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PubMed1999

Lymphoscintigraphy and intraoperative lymphatic mapping of sentinel lymph nodes in melanoma patients.

Identification of sentinel lymph nodes (SLNs) using lymphoscintigraphy, the blue dye technique and intraoperative lymphatic mapping with a gamma-detecting probe has become the standard of care in diagnosing and treating melanoma. Numerous clinical studies have proven the reliability of predicting the histology of remaining lymph nodes in the lymphatic basin from the histologic evaluation of the SLNs. Technical and clinical factors presented in this paper have been shown to increase the accuracy of localization of SLNs. The nuclear medicine technologist shares a vital role in the radiopharmaceutical preparation and administration for preoperative lymphoscintigraphy and intraoperative lymphatic mapping in patients with melanoma.

Humans↗
PubMed2000

[Locating the sentinel node in breast cancer by gamma probe and staining agent. Preliminary study].

OBJECTIVE: The sentinel node biopsy appears to offer an alternative to routine axillary lymph node dissection for staging patients with breast cancer. Various techniques have been studied for identifying the sentinel node, using vital blue or radioactive colloid. This study aimed to evaluate our preliminary results with these techniques. MATERIAL AND METHODS: In this work carried out in the Valencian Institute of Oncology, 21 women with T1- T2 breast cancer with an average age of 52 years (range: 42-73) underwent the sentinel node biopsy, which was immediately followed by standard axillary dissection. Both blue dye and radioisotope were used to identify the sentinel node. The radioactive axillary of sentinel node was localized by the gamma probe. RESULTS: The sentinel node was successfully identified by lymph node scintigraphy in 100%. It was localized by blue dye in 33% and by combination of blue dye and isotope in 95%. Of the 21 patients in this study in whom sentinel nodes were identified, 7 (35%) were histologically positive; in 6 cases, the sentinel was the only site of the metastases (86%). The histology of the sentinel node accurately predicted axillary node status in 95% of cases. CONCLUSIONS: The sentinel node biopsy technique is a promising and feasible procedure in patients with clinically T1-T2 N0M0 breast cancer, providing valuable axillary staging information.

Adult↗
PubMed2001

Sentinel lymph node detection and microstaging in vulvar carcinoma.

OBJECTIVE: To determine the efficacy of using complementary techniques for detecting sentinel lymph nodes (SLNs) in vulvar carcinoma and to evaluate the utility of microstaging techniques. STUDY DESIGN: Patients with invasive vulvar carcinoma underwent sentinel lymph node detection (SLND) using preoperative lymphoscintigraphy, intraoperative isosulfan blue dye injection and an intraoperative hand-held gamma-detecting probe. Eleven patients were included and a total of 16 groins evaluated. Sentinel nodes identified were excised, bisected and examined in surgical pathology using hematoxylin and eosin (H&E) staining. Pathologically negative SLNs were subjected to additional microstaging via serial sectioning and immunohistochemical staining for cytokeratin. Surgical management of the vulvar cancer and extent of inguinal-femoral lymphadenectomy were individualized based on clinicopathologic parameters, including depth of invasion, location of the tumor and patient performance status. RESULTS: Lymphoscintigraphy, dye and gamma-detector methods led to the total detection of 16, 19 and 17 SLNs, respectively. In two cases the isosulfan blue dye assisted in the isolation of an additional sentinel node over that of the gamma probe. Each method individually identified SLNs in 10/11 patients (91%). A total of 19 sentinel nodes were isolated. One SLN (5%) was positive for metastatic disease using H&E staining. Of the 18 negative SLNs, 2 (11%) had micrometastases (< 0.2 mm) upon serial sectioning and immunohistochemical staining. CONCLUSION: Combined-modality mapping enhances detection of SLNs in vulvar carcinoma. Histologic microstaging improves the detection of micrometastases within SLNs.

Adult↗
PubMed2003

Effect of low-viscosity resin-based composite on the microleakage of cervical restorations.

PURPOSE: To investigate the effect of a low-viscosity resin-based composite (LVC) liner on the microleakage of cervical resin-based composites bonded with new adhesive systems. METHODS: 180 wedge-shaped cervical cavities on bovine incisors were treated with AQ Bond (all-in-one), ABF (experimental two-step self-etching primer containing antibacterial monomer) or Clearfil SE Bond (two-step self-etching primer) systems according to the manufacturers' instructions. Half of the treated cavities in each adhesive system were lined with an LVC (Protect Liner F) and filled with a hybrid resin-based composite (Clearfil AP-X). Ten specimens from each group were immersed in 0.5% basic fuchsin solution immediately after finishing the restorations. The specimens to be subjected to thermocycling (5-60 degrees C, 15 seconds dwell time, 5,000 cycles) or to flexural load cycling (approximately 0.5 mm labio-lingual displacement at the incisal edge, 10,000 cycles, 1 Hz) prior to immersion in the dye solution were also prepared. The teeth were sectioned through the center of the restorations and the degrees of dye penetration were scored. The data were analyzed with the Mann-Whitney U-test or the Kruskal-Wallis test (P < 0.05). RESULTS: The use of the LVC liner significantly improved marginal sealing of AQ Bond. However, the LVC liner could not prevent the deterioration in the marginal integrity caused by thermocycling or flexural load cycling. In the case of ABF, the use of the LVC liner showed a trend toward less microleakage. For Clearfil SE Bond, the LVC liner prevented the deterioration in the marginal seal caused by thermocycling.

Animals↗
PubMed1988

A simple two-dye basic stain facilitating recognition of mitosis in plastic embedded tissue sections.

Semithin sections of buccal and palatal mucosa fixed in 2.5% glutaraldehyde followed by 1% osmium and embedded in Durcupan (an araldite-based resin) were stained with 2% malachite green in 50% ethanol at 80 C and poststained in 0.05% crystal violet in Sorensen's phosphate buffer (pH 6.4) at 45 C. Nuclear envelopes and chromatin stain vivid purple in contrast to the surrounding green cytoplasm and cell borders. Chromosomes of dividing cells stain bluish violet. Nucleoli, depending on their level in the epithelium, stain differing shades of greenish blue. The distinct and differential staining of each of these components facilitates recognition of mitoses in oral epithelium, where the small size and crowding of cells in the proliferative compartment renders more conventional stains for plastic sections inadequate.

Animals↗
PubMed1989

Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in Swiss 3T3 cells.

This paper describes two applications of the tandem scanning reflected-light microscope (TSM) for the observation of the structure of individual cells growing in tissue culture. First, the TSM is used as an alternative to interference reflection microscopy (IRM) or total internal reflection aqueous fluorescence microscopy (TIRAF) to observe cell-substratum adhesions in unstained living cells growing on a glass coverslip. Second, the TSM is used to produce improved images of cellular structures in 3T3 cells stained with various protein dyes including Napthol Blue Black (NBB) and Coomassie Brilliant Blue (CBB). More specifically, close contacts and focal contacts are resolved in living 3T3 cells, and features of the nucleus, the cytoskeleton and extracellular matrix are resolved in both NBB- and CBB-stained cells. The focal contacts and associated stress fibres are clearly imaged in NBB-stained cells. The TSM is an improvement over conventional incident light microscopy because of the confocal image excludes information from out-of-focus regions of the cytoplasm, and, unlike the laser-based confocal microscope, the actual colour of the specimen is viewed directly with TSM in almost real-time.

Amido Black↗
PubMed1985

Survival of myocutaneous flaps. Experimental evaluation by intra-arterial injection of a dye.

Ninety-four myocutaneous island flaps of the pectoralis major were studied in the rabbit using sulfan blue (Disulphine-blue). In 13 cases, the dye was injected intra-arterially in the acromiothoracic artery before the flap was lifted, and 47 more were injected after the lifting of the flap. In 34 further cases, sulfan blue was injected intravenously once the flap had been lifted. In another 16 flaps, vascularization was studied by means of "diaphanization" (ie, making the tissue transparent or diaphenous in nature). In all groups, the surviving length of flap was observed to be greater than the stained length. Intra-arterial administration of sulfan blue was associated with reduced flap survival. Viable flap length was not related to the anatomy of the vascular base.

Animals↗
PubMed1985

A method to quantitate Coomassie blue-stained proteins in cylindrical polyacrylamide gels.

A method for the quantitation of Coomassie blue-stained proteins in cylindrical polyacrylamide gels is described. It involves an elution of the dye with an 80% methanol solution in a sealed Pyrex tube at 100 degrees C for 3 h and a measurement of its concentration at 585 nm. Using a 6.5% polyacrylamide gel and bovine serum albumin as a protein standard, the curve of absorbance of the dye solution as a function of the amount of protein was observed to be linear up to 30-40 micrograms of protein and as little as 0.8-1.0 micrograms of protein could be measured. The validity of the method was indicated by the values obtained for the relative proportions of the human erythrocyte membrane proteins. Using this method, the color yields of several proteins varying widely with respect to their size, amino acid composition, and carbohydrate content were determined in a 6.5% polyacrylamide gel. The results showed that they were generally the same except for proteins having a high carbohydrate content which were significantly lower.

Animals↗
PubMedDate not supplied

Bonding to dentin: evaluation of three adhesive materials.

Dye penetration was observed in all specimens. SEM demonstrated isolated areas with no gap formation, suggesting a partial bond with dentin. A correlation is evident from the results of both techniques. Since dye-penetration was found to be similar in all the specimens, it was difficult to assess the effect of thermocycling on the amount of dye penetration. The use of posterior composites should be considered as a short-term tested procedure. It should be utilized carefully, following the manufacturer's instructions, and monitored routinely. Undoubtedly, the utilization of posterior composite materials is a very technique-sensitive procedure. Comparing the results of this in vitro study with those previously reported suggests that little improvement has been made in the bonding of the materials tested. Development of new materials and improved techniques are necessary.

Adhesives↗
PubMed1990

Increased uniformity in the response of the coomassie blue G protein assay to different proteins.

Coomassie blue G dye-based protein assays are exceptionally convenient because of their simplicity, sensitivity, speed, and resistance to interfering chemicals, notably reducing agents and most buffers. A major problem with the assay is the variation in response to different proteins. The addition of NaOH to the protein assay reagent reduced the variation in the response of this assay to different proteins. In addition, the sensitivity of the assay is increased. The NaOH can be added either in a separate step to solubilize cells or membranes or directly to the reagent. Linear standard curves were obtained when the log of the absorbance was plotted against the log of the protein quantity.

Animals↗
PubMed2006

In vitro toxicity evaluation of single walled carbon nanotubes on human A549 lung cells.

This paper describes the in vitro cytotoxicity assessment of single walled carbon nanotubes (SWCNT) on A549 cells, a human lung cell line. Cellular viability was determined using the alamar blue (AB), neutral red (NR) and MTT assays, which evaluated metabolic, lysosomal and mitochondrial activity respectively. In addition, the total protein content of the cells was measured using the coomassie brilliant (CB) blue assay. Supernatants were also assayed for Adenylate Kinase (AK) release and Interleukin 8 (IL-8) which indicated a loss of cell membrane integrity and an inflammation response respectively. To investigate the interactions between serum components in the test medium and the test materials, exposures were conducted both in serum containing (5%) and serum-free medium. Results from the cytotoxicity tests (AB, CB, MTT) revealed the SWCNT to have very low acute toxicity to the A549 cells as all but one of the reported 24h EC(50) values exceeded the top concentration tested (800 microg/ml). The SWCNT were found to interfere with a number of the dyes used in the cytotoxicity assessment and we are currently conducting a comprehensive spectroscopic study to further investigate these interactions. Of the multiple cytotoxicity assays used, the AB assay was found to be the most sensitive and reproducible. Transmission electron microscopy (TEM) studies confirmed that there was no intracellular localization of SWCNT in A549 cells following 24h exposure; however, increased numbers of surfactant storing lamellar bodies were observed in exposed cells.

Adenylate Kinase↗
PubMed2002

Binding to an RNA aptamer changes the charge distribution and conformation of malachite green.

RNA plays a central role in many biological processes and is therefore an important target for drug development. In recent years an increasing wealth of structural and functional information about RNA-ligand complexes has been obtained using in vitro selected RNAs (aptamers). However, all those studies focused on structure and changes of the nucleic acid and mostly considered the ligand as a rigid target. To develop a detailed picture of ligand structure and dynamics in RNA-small molecule complexes, the malachite green binding aptamer was studied. Isotopically labeled ligand in complex with RNA was analyzed by NMR spectroscopy in solution. The surprisingly asymmetric changes in the (13)C chemical shift of the ligand methyl groups indicate that the dye undergoes changes in its conformation and charge distribution upon binding. The role of the RNA electrostatic field in this interaction was explored using ab initio calculations of the ligand structure and charge distribution. The results indicate that the uneven charge distribution in the RNA binding pocket provides a major contribution to the driving force of the ligand structural changes. The observation that not only the RNA adapts to the ligand, in what is called adaptive binding, but that the ligand itself also undergoes conformational changes ("induced fit") is crucial for the rational design of RNA ligands and for understanding the properties of RNA-ligand complexes.

Molecular Conformation↗
PubMed1996

Intraoperative radio-lympho-scintigraphy improves sentinel lymph node identification for patients with melanoma.

BACKGROUND: The sentinel lymph node (SLN), the first node draining the primary tumor site, has been shown to reflect the histologic features of the remainder of the lymphatic basin in patients with melanoma. Intraoperative localization of the SLN, first proposed by Morton and colleagues, has been accomplished with the use of a vital blue dye mapping technique. Technical difficulties resulting in unsuccessful explorations have occurred in up to 20% of the dissections. OBJECTIVES: The authors aimed to define the SLN using gamma detection probe mapping and to determine whether intraoperative radiolymphoscintigraphy using technetium sulfur colloid and a hand-held gamma-detecting probe could be used to improve detection of all SLNs for patients with melanoma. METHODS: To ensure that all initial nodes draining the primary site were removed at the time of selective lymphadenectomy, the authors used intraoperative radiolymphoscintigraphy to confirm the location of the SLN, which was determined initially with the preoperative lymphoscintigram and the intraoperative vital blue dye injection. PATIENT POPULATION: The patient population consisted of 106 consecutive patients who presented with cutaneous melanomas larger than 0.75 mm in all primary site locations. RESULTS: The preoperative lymphoscintigram revealed that 22 patients had more than one lymphatic basin sampled. Two hundred SLNs and 142 neighboring non-SLNs were harvested from 129 basins in 106 patients. After the skin incision was made, the mean ratio of hot spot to background activity was 8.5:1. The mean ratio of ex vivo SLN-to-non-SLN activity for 72 patients who had SLNs harvested was 135.6:1. When correlated with the vital blue dye mapping, 139 of 200 (69.5%) SLNs demonstrated blue dye staining, whereas 167 of 200 (83.5%) SLNs were hot according to radioisotope localization. With the use of both intraoperative mapping techniques, identification of the SLN was possible for 124 of the 129 (96%) basins sampled. Micrometastases were identified in SLNs of 16 of the 106 (15%) patients by routine histologic analysis. CONCLUSION: The use of intraoperative radiolymphoscintigraphy can improve the identification of all SLNs during selective lymphadenectomy.

Adolescent↗
PubMed2000

Microleakage of a condensable resin composite: an in vitro investigation.

OBJECTIVE: The purpose of this investigation was to evaluate microleakage in Class II cavities restored with the condensable resin composite SureFil, placed with or without a liner and 1 of 2 different dentin bonding agents. METHOD AND MATERIALS: Extracted noncarious human teeth were randomly divided into 4 groups of 15 each and restored according to manufacturer's instructions: group 1, SureFil and Prime & Bond NT; group 2, SureFil and PQ1; group 3, SureFil, PermaFlow, and PQ1; group 4, Heliomolar and Syntac Single-Component (control). Specimens were stored in 37 degrees C water for 24 hours. All specimens were thermocycled between 5 degrees C and 55 degrees C for 500 cycles. Each specimen was immersed in 0.2% basic fuchsin dye for 24 hours and then sectioned longitudinally and horizontally. Dye penetration at enamel and cementum margins were viewed at x30 magnification by 2 independent, calibrated evaluators. Specimens exhibiting dye penetration > or = 0.1 mm beyond the cavosurface margin were considered to have leakage. RESULTS: Chi-square tests revealed no statistically significant differences in the presence of microleakage when all groups were analyzed together. However, 2 by 2 comparisons revealed that group 3 had significantly fewer specimens with microleakage than did group 1, 2, or 4. CONCLUSION: To limit microleakage, a flowable composite should be used as a liner when a condensable composite material is used.

Acid Etching, Dental↗
PubMed1998

Quantitation of Cremophor EL in human plasma samples using a colorimetric dye-binding microassay.

This paper describes an analytical procedure for the quantitative determination of the pharmaceutical vehicle Cremophor EL in human plasma samples. The procedure is based on rapid binding of Coomassie brilliant blue G-250 to Cremophor EL following plasma protein precipitation with acetonitrile and analyte extraction with n-butylchloride. The binding of the dye to Cremophor EL causes a shift in the absorption maximum from 465 to 624 nm, which is monitored using an automated microplate-absorbance reader. The assay permits estimation of Cremophor EL concentrations in the range 0.05-1.00% (v/v) in 50 microL of human plasma, with percentage deviation and precision of < or = 12 and < or = 15%, respectively. The assay was subsequently used to measure Cremophor EL concentrations in plasma samples in support of a project to develop a pharmacokinetic model for this compound in patients receiving paclitaxel.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Preparative binding of Coomassie brilliant blue to bovine serum.

Laboratory scale preparation of bovine serum albumin (BSA) stained with Coomassie brilliant blue (CBB) at alkaline pH is first described. Physical-chemical analyses of CBB-BSA showed that the unprotonated (anion) CBB dye binds tightly to BSA in buffered media of pH 8.2. Characteristic differences in spectra lambda(max) and molar absorptivities were found for the free anion CBB dye versus the CBB-BSA complex. Binding studies with low versus high dye/protein concentration ratios at alkaline pH gave values for n, binding site numbers, and K, intrinsic binding coefficient, consistent with those reported in analytical studies under acidic pH, but higher than values for neutral pH. Comparative analyses of Beer's law plots for the alkaline CBB-BSA complex under different experimental conditions showed its high stability toward various interferences, such as pH, strong detergents, temperature, light, prolonged storage, as well as high affinity for tannins. The hydrophobic nature of the CBB-BSA association at alkaline pH was tested.

Animals↗
PubMed1990

Assessment of marginal leakage around Class II composite restorations in retrieved primary molars.

The aim of the present investigation was to evaluate, by means of dye penetration, the microleakage around Class II composite restorations, in retrieved primary molars that functioned in the mouth for at least one year. The experimental material consisted of 13 exfoliated primary molars that had been restored with Herculite (Kerr Corporation, Romulus, Michigan 48174 USA) at least one year previously, utilizing an incremental or a bulk filling technique. The retrieved teeth were insulated with utility wax and nail polish, immersed in 2% basic fuchsin, embedded in acrylic resin, and ground off to various depths. The marginal leakage was assessed according the degree of dye penetration at the occlusal and cervical margins. No difference was observed between the two filling techniques. In most teeth, no leakage at the occlusal margins was observed; minimal leakage, limited to the enamel, was observed at the occlusal margins of two teeth, one of each filling technique. Severe penetration was evident at the cervical margin of three restorations, two of them filled incrementally and the third using the bulk technique. Mild to moderate penetration was observed at the cervical margin in the majority of the other restorations. It was concluded that an incremental filling technique could not eliminate microleakage at the cervical margins of Class II composite restorations.

Child↗
PubMed2001

Sentinel lymph node identification and sampling in women with early breast cancer using 99m Tc labelled dextran 500 and patent blue V dye.

The status of the homolateral axillary lymph nodes is still the most important prognostic factor in early stage breast cancer. The information obtained from the pathological examination of the lymph nodes guides is of critical importance in the decision process regarding the use of postoperative adjuvant therapy. However, lymph node axillary dissection can be followed by significant locoregional morbidity. The sentinel lymph node (SLN) technique was developed as a means of avoiding the full exploration of the axilla and consists in the identification of the first lymph node in the lymphatic drainage system of the breast tumour in the homolateral axilla. It has been demonstrated that the status of the SLN is highly predictive for the presence or absence of tumour involvement in the remaining lymph nodes in the axilla. In this study we evaluated the SLN technique using both 99mTc labelled dextran 500 and patent blue V dye in relation to the classical lymph node resection a series of 56 women with early breast cancer who attended the Breast Unit of the Academic Hospital of the Federal University of Rio Grande do Sul, Brazil. To our knowledge this is the first report in the literature of the utilization of 99mTc dextran 500 for the SLN technique. As there are no similar commercially available dedicated radiopharmaceuticals labelled for use in lymphoscintigraphy studies, we report on an effective method to label dextran 500 with 99mTc which proved to be simple, inexpensive and yielded similar results for SLN identification compared with those given in the literature. The median age of the patients was 57 years (range 32-82 years). Seventeen patients were age 50 years or less, and 39 patients were older than 50 years. The median tumour size was 2.0 cm (range 0.8-7.0 cm). The mapping of the SLN was possible in all cases during the transoperative period by using a hand-guided gamma probe and a blue dye. A median of 2.0 (range 1-5) SLN were excised per patient. The median of axillary lymph nodes excised per patient was 21 (range 10-36). The calculated sensitivity and specificity of the method were 95.6% and 100%, respectively. The negative predictive value and overall accuracy were 97% and 98.2%, respectively. In conclusion, the SLN technique was feasible and produced similar positive results as previously reported in the literature.

Adult↗