而与淋巴结转移和临床分期呈负相关,差异均有统计学意义。CDX2阳性表达组的五年生存率明显高于阴性表达组(P=0.038);多种变量对胃癌患者术后生存时间影响的分析显示,CDX2的表达和淋巴结转移是影响胃癌预后的独立因素(P0.05)。结论 CDX2可能在胃粘膜肠化生的发生及胃癌的发生发展中起重要作用,可作为胃癌诊断、判断胃癌的恶性程度及患者预后的有用指标。     

Earliest detection of oral cancer using non-invasive brush biopsy including DNA-image-cytometry: report on four cases.

OBJECTIVE: We describe four patients presenting early oral cancers, detected cytologically on non-invasive brush biopsies including DNA-image cytometry as an adjunctive method before histology on scalpel biopsies confirmed the evidence of malignancy. METHODS: Brush biopsies were performed and smears thereof investigated cytologically. After Feulgen restaining, DNA-measurements were performed using a DNA-Image-Cytometer. CASE REPORTS: Oral squamous cell carcinomas were diagnosed cytologically in macroscopically suspicious lesions and malignancy confirmed by DNA-cytometry. The initially performed scalpel biopsies did neither supply evidence of oral cancer nor of severe dysplasia. After at least one to 15 months the occurrence of cancer was finally proven histologically on a second scalpel biopsy each (three microinvasive and one in situ carcinoma). CONCLUSION: Non-invasive brush biopsies are a suitable instrument for early cytologic detection of cancer of the mouth. DNA-image-cytometry, as an adjunctive method, can be used to confirm the cytologic diagnosis or suspicion of cancer in patients with doubtful lesions (dysplasias). DNA-aneuploidy is a marker for (prospective) malignancy in smears of the oral cavity, which may detect malignancy months prior to histology. In future this method could be used as a mass screening tool in dentists practice.     

Mutagenic potential of toluidine blue evaluated in the Ames test.

Toluidine blue is a vital, metachromatic thiazine dye which is used as an adjunct in clinical examination for the early detection of asymptomatic recurrent or secondary primary carcinoma in individuals who are at high risk for developing oral cancer. Because available data on the mutagenicity of toluidine blue was limited and contradictory, this study was conducted to evaluate the mutagenic potential of toluidine blue in the in vitro Ames Salmonella test. Tester strains TA97a, TA98, TA100 and TA102 were used. Toluidine blue was tested at concentrations of 0.1, 1.0, 10, 50, 100, 250 and 500 micrograms/plate, with and without S9 microsomal activation, and positive and negative controls were included. Results from tests without S9 showed a significant increase (p less than 0.05) in number of revertants in TA102 and in TA97a with 50 and 100 micrograms toluidine blue/plate, respectively. In tests with S9 activation, doses of toluidine blue ranging from 10 to 250 micrograms/plate induced dose-related increases in the number of revertants in all 4 strains. The results of this study indicate that toluidine blue has a mutagenic effect in the Ames test.     

Improvements in dehydration and cement line staining for methacrylate embedded human bone biopsies

Undemineralized methacrylate embedded bone biopsies and other bone specimens can be processed much more rapidly by application of acidified 2,2-dimethoxypropane (DMP) dehydration, which requires two hours, than by traditional graded ethanol dehydration, which requires at least four days. This shortened processing time is valuable when biopsy results are urgently needed to detect osteomalacia or to determine bone features prior to possible parathyroidectomy. We have processed over 200 bone specimens with DMP and have compared DMP dehydration to graded ethanol dehydration in 11 biopsies in which two plugs were available from the same patient. DMP dehydration does not compromise the following: tetracycline retention, Goldner's stain, acid phosphatase localization or histochemical identification of aluminum. Cement lines, which provide a record of past remodelling, are useful in clinical interpretation of bone biopsies. We have adapted two stains, toluidine blue and methylene blue/basic fuchsin, for improved cement line identification. Five-micrometer sections individually demineralized in acetate buffer prior to cement line staining show best results with toluidine blue at pH 5.5 and with methylene blue/basic fuchsin at pH 2.5-3.5.     

儿童皮肤肥大细胞瘤的临床病理分析

目的探讨儿童皮肤肥大细胞瘤的临床病理特征。方法对5例皮肤肥大细胞瘤进行临床病理观察和免疫组化检测。结果患者年龄3个月-8岁,3例出生时即有病变,1例在出生后半年内,另一例在7岁时发现病变,均无明显症状,免疫组化示CD43、CD117、LCA、VIM阳性,甲苯胺蓝染色见胞浆内有紫红色颗粒。结论皮肤肥大细胞瘤好发于儿童,大多数在出生时即有或6个月内发病,临床医生易诊断为脉管瘤。组织病理学上关键在于认识肥大细胞,结合免疫组化和甲苯胺蓝染色,并通过询问病史可以确诊。儿童皮肤肥大细胞瘤预后良好,在青春期会自动消退。     

How often is dysplasia diagnosed by biopsy or smear examination? Application of a maximum likelihood based method to the assessment of detection rates in the nasal mucosa of nickel workers.

In view of the known increased risk of nasal carcinoma and the high prevalence of dysplastic lesions of the nasal mucosa among nickel workers, regular screening for the existence of possibly precancerous dysplastic lesions is offered to workers in a Norwegian nickel refinery. Unfortunately, available sampling techniques do not allow the identification of all subjects in whom dysplastic changes are present. Independent histological and cytological (brush cytology) diagnoses, obtained for each of a group of 90 workers, have been used to estimate, by a maximum likelihood method, the probabilities that existing dysplastic lesions will be detected by each of these two screening methods. In the group studied, cytology performed rather less well than histology in unambiguously detecting dysplasia. However, when cytological specimens showing irregular (possibly dysplastic) epithelial cells were grouped with those showing clear dysplastic changes, detection probabilities were estimated at 0.52 by histology and 0.57 by cytology. Detection probabilities were estimated to be higher among subjects with a previously known history of dysplasia, particularly by histology (P < 0.01), probably due to larger dysplastic areas. In view of both its greater facility and speed of sampling, and its greater acceptability, brush cytology may be preferable to biopsy sampling for the screening of large numbers of workers at risk.     

Investigation of the anisotropy of glycocalyx stained with 1.9-dimethyl methylene blue and N,N'-diethylpseudoisocyanine chloride (author's transl)]

In the present study the anisotropic staining of the erythrocyte membrane with 1.9-dimethyl methylene blue and N,N'-diethylpseudoisocyanine chloride was studied and simultaneously compared with the toluidine blue topo-optical staining. The difference between anisotropic toluidine blue and 1.9-dimethyl methylene blue staining, except after KMnO4-oxidation, was only of quantitative nature. On the contrary, striking differences were observed between N,N'-diethylpseudoisocyanine chloride staining, and toluidine blue or 1.9-dimethyl methylene blue staining. Enzymatic and chemical degradation resulted the disappearance of N,N'-diethylpseudoisocyanine chloride staining. Following these treatment membrane birefringence could be restored by aldehyde bisulfate and/or KMnO4-oxidation, while the N,N'-diethylpseudoisocyanine chloride staining was restored only after KMnO4-oxidation. After methylation or acetylation the membrane birefringence disappears, while after KMnO4-oxidation both topo-optical reactions return. The digitonin reaction brought about a rearrangement of the glycocyalyx components. The results draw attention to the spatial orientation of the glycoprotein of the erythrocyte membrane. The role of glycocalyx in the three topo-optical reactions was thus clearly demonstrated.     

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Unmasking of Proteins by in Vitro Methylation of Rat Liver Rna During Azo Dye Carcinogenesis

In liver parenchyma of rats fed the azo dye 4-dimethylaminoazobenzene cytoplasmic ribonucleic acid (RNA) stains intensely with toluidine blue in sites of neoplastic transformation and hepatomas. With the mercury-bromphenol blue method, the staining intensity of proteins was found to be much weaker in these areas than in surrounding tissue. A 4 hr treatment at 60 C in a mixture of methanol and HCI completely abolished the increased staining of RNA with toluidine blue, but restored protein stainability with bromphenol blue in preneoplastic foci and tumors; the staining intensity of proteins was then comparable to that in surrounding liver. These results suggest that proteins were masked by the RNA responsible for hyperbasophilia, and that in vitro addition of methyl groups to such RNA is equal in effect to RNase extraction which was previously found to unmask cytoplasmic proteins.     

Laminin-5 immunocytochemistry: a new tool for identifying dysplastic cells in oral brush biopsies

BACKGROUND: The brush biopsy technique is not only a seminal technique but also a critically discussed method for detection of oral pre-cancerous stages and manifest carcinomas. The gamma2 chain of laminin-5 and its proteolytic fragments comprise an invasion factor for many carcinomas. OBJECTIVES: The aim of this study was to determine whether the immunocytochemical presentation of the laminin gamma2 chain identifies pre-invasive or invasive squamous cells in brush biopsies. METHODS: The value-based identification of atypical epithelia was analysed in 93 consecutive brush biopsies with histopathological diagnoses: standardized haematoxylin and eosin staining; standardized immunocytochemistry: monoclonal antibodies against laminin gamma2 chain: D4B5, 4G1, detection using ChemMate and Autostainer. RESULTS: Conventional cytology did not result in any false-positive cases, i.e. atypical cells in normal, inflamed or benignly hyperproliferative mucosa (specificity, 100%), whereas immunocytochemistry revealed one false-positive case (specificity, 98%). In brush biopsies of oral squamous cell carcinomas, the following immunocytochemical patterns were possible: (1) staining of the cytoplasm, (2) banded markings between clumped carcinoma cells and (3) positive hazes surrounding atypical cells. Bacterial colonies appeared as false-positive results. Four of 27 carcinomas and one of three recurrences were not cytologically identified (sensitivity of conventional cytology, 79%). Three of the five carcinomas not identified by cytology were immunocytochemically stained with laminin gamma2 chain antibody (sensitivity of laminin gamma2 chain immunocytochemistry, 93%). The positive predictive value was 100% for conventional cytology and 97% for laminin gamma2 chain immunocytochemistry. The negative predictive value attained was 92% for conventional cytology and 97% for laminin gamma2 chain immunocytochemistry. CONCLUSIONS: The high sensitivity level observed for method-enhanced brush cytology suggests that this technique be used as an initial diagnostic step.     

Endometrial and endocervical secretion: the search for histochemical differentiation

OBJECTIVE: To determine the ideal histochemical stain to differentiate between non-neoplastic and neoplastic endocervix and endometrium. STUDY DESIGN: A total of 90 cases representing nonneoplastic cervix, non-neoplastic endometrium, endocervical adenocarcinoma and endometrial adenocarcinoma were stained with toluidine blue (TB); methylene blue (MB); mucicarmine (MUC); periodic acid-Schiff before and after diastase digestion (PAS, PAS-D); Alcian blue, pH 2.5 (AB); and periodic acid-Schiff after Alcian blue, pH 2.5 (PAB). Cases were blinded and randomly divided between two pathologists for evaluation of the staining and the staining distribution of the glandular epithelium by means of a 36-color scheme. RESULTS: The majority of non-neoplastic endocervix samples stained blue with MB (57%), fuchsia with MUC (70%), magenta with PAS (77%) and PAS-D (73%) and dark turquoise with AB (70%). The majority of non-neoplastic endometrium samples stained slate blue with TB (60%) and pink with PAS-D (53.3%). There is statistical difference (p < 0.05) in the color of the epithelium and secretions between the non-neoplastic cervix and endometrium. The malignant glands of endocervical origin could be differentiated significantly (p = 0.043) from non-neoplastic endocervical epithelium by MUC. The epithelium of the non-neoplastic endometrium is significantly differentiated from malignant endometrium using TB (p = 0.015) and MB (p = 0.038). Endocervical carcinoma could be significantly differentiated from endometrial carcinoma by MB. The staining in endocervical adenocarcinoma and endometrial carcinoma was predominantly present in both apical and cytoplasmic locations compared to their non-neoplastic counterparts (endocervix, p = 0.003; endometrium, p = 0.049). CONCLUSION: This study showed that a panel of histochemical stains could differentiate glandular cells of endocervical epithelium from endometrium.     

Cytologic screening for esophageal cancer in a high-risk population in Anyang County,China

OBJECTIVE: Anyang County, China, is one of the areas with the highest incidence of esophageal cancer in the world. Esophageal cancer has a poor prognosis because most tumors are unresectable at the time of diagnosis. We launched a screening study for early esophageal carcinoma in western Anyang County in 1997. The scope was to identify patients with in situ and early invasive carcinoma, applying esophageal balloon cytology and treating with photodynamic therapy (PDT). STUDY DESIGN: The study cohort consisted of all inhabitants over 35 years of age in 10 communes. Screening was performed by balloon cytology. Grade 2 dysplasia and more advanced lesions were examined with endoscopy, including biopsy and brush cytology, followed by PDT for early cancer. RESULTS: In total, 20,049 persons participated in the screening program, and 1,018 were diagnosed with a grade 2 dysplasia or higher, including 164 invasive cancers and 169 near-cancers. Ninety-four percent of atypical lesions were of squamous cell type. Seventy-two percent of cases showing severe dysplasia and cancer were located to the middle esophageal segment. The prevalence of dysplasia and cancer increased significantly with age. The balloon cytology results were confirmed by brush cytology and histology. CONCLUSION: Balloon cytology is a reliable method for esophageal cancer screening. Positive cytology must be verified by endoscopy and biopsy.     

Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology

Sperm chromatin alteration is an important feature that can affect fertility of the male rabbit. This study compared toluidine blue staining with Feulgen reaction (as methods for evaluating chromatin alteration) and investigated the relationship between sperm morphology and chromatin alteration. Seven hundred rabbit ejaculates of animals with unknown fertility were used. Primary and secondary morphological sperm abnormalities were evaluated in semen smears with phase-contrast microscopy. Chromatin alterations were evaluated in semen smears stained with toluidine blue (pH 4.0 and 5.0) and with the Feulgen reaction. While the three methods were equally efficacious for identification of chromatin alterations, toluidine blue staining was more appropriate to characterize the intensity of chromatin alterations. The correlation between primary sperm defects and chromatin alteration was high and positive, suggesting that sperm chromatin structure affected sperm head morphology. The correlation between secondary sperm defects and chromatin alteration was also positive, but lower. The final chromatin compaction occurs in the epididymus, where secondary sperm defects originate. Therefore, the causes of secondary sperm defects could also intervene with final chromatin compaction. In summary, the toluidine blue stain was an effective means of evaluating the sperm chromatin alteration in rabbit spermatozoa.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

Summary The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes showed higher electron density than control chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82 — for euchromatic arms — and 0.85 — for centromeric heterochromatin — were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Differential use of Alcian blue and toluidine blue dyes for the quantification and isolation of anionic glycoconjugates from cell cultures: application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes

Alcian blue and toluidine blue dyes form complexes with anionic glycoconjugates (AG) such as proteoglycans (PG) and glycosaminoglycans (GAG). However, the Alcian blue-AG complexes do not readily dissociate, while the toluidine blue-AG complexes do so in salt solutions. This differential dissociation of the dye-AG complexes has been utilized in the analysis and isolation of radiolabeled AG elaborated by articular chondrocyte cultures incubated with the radiolabeled precursors of AG. For the rapid quantification of newly synthesized (35)S-labeled PG, small replicate aliquots of the radiolabeled culture media were applied directly to cellulose acetate strips, stained with Alcian blue and the stained immobilized radiolabeled PG was quantified by liquid scintillation counting. Comparison of anionic glycoconjugates quantified in the culture media employing toluidine blue and Alcian blue staining on cellulose acetate trips gave similar results. Staining on cellulose acetate strips using these two dyes is particularly suited for the simultaneous processing of large numbers of samples, as illustrated by the screening of the effects of biological materials and drugs on AG synthesis, in cultures labeled with [(35)S]-sulfate and [(3)H]-glucosamine. The Alcian blue and toluidine blue precipitation methods yielded similar results for the total AG recovered from the media of TGF-beta-stimulated chondrocytes. Electrophoretic analysis of toluidine blue- and Alcian blue-precipitated AG followed by autoradiography and Alcian blue staining in combination with silver nitrate demonstrated that both dyes yielded similar pattern of bands on gels. However, some AG from Alcian blue precipitate did not enter the gel, suggesting incomplete dissociation of Alcian blue-AG complex. The application of the toluidine blue precipitation method, in combination with enzymatic digestion of the GAG chains of the PGs, is illustrated by the isolation of a non-PG high-molecular-weight AG, as well as the PGs from the media of chondrocyte cultures stimulated by TGF-beta.     

Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates.

Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS)