biotinyltyramide and Carcinoma--Squamous-Cell

We investigated non-specific staining in a catalyzed reporter deposition (CARD) reaction and improved its blocking methods in supersensitive immunohistochemistry, based on our simplified catalyzed signal amplification (CSA) system (Hasui et al. 2002). In the CARD reaction using biotinyl tyramide, non-specific staining could be reduced by pretreatment with a casein solution or 3% bovine serum albumin (BSA)-phosphate buffer saline (PBS) with 0.1% Tween 20. In the CARD reaction using FITC-labeled tyramide, non-specific staining could be blocked by pretreatment with 0.3% BSA-PBS with 0.1% Tween 20 or 3% polyethylene glycol-PBS with 01% Tween 20. Thus, our new simplified CSA system features: 1) destruction of the endogenous peroxidase activity; 2) blocking of the nonspecific reaction of the primary antibody; 3) a primary antibody reaction; 4) blocking of the non-specific reaction of the polymer reagent by casein treatment; 5) a polymer reaction; 6) blocking of the non-specific reaction of CARD reaction by casein treatment; 7) a CARD reaction; and 8) detection of deposited tyramide. This new system proved useful for detecting an extremely low amount of antigen in the endogenous biotin-rich tissues such as the gastrointestinal tract and liver. By this method, the Ki67 antigen in the G1 phase cell cycle could be detected and a metabolic disorder of the Ki67 antigen was implicated in a carcinoid tumor in the stomach. We believe that this new simplified CSA system represents a new standard of supersensitive immunohistochemistry for use in light-microscopic investigation.

Topics: Appendix; Biotin; Carcinoid Tumor; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Caseins; Fluorescein-5-isothiocyanate; Fluorescent Dyes; G1 Phase; Humans; Immunohistochemistry; Ki-67 Antigen; Liver Neoplasms; Lymph Nodes; Polyethylene Glycols; Sensitivity and Specificity; Serum Albumin, Bovine; Stomach Neoplasms; Tongue Neoplasms; Tyramine

With regard to the cellular origin of bronchial squamous-cell carcinomas, there are some clinicopathologic and experimental data indicating a link between neuroendocrine (NE) bronchial tumors and the traditionally non-NE squamous-cell carcinomas. Against this background, 29 consecutively resected bronchial squamous-cell carcinomas were examined immunohistochemically (IHC) by means of the specific NE cell marker chromogranin A (CgA), using not only conventional IHC methods, but also the technique with increased sensitivity, offered by the tyramide signal amplification (TSA) procedure. Whereas none of the 29 tumors displayed CgA immunoreactive (IR) cells using the conventional IHC procedure, 10 were found to display a fine granular CgA IR in the neoplastic parenchymal cells using the TSA technique. This incidence is higher than previously reported. However, the CgA IR cells never formed any majority cell population of the neoplastic parenchyma; when present, most of them occurred as micronodules or larger confluent areas in the peripheral most undifferentiated parts of the carcinomatous sheets. Single CgA IR cells were detected only rarely in the spinocellular or keratinized areas. It can be speculated that the observations conform with the recently proposed hypothesis that there is a reservoir of NE progenitor cells in the bronchial mucosa capable of proliferation.

Topics: Biomarkers, Tumor; Biotin; Bronchial Neoplasms; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Differentiation; Chromogranin A; Chromogranins; Humans; Immunohistochemistry; Lung Neoplasms; Neurosecretory Systems; Tyramine

Three different in situ hybridization (ISH) methods were compared for their clinical relevance and suitability in detecting human papillomavirus (HPV) 16/18 in 55 cases of squamous cell carcinoma (SCC) of the uterine cervix. After the initial biopsy, surgery, and/or radiation therapy, patients were followed for 5 to 8 years. A biotinylated cDNA probe for HPV 16/18 was applied to serial sections in combination with conventional streptavidin-biotin-peroxidase ISH (a widely applied routine procedure), streptavidin-Nanogold-silver ISH, and tyramide-signal amplified (TSA) streptavidin-Nanogold-gold ISH. The TSA principle is also known as catalyzed reporter deposition and is, apart from in situ PCR, probably today's most sensitive technique for detecting papillomavirus infection by microscopic means. Nearly 65.5% of the cases showed specific HPV 16/18 detection with TSA ISH, whereas 43.6% were positive with streptavidin-Nanogold-silver-ISH, and only 40.0% with peroxidase-based ISH. Statistical analyses comparing early and advanced stages in both HPV-positive and -negative groups revealed a significantly better outcome for early disease patients; statistical significance was most pronounced with TSA ISH. In a subgroup of patients who had received radiation therapy without prior surgery (n = 35), those with advanced disease were significantly less likely to have HPV 16/18 infection than those with early disease. A significantly better overall survival was observed in those women with HPV 16/18-positive carcinomas who had undergone surgery before radiation therapy (seen with all three methods). We conclude that TSA, in addition to being the most sensitive HPV in situ method applied in this study, gave the most significant and clinically relevant statistical results.

Topics: Adult; Aged; Aged, 80 and over; Biotin; Carcinoma, Squamous Cell; Disease-Free Survival; DNA, Complementary; Female; Follow-Up Studies; Humans; In Situ Hybridization; Indicators and Reagents; Middle Aged; Papillomaviridae; Peroxidase; Reproducibility of Results; Sensitivity and Specificity; Silver; Streptavidin; Time Factors; Tyramine; Uterine Cervical Neoplasms