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PubMed2004

Immunohistochemical localization of cerebrovascular cannabinoid CB1 receptor protein.

Cannabinoids are powerful hypotensives and vasodilators. However, their mode of action is controversial. This study is the first to investigate the distribution of vascular CB1 receptor protein expression in situ. We used double-fluorescence and chromogenic immunohistochemistry to investigate patterns of CB1 protein expression in cerebrovascular tissue in rat brain sections. We found a layer of intense CB1 labeling immediately adjacent to the internal elastic lamina, consistent with myointimal and vascular smooth muscle cells, and diffuse labeling adventitial to this layer. We concluded that CB1 receptor are most intensely expressed in the vascular smooth muscle layer in cerebral arteries, and are likely to be chiefly responsible for the potent vasodilatory effect of cannabinoids.

3,3'-Diaminobenzidine↗

PubMed1988

Early diagnosis of invasive candidiasis and rapid evaluation of antifungal therapy by combined use of conventional chromogenic limulus test and a newly developed endotoxin specific assay.

Since beta-1,3-glucan is a common component of fungal cell wall, its detection might be useful in diagnosing invasive candidiasis. Not only endotoxin but beta-1,3-glucan activates proclotting enzyme contained in a conventional chromogenic limulus test (CCLT). Endotoxin activates this enzyme through factor C, while the beta-1,3-glucan activates through factor G. Since endotoxin specific test (EST) contains factor C, endotoxin would be quantified. By subtracting EST value from CCLT value, beta-1,3-glucan would be quantified. We named this value Fungal Index (FI), and examined if it actually reflects the candidal infection. Ninety-two patients were tested for CCLT and EST prospectively. FI increased significantly in candidal infection (p less than 0.05) but remained low in GNR infection. Moreover, FI increased proportionally to the severity of candidal infection. Elevated FI decreased when antifungal therapy was successful. Thus FI was a useful index not only in the diagnosis of invasive candidiasis but also in the evaluation of antifungal therapy.

Antifungal Agents↗

PubMed2001

Overexpression of p53-related proteins predicts rapid growth rate of head and neck cancer.

OBJECTIVES/HYPOTHESIS: The p53 tumor suppressor gene plays an important role for cell cycle regulation and is the most frequent mutated gene in head and neck cancer. Controversy remains regarding the biological and clinical value of immunohistochemical identification of the proteins accumulated in association with inactivation of the p53 gene and increased tumor growth. Therefore, the objective of the present study was to perform a cell kinetic analysis of cases with untreated squamous cell carcinoma and to compare the result with immunostaining for p53-related proteins in the tumor cells. STUDY DESIGN: A prospective series of 32 patients presenting with various stages of untreated squamous cell carcinoma of the head and neck were included. Bromodeoxyuridine (BrdU) was injected as a tracer dose before tumor biopsy for cell kinetic analysis, and p53 protein accumulation was detected using two antibodies (DO7 and PAb 1801). RESULTS: Antibody DO7 showed the highest and the optimal immunoreactivity. Diploid tumors were found in 27 cases (84%), and the mean potential doubling time (Tpot) was 55 +/- 7 hours for these tumors. Positivity of DO7 (>1%) was demonstrated in 85% of the cases. However, a discrimination level exceeding 20% was required to obtain a significant negative relationship (Spearman's rank correlation coefficient test, P < or = .03) between Tpot and DO7 positivity. At that level, 33% of the tumors remained DO7-positive. The corresponding Tpot was not significantly different from the overall mean. The rates of metastatic disease and survival were not dependent on DO7 immunoreactivity or cancer cell kinetics. CONCLUSION: Accumulation of p53-related proteins is associated with an unrestrained growth of head and neck cancer.

Adult↗

PubMedDate not supplied

Glutathione content of human malignant melanoma cell lines: correlation of flow cytometric and biochemical assays, and application to human tumour aspirates.

We measured the glutathione content of a panel of human malignant melanoma cell lines by flow cytometry after staining with mercury orange, using visible light (488 nm) excitation, and compared the values obtained with those measured biochemically using a modified Tietze assay. Glutathione levels were depleted by culturing cells with various concentrations of buthionine sulphoximine to provide a suitable spread of glutathione concentrations. The two assays showed good correlation (r = 0.93). We found a number of technical factors to be critically important. In particular, the conditions of staining, cell number, and method of mixing media, cells and stain were responsible for wide variations in fluorescence intensity. We applied the flow cytometric technique to cell suspensions obtained by fine needle aspiration biopsy of human malignant melanoma deposits, and observed a proportion of cells with high glutathione levels in many samples. The results confirm the feasibility of measuring glutathione content by visible light flow cytometry, and raise the possibility of monitoring glutathione content as an indicator of drug resistance in clinical therapy of human malignant melanoma.

Artifacts↗

PubMed2004

Evaluation of epidermal growth factor receptor (EGFR) by chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) in archival gliomas using bright-field microscopy.

Overexpression of EGFR secondary to EGFR gene amplification is a common feature in primary malignant gliomas. To correctly assess EGFR protein and gene level as possible prognostic and predictive markers in gliomas, straightforward assays, which can be used routinely in the pathology laboratory to evaluate EGFR status, becomes critical. EGFR gene amplification and chromosome 7 aneuploidy was detected in 34 formalin-fixed, paraffin-embedded benign and malignant gliomas by chromogenic in situ hybridization (CISH) using digoxigenin-labeled EGFR and biotin-labeled chromosome 7 centromeric probes. The results were evaluated by bright-field microscopy under a 40x objective lens. EGFR protein level was detected by immunohistochemistry (IHC) using monoclonal antibody 31G7. Five cases, 3 astrocytoma grade III (33%) and 2 glioblastoma multiforme (GBM) (33%), had EGFR amplification displayed as diaminobenzidine-stained multiple dots suggesting the pattern of double-minute chromosomes. Chromosome 7 polysomy was found in 68% gliomas, 100% GBM, 67% astrocytoma grade III, 42% astrocytoma grade II, 50% astrocytoma grade I, 100% ependymoma, and the 1 case of mixed glioma III. High expression of EGFR protein was present in 62% gliomas and displayed membrane and cytoplasmic staining. All tumors with EGFR gene amplification showed EGFR high expression. High expression of EGFR without gene amplification was observed in all grades of gliomas. Simultaneous detection of EGFR gene copies or chromosome 7 centromere signals along with tissue morphology allows us to compare CISH results easily with IHC results. Our results show that CISH is an objective, practical, and accurate assay to screen for EGFR gene status in gliomas.

Aneuploidy↗

PubMed2006

JC virus chromogenic in situ hybridization in brain biopsies from patients with and without PML.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC polyoma virus. Electron microscopy and immunohistochemistry are the traditional methods of confirming the presence of the virus in brain biopsies from these patients. We studied the brain biopsies from 7 patients with PML and 6 patients without PML with chromogenic in situ hybridization (CISH) for the JC polyoma virus using a commercially available probe. The biopsies from the patients with the PML cases were proven to contain the JC polyoma virus by traditional and molecular methods. The CISH findings were compared with the known state of infection. All (7/7) of the biopsies from patients with PML were positive for the presence of polyoma virus by CISH, whereas the biopsies from patients without PML were uniformly negative. CISH seems to be a useful tool for the detection of the JC virus in brain biopsies from patients with PML, and is more accessible because a commercial probe is available.

Brain↗

PubMed2005

Chromo- and magnifying endoscopy for colorectal lesions.

It is essential to identify patients with premalignant or early malignant changes during colonoscopy. Thus, curative resection can be offered. At present, endoscopy can be performed with new powerful high-resolution or magnifying endoscopes. Comparably to the rapid development in chip technology, the optic features of the newly designed endoscopes offer resolutions which allow new mucosal surface details to be seen. In conjunction with chromoendoscopy, the newly discovered tool video endoscopy is much easier and more impressive than with conventional fibre optics. This review summarizes the value of magnifying endoscopy in the lower gastrointestinal tract and focuses on colorectal lesions.

Adenoma↗

PubMed1992

Functional activity of protein C in newborn infants. A report of a study and a review of the literature.

Protein C is lower in newborn infants than in adults. There are conflicting reports regarding functional activity and the presence of des-gamma carboxylated species in the newborn. We have compared protein C activity and antigenic level in newborn infants and found the activity: antigen ratio to be lower than in adults (0.69 versus 1.0). We discuss this finding in relation to the previously published reports.

Adult↗

PubMed2004

Her2 amplification: correlation of chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization.

Detecting Her2 gene amplification has become routine in predicting therapeutic responsiveness in patients with breast carcinoma. Fluorescence in situ hybridization (FISH) is a common technique for detecting Her2 amplification, yet dark field fluorescence microscopy remains problematic for many pathologists. Thus, a technique such as chromogenic in situ hybridization (CISH), in which the more familiar light microscopy can be used, is appealing. Paraffin-embedded sections from 61 breast carcinomas were tested for Her2 amplification by immunohistochemistry (IHC) and CISH. FISH was used to confirm CISH results. Excellent correlation was found between IHC and CISH except in cases considered negative (1+ on the DAKO scale) by IHC. CISH detected low-level Her2 amplification in 4 of 9 of these cases. Amplification was subsequently confirmed by FISH in all but 1 case. When compared with FISH, CISH was more sensitive than IHC for detecting low levels of Her2 gene amplification. Moreover, excellent concordance was found between FISH and CISH, supporting the conclusion that the CISH assay for Her2 gene amplification provides an accurate, effective, and practical alternative to FISH.

Breast Neoplasms↗

PubMed2006

Development and clinical evaluation of two chromogenic substrate methods for monitoring fondaparinux sodium.

This study was designed to develop methods for monitoring of the selective factor Xa inhibitor fondaparinux sodium (ARIXTRA) based on standard laboratory methods for the chromogenic determination of the anti-factor Xa activity of low molecular weight heparin. To examine the biologic activity of fondaparinux in comparison to its plasma concentration, 2 methods were investigated: 1 working with the addition of antithrombin (AT), the other without exogenous AT. Both methods showed a linear relationship of fondaparinux concentration and OD/min on a log-lin scale in the range from 0.1 to 2 microg/mL. Inter- and intra-assay variability was <6% in all cases. The results of spiked samples from patients on vitamin K antagonists (VKA) were in good agreement with both methods, and the determination of the fondaparinux concentration was not influenced by reduced levels of factor X in plasma caused by VKA-intake. Ex vivo samples from orthopedic patients (n=18) on prophylactic treatment with fondaparinux showed concentrations between 0.2 to 0.7 microg/mL 3 hours after s.c. injection. No significant differences were detected between both methods with these samples. The presented methods are suitable tools for monitoring of fondaparinux. The linear calibration curve in the range 0.1 to 2 microg/mL is suitable for determination of prophylactic and therapeutic application of fondaparinux. Both methods, with and without addition of AT, can be performed fully automated in clinical routine on an automated coagulation analyzer (STA coagulation analyzer). No significant differences were detected between both methods with these samples.

Anticoagulants↗

PubMed2002

Chromogenic in situ hybridization: a novel approach to a practical and sensitive method for the detection of HER2 oncogene in archival human breast carcinoma.

The high incidence of HER2 overexpression on the cell surface of breast cancer cells and the recognized prognostic and potentially predictive value of HER2 render this cell surface receptor a novel and important therapeutic target. Although immunohistochemistry (IHC; HercepTest) and fluorescence in situ hybridization (FISH; PathVysion and INFORM)-both approved by the Food and Drug Administration-have emerged as the most viable assays for evaluation of HER2 status in routine clinical practice, there is still no consensus on which is the best method for assessing HER2 status. Therefore, our specific objective was to establish a chromogenic in situ hybridization (CISH) assay for the detection of HER2 amplification on a cohort of 173 archival invasive breast carcinomas. Results were compared with HercepTest, which is the most frequently used method for detecting HER2 alteration. Additionally, HER2 gene copy number was investigated using differential PCR (dPCR) as a testing system. HER2 overexpression was found by IHC in 24.3%; HER2 amplification was found by CISH in 19.1% and by dPCR in 9.2% of the tumors. The overall concordance rate was 95.9% between CISH and IHC and 85.0% between dPCR and IHC. Kappa statistics revealed an excellent agreement between IHC and CISH (kappa = 0.878), but only a moderate agreement was found between IHC and dPCR (kappa = 0.482). Discrepant cases between CISH and HercepTest and all IHC-positive cases (+2 and +3), a total of 42 cases, were analyzed with the FISH PathVysion (Vysis) assay. Among 25 HercepTest-positive cases (score +3), 2 showed no gene amplification by FISH or CISH. Four of 13 tumors with weak HER2 overexpression (score +2) were negative with both FISH and CISH. Concordance between CISH and FISH was 100% for the 38 cases analyzed. The current study showed that CISH represents a practical and simple assay for evaluating HER2 gene amplification in archival material, offering a promising alternative to IHC or FISH for the routine diagnostic setting.

Breast Neoplasms↗

PubMed2005

Effects of pre-analytical variables on the anti-activated factor X chromogenic assay when monitoring unfractionated heparin and low molecular weight heparin anticoagulation.

The purpose of this study was to determine whether the anti-activated factor X (anti-FXa) assay is less affected by pre-analytical variables in monitoring patients on unfractionated heparin (UFH) and low molecular weight heparin (LMWH) than the activated partial thromboplastin time (aPTT). Forty-six subjects receiving either enoxaparin (LMWH) or UFH were randomly selected. Each study subject had six vacutainer tubes (3.8% sodium citrate, 3.2% sodium citrate) drawn by an atraumatic venipuncture. One tube from each set had a blood to anticoagulant ratio of 9: 1. The other tube had an intentional "short-draw" of approximately 6: 1 blood to anticoagulant ratio. All specimens had an aPTT and a chromogenic anti-FXa assay performed on each specimen regardless of heparin type. The aPTT assay mean with the 3.8% sodium citrate tube short-draw tube was statistically different from the other aPTT assays (P = 0.06). However, all six of the mean anti-FXa assays for the UFH and LMWH heparin subjects were not statistically or clinically different (analysis of variance, P = 0.9878 for UFH and P = 0.9060 for LMWH). The intentional short-draw tube did not affect the anti-FXa assay regardless of the anticoagulant. The anti-FXa assay appears to be a better method for monitoring heparin subjects than the aPTT due to the lack of effect of pre-analytical variables.

Adult↗

PubMed2006

The intrinsic coagulation activity assay.

A new assay for the contact-phase-mediated generation of thrombin activity has been developed - the intrinsic coagulation activity assay (INCA). Citrated plasma (50 microl) is incubated with 5 microl SiO2, 250 mmol/l CaCl2 in polystyrole flat-bottom wells. After exactly 4 and 5 min (37 degrees C) coagulation reaction times (INCA-4 and INCA-5), 100 microl of 2.5 mol/l arginine, pH 8.6, is added to inhibit hemostasis activation in the important ascending part of the thrombin generation curve and to depolymerize fibrin. After 20 min, 50 microl of 1 mmol/l (final concentration 0.24 mmol/l) chromogenic thrombin substrate CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, is added. The increase in absorbance is determined at 405 nm using a microtiterplate photometer. The assay is calibrated against 1 IU/ml thrombin. The normal thrombin activity range of INCA-4 (main value) or INCA-5 (control value) is 100 +/- 30% of normal (mean value +/- 1 SD; 100% = 0.5 IU/ml for INCA-4 and 1.9 IU/ml for INCA-5). With the INCA the normal range of intrinsic hemostasis is reflected, low-molecular-weight heparins can be monitored, the plasma matrix is not changed significantly, and the assay results are a percentage of normal generated thrombin activity and not coagulation seconds.

Blood Coagulation Tests↗

PubMed2005

HER2 positivity in breast carcinoma: a comparison of chromogenic in situ hybridization with fluorescence in situ hybridization in tissue microarrays, with targeted evaluation of intratumoral heterogeneity by in situ hybridization.

An accurate and reproducible assay method for determining HER2 status is crucial, as a positive HER2 gene status is an eligibility requirement for Herceptintrade mark therapy. Although immunohistochemical (IHC) assessment is both practical and inexpensive, a worrying trend of high false-positive rates has been reported. Fluorescence in situ hybridization (FISH) is the universally accepted gold standard for confirming IHC 2+ cases and ambiguous results but is costly and requires specialized equipment and technical expertise. Chromogenic in situ hybridization (CISH) amalgamates the practical advantages of IHC with the reproducibility of FISH, and high concordance between the CISH and FISH methods has been reported in conventional sections. Tissue microarrays (TMAs) allow high throughput of specimens, and HER2 status assessment in TMA cores using IHC and FISH has correlated well with scores in conventional sections. The authors used TMA technology to compare the efficacy of ZYMED(R) CISH with PathVysiontrade mark FISH in a cohort of 119 archival breast resection cases and investigated possible intratumoral heterogeneity in a "mini-array" of 21 HercepTest "equivocal"/2+ cases. Concordance between FISH and CISH in TMA sections was 99%. All prescored 2+ HercepTest cases were nonamplified. Four 3+ HercepTest cases were classed as potential false-positives. The authors suggest that confirmatory ISH should be performed on all positive HercepTest cases. CISH was easier to perform and quicker to enumerate than FISH. The authors conclude that CISH is a practical alternative to FISH as a confirmatory tool for HER2 gene amplification status. Intratumoral heterogeneity did not affect the patient's HER2 status.

Aneuploidy↗

PubMed2005

Identification of MYCN gene amplification in neuroblastoma using chromogenic in situ hybridization (CISH): an alternative and practical method.

Chromogenic in situ hybridization (CISH) is a recently developed technique, which utilizes the general principles of in situ hybridization and a detection system similar to immunohistochemistry. To assess the utility of CISH for analysis of MYCN gene amplification, we compared this assay with established diagnostic assays such as Southern blot analysis (SB) and fluorescent in situ hybridization (FISH). CISH was performed on 67 cases of neuroblastoma using tissue microarray (65 cases) and whole tissue sections (2 cases). Unequivocal, high-level amplification (> or =10 gene copies per tumor nucleus) was identified in 19 of 67 (28.4%) tumors. Two (3%) tumors showed low-level amplification (6-9 gene copies per tumor nucleus). No amplification was seen in 46 of 67 (68.6%) tumors. SB data were available in 44 tumors. Forty-one of the 44 tumors (93%) showed concordant results between CISH and SB. Three tumors showed MYCN amplification by CISH but no amplification by SB, most likely due to dilution effect of nonneoplastic tissue in the test samples. Two of these three tumors also showed MYCN amplification by FISH, and the third tumor was not analyzed by FISH. FISH data were available in total of 30 tumors. All 30 tumors showed concordant results between CISH and FISH for classifying a tumor as MYCN amplified or not amplified. We conclude that CISH is an accurate method for determining MYCN gene amplification, with added advantages that make it a more practically useful method.

Child, Preschool↗

PubMed2006

Use of chromogenic in situ hybridization to identify MYCN gene copy number in neuroblastoma using routine tissue sections.

Amplification of MYCN in neuroblastoma is associated with a poor prognosis. However, methods for estimating the number of MYCN genes based on pooled cells do not address copy number heterogeneity at the cell level and can underestimate or even miss amplification. MYCN copy number can be directly assessed by fluorescence in situ hybridization, but evaluation of tissue histology is next to impossible. We have used a chromogenic method for in situ hybridization (CISH) that enables determination of MYCN copy number using routine light microscopy on routinely processed paraffin sections. Of 41 cases studied, CISH identified 100% of the 18 cases that were determined to be amplified by other techniques and was more sensitive than Southern blotting or quantitative DNA polymerase chain reaction. Because the technique evaluates individual tumor cells, heterogeneity of MYCN copy number was apparent from cell to cell. When defined as 50% or greater variation in copy number between cells in amplified tumors, almost 30% of cases were scored as heterogeneous. Heterogeneity reflects different tumor clones and its role has likely been under-recognized and underestimated in neuroblastoma biology. CISH will provide a valuable tool to assess this phenomenon in conjunction with other morphologic parameters in neuroblastoma specimens, to further our understanding of the biology of this childhood tumor.

Blotting, Southern↗

PubMed2002

Comparison of Candida ID medium with sabouraud-chloramphenicol agar for the isolation of yeasts from clinical haematology surveillance specimens.

Candida ID is a new chromogenic medium for the identification of yeasts from clinical specimens. C. albicans produces blue pigmentation, whereas pink pigmentation is produced by C. tropicalis, C lusitaniae, C. guilliermondii and C. kefyr; other Candida species appear white. In this study, 240 clinical samples (throat swabs and stool samples) from haematology patients were inoculated on to Candida ID and Sabouraud-chloramphenicol agar in parallel, yielding a total of 105 yeasts; the media had overall detection rates of 85.7% and 86.7% respectively. The sensitivity of Candida ID for identification of C. albicans by blue pigmentation was 52.9% at 24 h and 94.1% at 48 h. Specificity of the blue pigmentation was 100% at 48 h. Two strains of C. tropicalis were identified, one produced pink pigmentation at 72 h, the other strain did not produce any pigmentation after 5 days. Candida ID was superior in detecting mixtures of yeasts compared with Sabouraud-chloramphenicol agar. Candida ID is a suitable primary isolation medium for yeasts from clinical specimens, providing rapid direct identification of C. albicans and enhanced detection of mixtures.

Agar↗

PubMed2005

Use of chromogenic medium in the isolation of yeasts from clinical specimens.

Over a 1 year period 3296 specimens submitted for fungal culture were plated onto routine mycological media (RM) and CHROMagar Candida (CaC) to evaluate the capability of CaC to improve on RM. With RM, cultures producing single yeast isolates were identified from 802 specimens. CaC produced similar results, with 76 % agreement. Of 761 specimens that yielded a single Candida species by RM, 615 (81 %) produced one or more yeast isolates using CaC. Of concern, 132 negative CaC cultures corresponded to specimens that yielded C. albicans alone on RM. When yeasts were recovered, CaC correctly identified 98 % of C. albicans, 93 % of Candida tropicalis, 96 % of Candida glabrata and 100 % of Candida krusei based on typical colours. CaC did potentially improve on RM by detecting yeasts in 91 specimens that yielded none by routine methods. CaC was noted to recover more yeast isolates than RM when mixed cultures were detected. Overall, the role of CaC in improving RM appears limited.

Blood↗