大鼠不同部位疏松结缔组织撕片的制备和观察

目的　对比大鼠皮下取材和肠系膜取材制备疏松结缔组织撕片的差异;观察同一部位取材的疏松结缔组织HE染色和醛复红-亮绿-橘黄G染色差异;分析不同部位取材的疏松结缔组织两种染色方法的结果差异。 方法　Wistar大鼠腹腔注射10 g/L苔盼蓝生理盐水溶液2.5 ml,1次/d,连续3 d,分别在皮下、肠系膜取疏松结缔组织,铺片。两个部位的铺片分别采用HE染色、醛复红-亮绿-橘黄G染色。 结果　皮下取材疏松结缔组织经HE染色可见大量成纤维细胞,肥大细胞明显,巨噬细胞可见,弹性纤维和胶原纤维可见,但不明显;皮下取材疏松结缔组织经醛复红-亮绿-橘黄G染色弹性纤维呈紫红色、胶原纤维呈橙色,细胞不易着色;肠系膜取材疏松结缔组织经HE染色,可见成纤维细胞、肥大细胞、巨噬细胞明显,弹性纤维呈蓝紫色、胶原纤维呈淡红色;肠系膜取材疏松结缔组织经醛复红-亮绿-橘黄G染色,弹性纤维被染成紫红色、胶原纤维染成鲜艳的绿色,肥大细胞被染成紫红色,核呈圆或椭圆形、棕黄色,巨噬细胞清晰可见、形态不规则,胞质中可见粗大呈蓝紫色的苔盼蓝颗粒,细胞核呈圆形、棕黄色;成纤维细胞胞质无着色,核呈棕黄色。 结论　大鼠肠系膜取材制备的疏松结缔组织撕片经醛复红-亮绿-橘黄G染色能够更好的显示各种类型细胞和纤维,各结构间对比明显。     

Immunohistochemical Expression of the Intracellular Component of Galectin-8 in Squamous Cell Metaplasiaof the Bronchial Epitheliumin Neoplastic and Benign Processes

Specified galectins are known to play a role in regulating cell proliferation, differentiation, adhesion and migration. Po66, a mouse IgG1 monoclonal antibody produced by immunization against squamous cell cancer, reacts against a carbohydrate-binding protein (Po66-CBP), recently shown to be a member of the galectin family with a strong homology with galectin-8 (PCTA-1), identified as a human tumor-associated antigen.

We studied Po66 in squamous metaplasia of the bronchi in order to determine whether it could be specifically involved in neoplastic conditions and if so, if it would be helpful in distinguishing metaplasia at risk of cancer.

Twenty-eight formalin-fixed, paraffin-embedded archival tissues of 17 metaplasias with SCC, 3 metaplasia with distant neoplastic disease and 8 metaplasias with an inflammatory process, were immunostained using a streptavidin biotin peroxydase method.

The squamous metaplasias were positively stained in non-neoplastic disease as well as in neoplastic processes. Expression was also observed in stromal and normal cells.

Po66-CBP was not associated with a pre-neoplastic character. We discussed the expression of this intra-cellular component of galectin-8 according to the functions of galectins in cellular differentiation, host reaction against tumor, and inflammation.     

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.     

精液中生精细胞5种染色方法的探讨与评价

对脱落于精液中的生精细胞，用瑞－吉染色法可使细胞核着色清晰度好；用伊红－瑞－吉染色法能区别死亡和活的精子，并使精子顶体着色清晰；用苯胺黑－伊红－吉染色法精子核染成红色，精子鞭毛不着色；用煌绿－瑞－吉染色法，成熟精子着色浅，不成熟精子头部着紫红色；过氧化物酶－瑞－吉混合染色，中性白细胞核呈紫红色，胞浆呈浅绿色。５种染色方法中瑞－吉法对生精细胞染色效果最好，可视精液检查的不同要求选用不同方法。     

Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology.     

Differential expression patterns of MMPs and their role in the invasion of epithelial premalignant tumors and invasive cutaneous squamous cell carcinoma

Co-expression of several members of the matrix metalloproteinase (MMP) family is characteristic of human malignant tumors. MMP-2, MMP-9, TIMP-2, and MT1-MMP are thought to be involved in the process of destruction of basement membranes and stromal invasion by neoplastic epithelial cells. In this study, we investigated the expression and role of MMPs in cutaneous oncogenesis. Tissue microarray consisting of 62 squamous cell carcinomas (SCC), 32 Bowen's disease (BD) samples, 25 normal epidermis samples were obtained for the study. MMP-2,-9, MT1-MMP and TIMP-2 proteins were examined by immunohistochemical staining and mRNA level was detected by quantitative RT-PCR in fresh tissues consisting of 5 cutaneous SCCs and paired normal epidermis samples. Gelatinase activity of MMP-2 and MMP-9 was investigated by gelatin zymography and protein levels of MT1-MMP and TIMP-2 were measured by western blot in 2 human SCC cell lines. The invasive property was evaluated with invasion assays using Transwell filters. SCC exhibited significantly increased MMP-2, MT1-MMP and decreased TIMP-2 mRNA and protein expression compared to that of the normal epithelium. Immunohistochemical staining revealed that MT1-MMP was strongly expressed on the invasive front of SCCs, whereas BD exhibited higher expression around the dyskeratotic cells in the epithelium. In comparison with the expression observed in BD, SCC exhibited significantly increased MMP-2 expression. In addition, high MMP-2 and MT1-MMP expression and low TIMP-2 expression had a significant positive correlation with the invasiveness of SCC cell lines in vitro. Our results revealed significantly increased MT1-MMP and MMP-2 expression and decreased TIMP-2 expression in cutaneous SCC, and the expression correlated with the invasiveness of SCC cell lines. Therefore, the expression of these factors in cutaneous tumors may serve as an indicator of tumor aggressiveness and invasion.     

Serpin peptidase inhibitor clade A member 1 (SerpinA1) is a novel biomarker for progression of cutaneous squamous cell carcinoma

The incidence of keratinocyte-derived nonmelanoma skin cancers is increasing worldwide because of cumulative recreational exposure to sunlight. At present, no specific molecular markers are available for assessing the progression of premalignant actinic keratoses to invasive cutaneous squamous cell carcinoma (SCC). We examined the role of the Serpin family in skin SCCs. Expression profiling of cutaneous SCC cell lines (n = 8) revealed up-regulation of SerpinA1 compared with normal epidermal keratinocytes (n = 5). Analysis with quantitative RT-PCR showed that the mean level of SerpinA1 mRNA was markedly up-regulated in cutaneous SCC cell lines (n = 8) compared with in normal keratinocytes. SerpinA1 production by SCC cells was dependent on p38 mitogen-activated protein kinase activity and was up-regulated by epidermal growth factor, tumor necrosis factor-α, interferon-γ, and IL-1β. Immunostaining of tissue arrays with 148 human tissue samples revealed tumor cell-associated expression of SerpinA1 in 19 of 36 actinic keratoses, 22 of 29 Bowen's disease samples, 67 of 71 sporadic SCCs, and all 12 recessive dystrophic epidermolysis bullosa-associated SCCs examined. Moreover, tumor cell-associated SerpinA1 staining was detected in all chemically induced mouse skin SCCs studied (n = 17). Overexpression of SerpinA1 mRNA was also detected by quantitative RT-PCR in chemically induced mouse skin SCCs (n = 14) compared with control tissues (n = 14). These data identify SerpinA1 as a novel tumor cell-associated biomarker for progression of cutaneous SCCs.     

Immunohistochemical study of the inflammatory infiltrate associated with equine squamous cell carcinoma.

The distribution of T (CD3), B (CD79) lymphocytes, immunoglobulin (IgG, IgM and IgA)-producing plasma cells, macrophages (lysozyme, Mac387) and MHC Class II antigen was analysed in the inflammatory infiltrate associated with 19 equine squamous cell carcinomas (SCCs) and six cases of precancerous lesions (actinic keratosis). The SCCs came from the penis (11 cases), conjunctiva (four), skin (two), nasal cavity (one) and oral cavity (one). Seven cases were well-differentiated and 12 moderately differentiated. Nine cases showed no invasion of peritumoral deep tissues (locally invasive), whereas the remaining 10 cases were highly invasive. An abundant inflammatory infiltrate was associated with the majority of the SCCs and with lesions of actinic keratosis. This infiltrate was composed mainly of CD3(+)T lymphocytes, CD79(+)B cells and numerous IgG(+)plasma cells; IgM- and IgA-producing plasma cells were scarce and variable, respectively. Macrophages were usually numerous. Macrophages, lymphocytes, intra-epithelial dendritic cells and fibroblasts expressed MHC Class II antigen. No significant correlation was found between the nature of the inflammatory infiltrate and the SCC histological grade or degree of invasion, suggesting that the local anti-tumour immune response failed to prevent tumour invasion or metastasis. MHC Class II was expressed by a variable number of neoplastic epithelial cells in four SCCs, all of which were only locally invasive. In addition, in areas where SCC cells expressed Class II antigen, numerous CD3(+)T lymphocytes were present and some of them were associated with degenerate tumour cells. These findings suggest that the expression of MHC Class II by neoplastic cells induces an improved local anti-tumour immune response.     

Superficial basaloid squamous carcinoma of the esophagus. A clinicopathological and immunohistochemical study of 12 cases

Basaloid squamous carcinoma (BSC) is a rare variant of squamous cell carcinoma (SCC). In this study, clinicopathological and immunohistochemical characteristics of 12 superficial esophageal BSCs were examined and compared with those of typical superficial SCCs. Eight cases were classified into an elevated type, and the other four into a depressed type. High-grade intraepithelial neoplasia was not observed around the invasive lesions in five cases, and only BSC components were apparent. High-grade intraepithelial neoplasia was demonstrated in seven cases, five of which had both BSC and SCC components in the invasive lesion. A cribriform growth pattern, comedo-type necrosis, and hyaline deposits were conspicuous histological findings. CK14 was positively stained in 90% of the series, but the proportion of positive cells was small in most cases. Type IV collagen was increased or well preserved in the basement membrane in 70% of cases, but heparan sulfate was decreased in the majority. In comparison with SCCs, lymphatic permeation was observed less frequently. However, regarding the frequencies of venous permeation, nodal metastasis, p53 protein expression, and Ki-67 labeling index, no significant differences were noted. Thus, esophageal BSCs demonstrate the pathological features characteristic of an early stage, but pathological parameters related to biological behavior do not significantly vary from those typical of SCCs.     

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。     

Collagenase-3 (MMP-13) is expressed by tumor cells in invasive vulvar squamous cell carcinomas

Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by invading tumor cells in squamous cell carcinomas (SCCs) of the head and neck. Here, we have further elucidated the role of MMP-13 in tumor invasion by examining its expression in invasive malignant tumors of the female genital tract. Using in situ hybridization, expression of MMP-13 mRNA was detected in 9 of 12 vulvar SCCs, primarily in tumor cells, but not in intact vulvar epithelium, in cervical SCCs (n = 12), or in endometrial (n = 11) or ovarian adenocarcinomas (n = 8). MMP-13 expression was especially abundant in vulvar carcinomas showing metastasis to lymph nodes and was associated with expression of membrane type 1 MMP by tumor cells and gelatinase-A (MMP-2) by stromal cells, as detected by immunohistochemistry. MMP-13 mRNAs were detected in 9 of 11 cell lines established from vulvar carcinomas and in 4 of 6 cell lines from cervical carcinomas, whereas endometrial (n = 10) and ovarian (n = 9) carcinoma cell lines were negative for MMP-13 mRNA. No correlation was detected between MMP-13 expression and p53 gene mutations in vulvar SCC cell lines. However, MMP-13 expression was detected in 5 of 6 vulvar and cervical SCC cell lines harboring HPV 16 or 68 DNA. These results show that MMP-13 is specifically expressed by malignantly transformed squamous epithelial cells, including vulvar SCC cells, and appears to serve as a marker for their invasive capacity.     

Proteomic signatures reveal a dualistic and clinically relevant classification of anal canal carcinoma

Aetiologically linked to HPV infection, malignancies of the anal canal have substantially increased in incidence over the last 20 years. Although most anal squamous cell carcinomas (SCCs) respond well to chemoradiotherapy, about 30% of patients experience a poor outcome, for undetermined reasons. Despite cumulative efforts for discovering independent predictors of overall survival, both nodal status and tumour size are still the only reliable factors predicting patient outcome. Recent efforts have revealed that the biology of HPV‐related lesions in the cervix is strongly linked to the originally infected cell population. To address the hypothesis that topography also influences both gene expression profile and behaviour of anal (pre)neoplastic lesions, we correlated both proteomic signatures and clinicopathological features of tumours arising from two distinct portions of the anal canal: the lower part (squamous zone) and the more proximal anal transitional zone. Although microdissected cancer cells appeared indistinguishable by morphology (squamous phenotype), unsupervised clustering analysis of the whole proteome significantly highlighted the heterogeneity that exists within anal canal tumours. More importantly, two region‐specific subtypes of SCC were revealed. The expression profile (sensitivity/specificity) of several selected biomarkers (keratin filaments) further confirmed the subclassification of anal (pre)cancers based on their cellular origin. Less commonly detected compared to their counterparts located in the squamous mucosa, SCCs originating in the transitional zone more frequently displayed a poor or basaloid differentiation, and were significantly correlated with reduced disease‐free and overall survivals. Taken together, we present direct evidence that anal canal SCC comprises two distinct entities with different cells of origin, proteomic signatures, and survival rates. This study forms the basis for a dualistic classification of anal carcinoma, with implications for management, outcome expectations, and possibly therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

A new histochemical technique of use in the interpretation and diagnosis of adenocarcinoma and villous lesions in the large intestine.

The periodic acid-thionin Schiff/potassium hydroxide/periodic acid-Schiff (PAT/KOH/PAS) procedure has been used to investigate the histochemical staining characteristics of the mucins found in adenocarcinoma and villous lesions of the large intestine. The 46 blocks examined represented 58 lesions from 37 patients, all of whom had had resections for carcinoma of the colon. tin sharp contrast to normal colon, none of the adenocarcinomas stained red with the PAT/KOH/PAS. With two exceptions the poorly and moderately differentiated adenocarcinomas stained blue, whereas of the well differentiated lesions half were blue and half purple. The malignant villous lesions demonstrated the same trends, although a larger percentage were purple. None of the benign lesions stained blue. It is suggested that malignancy in the colon is accompanied by an increase in blue staining in the PAT/KOH/PAS technique and that such staining may be of value in the interpretation of highly atypical adenoma where it might identify the onset of malignancy. This change in staining indicates a distinct alteration in the chemistry of the mucins which we interpret as a reduction in the degree of side chain O-acylation of their constituent sialic acids.     

If it's not CK5/6 positive, TTF-1 negative it's not a squamous cell carcinoma of lung

Novel targeted treatment of non-small cell lung cancer (NSCLC) requires accurate classification of NSCLC as squamous cell carcinoma (SCC) and adenocarcinoma (AC). This study details the CK5/6 and TTF-1 immunoprofile of surgical resections of 45 NSCLCs (24 ACs and 21 SCCs) in tissue microarrays. All SCCs were CK5/6 positive, TTF-1 negative. 20 of 24 adenocarcinomas had the reverse pattern. In conclusion, all SCCs in this study were CK5/6 positive and TTF-1 negative, and therefore tumours that do not display this phenotype are unlikely to be SCCs. CK5/6 and TTF-1 is therefore a practical panel for the distinction between pulmonary SCC from AC in routine histopathology practice.     

Diagnostic value of GLUT-1 immunoreactivity to distinguish benign from malignant cystic squamous lesions of the head and neck in fine-needle aspiration biopsy material

The distinction of cystic squamous-cell carcinoma (SCC) from benign cystic squamous lesions (BCSLs) of the head and neck can be problematic on fine-needle aspiration biopsy (FNAB) material, particularly when BCSLs display epithelial reactive atypia or when SCC is well differentiated. Glucose transporter 1 (GLUT-1), a facilitative cell surface glucose transport protein, is aberrantly expressed in many cancers including oral and hypopharyngeal SCC. We evaluated the expression of GLUT-1 by immunochemistry on FNAB material to determine its value in distinguishing cystic SCC from BCSL of the head and neck. A 5-yr retrospective review of all head and neck cystic squamous lesions having FNAB specimens with cell block material, radiological studies, and histological confirmation was performed at our institution. Cell block material from 24 cystic squamous lesions, including 8 (33%) BCSL (7 branchial cleft cysts and 1 thyroglossal duct cyst[TDC]) and 16 (67%) metastatic SCCs with cystic/liquefactive degeneration, was retrieved and immunostained with anti-GLUT-1. GLUT-1 expression was considered positive when at least 10% of squamous cells exhibited distinct cell membrane reactivity. Positive GLUT-1 immunostaining was detected in all 16 SCCs and in none of the 8 BCSLs. In the carcinoma cases, the majority of malignant cells exhibited GLUT-1 reactivity; only a minor population of well-differentiated SCC cells displaying keratinization and arranged as squamous pearls did not express GLUT-1. GLUT-1 expression in cell block material can help to distinguish cystic SCCs from BCSLs of the head and neck. In conjunction with clinical and radiological correlation, GLUT-1 immunoreactivity can be an important diagnostic aid when the cytological findings are ambiguous.