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PubMed1986

Nucleic acid staining with the methyl green-pyronin method. A comparison of the use of pure dyes and commercially available dyes.

We compared the staining obtained using commercially available pyronin Y samples with that obtained using pure pyronin Y in a standardized methyl green-pyronin procedure. In addition, the importance of the dye content of the anhydrous dye was investigated by varying the dye content by the addition of pure pyronin Y to one of the commercially available pyronin Y samples. We found that, for routine histological work, commercially available pyronin Y samples may produce acceptable results provided the sample can be shown by spectrophotometry to contain at least 43% pyronin Y.

Animals↗
PubMed2004

Sentinel lymph node biopsy assessment using intraoperative imprint cytology in breast cancer patients: results of a validation study.

OBJECTIVE: Sentinel lymph node biopsy (SLNB) in breast cancer patients is emerging as a promising minimally-invasive tool. There has been an exponential increase in the literature related to sentinel lymph nodes (SLN) in breast cancer patients, mainly from Western centres. This study was carried out to address issues relevant to breast cancer patients in developing countries, including the method of SLN detection, the role of imprint cytology in the assessment of SLN, and the role of SLNB in locally advanced breast cancer (LABC). METHODS: This study included 76 women with breast cancer. The blue-dye method was used to identify the sentinel node. Touch imprint smears were prepared from the sectioned node, stained using the Jenner-Geimsa technique, and examined for tumour deposits. RESULTS: Sentinel nodes were identified in 69 of 76 patients. The sensitivity, specificity and accuracy of SLNB in predicting axillary node status were 84.2%, 100% and 91.3%, respectively. The sensitivity, specificity and accuracy of intraoperative imprint cytology were 96.9%, 100% and 98.6%, respectively. CONCLUSIONS: These results prove that high levels of SLN detection can be achieved using the blue-dye method alone. Its role in LABC patients needs further evaluation. In view of promising results, imprint cytology should be used more frequently as an alternative to frozen section for the assessment of sentinel nodes.

Axilla↗
PubMed1955

Buffered osmium tetroxide (OsO4) fixation for cytological and Feulgen microspectrophotometric studies of human rectal polyps.

Evidence has been presented that when human rectal mucosa and polyps are fixed with buffered OsO(4) according to the method of Palade, staining of paraffin sections by basic and acidic dyes results in a distinct color difference between chromatin and cytoplasm. Furthermore, the Feulgen reaction also can be carried out successfully and such OsO(4)-fixed Feulgen-stained sections are especially suitable for microspectrophotometric determinations of DNA in individual nuclei.

Buffers↗
PubMed1977

Allergic reaction to Patent Blue Violet during lymphography.

A case of an allergic reaction to Patent Blue Violet dye used for the purpose of lymphangiography is reported. The clinical findings were urticarial reaction, edema of the eyelids and lips, and hypotension. Previous sensitization to triphenylmethane dyes, in drugs and other chemicals was likely, but not proven. Such commonly used compounds have been compiled in a Table. History of a previous hypersensitivity reaction to these compounds is not a contraindication for lymphangiography but should call for emergency planning and possible premedication.

Adult↗
PubMed2001

Intraoperative decrease in pulse oximeter readings following injection of isosulfan blue.

PURPOSE: Sentinel lymph node mapping, involving injection of isosulfan blue dye around a tumour, is beginning to be used in patients with carcinoma of the breast. Absorption of the dye into the circulation may interfere with pulse oximetry, causing falsely low readings. This report describes changes in pulse oximeter readings following injection of isosulfan blue for sentinel lymph node mapping in a patient with carcinoma of the breast. CLINICAL FEATURES: An 83-yr-old female patient underwent sentinel node biopsy of the axilla followed by partial mastectomy for carcinoma of the left breast. Isosulfan blue was injected in the area of the tumour in the left breast. The SpO2 began to decrease 15 min after dye injection, reaching a nadir of 89-90% 30 min after injection. Arterial blood gas analysis showed normal arterial partial pressure of oxygen. Pulse oximeter readings did not return to normal until more than six hours after dye injection. CONCLUSION: Review of the literature reveals a small number of case reports of similar occurrences of low pulse oximeter readings following injection of isosulfan blue or patent blue dye for lymphatic mapping. Data from these reports and the case described here suggest that the latency, magnitude and duration of effect on pulse oximeter readings following injection of these dyes is highly variable. It is important to rule out other causes of low pulse oximeter readings when this effect occurs; normal oxygenation can be verified with arterial blood gas analysis. Co-oximetry can be done to rule out methemoglobinemia as a cause of decreased SpO2.

Adenocarcinoma↗
PubMed1987

Inorganic and organic phosphate measurements in the nanomolar range.

A procedure, based on the complex formation of malachite green with phosphomolybdate under acidic conditions, to measure inorganic orthophosphate in the nanomolar range is described. The addition of polyvinyl alcohol is required to stabilize the dye-phosphomolybdate complex. The advantages of the assay are simplicity, stability of the reagents, and high sensitivity. Due to the high permissible acidity in the assay (0.9 N H2SO4), the method can be adapted easily to measure nanomolar amounts of phosphate, liberated from organic compounds like phosphoproteins and phospholipids after wet digestion.

Microchemistry↗
PubMed1998

Fuchsin fluorescence and autofluorescence in Cryptosporidium, Isospora and Cyclospora oocysts.

Cryptosporidium parvum and Isospora belli oocysts stained with carbol-fuchsin, as in a modified Ziehl Neelsen technique, fluoresce bright red under green light (546nm). Cryptosporidium oocysts tend to fluoresce more brightly the less intensely stained they appear under transmitted light; this is not the case with Isospora. Fuchsin-stained Cyclospora cayetanensis oocysts fluoresce rather dimly, but those not taking the dye retain their typical autofluorescence. Cryptosporidium and Isospora oocysts are also autofluorescent, appearing violet under u.v. light (365 nm), and green under violet (405 nm) and blue-violet light (436 nm). Their autofluorescence does not survive the staining procedure.

Animals↗
PubMed2002

Direct quantification of autologous serum albumin leakage after burn injury in mice.

The current technique determining the extent of capillary leakage after injury is to measure the leakage of dye-labeled foreign albumin. A recent report, however, demonstrated that albumin leakage is dependent upon the type of fluorescent dye used for labeling. We chose to develop and test a technique for determining the extent of vascular albumin leakage after burn injury without the use of dyes. Skin and blood samples were harvested at 3h and 7 days after burn injury in mice. Total skin lysates and extracts as well as sera were analyzed for albumin leakage. Coomassie staining and Western blot analyses of skin preparations followed by the densitometric measurement revealed increased levels of albumin, suggesting that the leakage of serum albumin started within 3h after burn injury. Instead of employing dye-labeled foreign albumin, the use of SDS-polyacrylamide gel electrophoresis of tissue extracts followed by Coomassie staining will allow for a simple and direct quantification of autologous albumin leakage due to burn as well as other types of injury.

Animals↗
PubMed1998

Experience with lymphatic mapping in breast cancer using isosulfan blue dye.

Sentinel (first tumor-draining) lymph node (SLN) biopsy directed by the blue dye technique may be as accurate as complete axillary lymph node dissection (ALND) in determining whether breast cancer has metastasized to the lymph nodes and may have fewer surgical complications because it is less invasive. Breast cancer patients scheduled for ALND between February and June 1997 who did not have prior axillary surgery, prior radiation therapy, or preoperative chemotherapy were included. Isosulfan blue dye was injected around the primary tumor or the biopsy cavity just before ALND. Operations were performed in a tertiary breast center by two breast surgeons who did not have experience with the technique before this study. The results of blue stained nodes were compared to those of the ALND. Blue-stained nodes were identified in 35 of 40 patients (88%), and the results were concordant with ALND in 33 of 35 (94%), 7 patients were concordant for positive results and 26 for negative results. We identified SLNs in patients whose cancers were either in the medial or lateral halves of the breast. Average time for SLN dissection was 19 +/- 9 minutes, and there were no complications. The diagnostic accuracy of the isosulfan blue dye technique for SLN biopsy, 94%, is high enough to warrant further research. The lack of complications and the short time needed to perform the technique are attractive features. Broader experience with the technique is required to evaluate the reliability and reproducibility of this method.

Adult↗
PubMed1982

Bulk preparation of CNS cytoskeleton and the separation of individual neurofilament proteins by gel filtration: dye-binding characteristics and amino acid compositions.

The three major proteins of mammalian neurofilaments, of molecular weight 70,000, 160,000, and 210,000, have been resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and more recently, by ion-exchange chromatography in urea solution. We describe here a method to separate the neurofilament proteins by gel filtration without the use of SDS. A bulk preparation of cytoskeleton from rat spinal cord was first characterized. This preparation was then solubilized in a buffer containing 8 M urea and subjected to gel filtration. Individual neurofilament proteins, in milligram quantities, were harvested following the pooling of appropriate fractions. Gel electrophoresis showed a high degree of homogeneity in each of the three pooled fractions. Dye binding studies demonstrated that the protein of molecular weight 210,000 was relatively underrepresented when stained with Coomassie Blue, while all three neurofilament proteins showed similar dye binding properties with Fast Green. Amino acid analysis indicated that (1) all three neurofilament proteins contained a high content of acidic residues; (2) the molecular weight 210,000 protein contained greater than 8 mol% proline; and (3) no simple oligomeric relationship existed among the neurofilament triplets.

Amino Acids↗
PubMed2004

Microleakage and sealant penetration using a vibrating probe.

PURPOSE: To evaluate the effect of a vibrating probe on sealant penetration into pits and fissures. METHODS: Thirty-six extracted human third molars were used. The occlusal surface of each molar was cleaned with a rubber cup and pumice slurry. Scotchbond Etchant (35% phosphoric acid gel) was applied to the occlusal fissures of each molar for 20 seconds. Fissures were then sealed as follows: Group 1: Concise Light Cured White Sealant was applied without vibration; Group 2: same as in Group 1 but with vibration; Group 3: Single Bond Adhesive was applied, then Concise Light Cured White Sealant was applied without vibration; Group 4: same as in Group 3, but sealant was applied with vibration; Group 5: Concise Light Cured White Sealant was applied without vibration, then Z-100 composite was burnished over the sealant and light-cured; Group 6: same as in Group 5, but sealant was applied with vibration. Microvibe vibrating probe was used for sealant vibration. After 24-hour immersion in 2% basic fuchsin dye solution, specimens were sectioned, and dye and sealant penetration were scored using a stereomicroscope and color slides. Selected sectioned samples were observed under the SEM. RESULTS: No statistically significant differences between the groups were observed for either dye or sealant penetration scores. In all groups, sealant penetration was more influenced by the shape, depth and residual debris of fissures than by the use of a vibrating probe.

Acid Etching, Dental↗
PubMedDate not supplied

Quantitation of immobilized proteins.

A method for the quantitative determination of immobilized proteins based on the binding and subsequent elution of Coomassie Blue R is presented. Also presented is a method for the immobilization of proteins in solution by entrapment in polyacrylamide. These entrapped proteins are then available for use in the assay method presented. Other analytical procedures can also be performed on the entrapped proteins, either alone or in combination with the protein quantitation. The dye binding and elution method presented provides a sensitive and, in most applications, rapid method for the quantitative detection of immobilized proteins. Rather than immobilization being an obstacle to the assay method, this approach utilizes the advantages of immobilization for the removal of excess reagents. Application of this approach to several types of immobilized protein are presented.

Acrylic Resins↗
PubMedDate not supplied

Combination probe and dye-directed lymphatic mapping detects micrometastases in early colorectal cancer.

Nodal metastasis is the single most important prognostic factor in early colorectal cancer (CRC). Lymphatic mapping can identify sentinel nodes for focused histopathologic examination and thereby improve the nodal staging of CRC; however, the optimal technique for identifying sentinel nodes in CRC is unclear. We hypothesized that a combination of radiotracer and blue dye would more accurately identify tumor-positive sentinel nodes than blue dye alone. Lymphatic mapping was performed in 48 consecutive patients undergoing resection for CRC and in two original patients who underwent sentinel node mapping in 1996. Prior to resection, 1% vital blue dye and radiotracer were injected around the tumor in the subserosal layer. Nodes were designated as sentinel by blue coloration and/or radioactivity. Lymphatic mapping identified at least one sentinel node in 49 patients. Focused examination of multiple sentinel node sections by means of hematoxylin and eosin and immunohistochemical analysis showed that sentinel nodes accurately predicted the status of the nodal basin in 93.8% (46 of 49) of patients. Of the 19 patients with nodal metastases, 11 had macrometastases (>.2 mm), three had micrometastases (between 2 mm and 0.2 mm), and five had isolated tumor cells or clusters (<.2 mm) identified by immunohistochemical analysis only. Patients had significantly fewer blue/radioactive ("hot") nodes than blue-only nodes (1.38 vs. 2.48 per patient; P = 0.0001). It is important to note that nodal metastases were more common in blue/hot nodes than in blue-only nodes (27.3% [19 of 68] vs. 8.8% [11 of 124]; P = 0.005). Dual-agent lymphatic mapping more accurately identifies sentinel node metastases than blue dye alone. In addition, this technique allows a more focused histopathologic examination of these nodes, in conjunction with the revised American Joint Committee on Cancer guidelines, and thereby offers the potential for significant upstaging of CRC.

Aged↗
PubMed2004

A prospective analysis of the effect of blue-dye volume on sentinel lymph node mapping success and incidence of allergic reaction in patients with breast cancer.

BACKGROUND: This study examined whether the volume of isosulfan blue dye used in sentinel lymph node (SLN) mapping in breast cancer is related to the SLN identification rate or to the incidence of allergic reactions. METHODS: From January 2001 to November 2002, 1728 breast cancer patients underwent 1832 SLN mapping procedures with the combined technique of intraparenchymal blue dye and intradermal radioisotope. Details of each procedure and all allergic reactions were prospectively recorded. Bilateral synchronous SLN procedures were considered as one dye exposure but as two distinct procedures for determining mapping success. Dye-only success was defined as the proportion of cases in which the SLN was identified by blue dye alone. Overall dye success was defined as the proportion of cases in which the SLN was identified by blue dye with or without isotope. RESULTS: When stratified by volume of blue dye, there were no significant differences in dye-only successes, overall dye successes, or mapping failures. Allergic reactions were documented in 31 (1.8%) of 1728 patients. Hypotensive reactions occurred in 3 (.2%) of 1728 patients; 2 (.1%) required pressor support. There was a nonsignificant trend toward fewer allergic reactions with smaller volumes of blue dye. CONCLUSIONS: In combined-technique SLN mapping protocols for breast cancer, using smaller volumes of blue dye may represent a means of optimizing the safety of the procedure without compromising its success.

Adult↗
PubMedDate not supplied

[Sentinel lymph node identification in the staging of cutaneous melanoma. Blue dye vs. radioguided localization].

BACKGROUND: The purpose of this study is to emphasize the usefulness of combined intraoperative gamma-detecting-probe (C-Trak) and blue dye guided research of sentinel nodes (SN) in the treatment of cutaneous melanoma. METHODS: At the Department of General Surgery of Macerata Hospital, after informed consent, 22 consecutive patients (10 males and 12 females) with mean age 53 years (20-78 years) affected by histologically proved cutaneous malignant melanoma in stage I (TC, ultrasonography and bone scintigraphy) were studied by dynamic lymphoscintigraphy with 10.8-22.2 MBq of 99mTc albumin microcolloids 18-22 hours before surgery and by intradermal injection of blue dye at induction of anaesthesia. Intraoperative mapping technique to localize SN has been done by using a combination of a vital blue dye and a radioactive tracer. RESULTS: A total of 42 SN were identified. Micrometastases were found in 2 (9.1%) patients; 13 SN were well-coloured (31%), 23 SN were poorly-coloured (55%), and 6 SN were not coloured (14%). Overall localization with blue dye was 86%. All SN were radiolabeled, but identification with gamma detecting probe was possible only in 41 cases (95%). Combined techniques was effective in 100% of cases. CONCLUSIONS: Combined use of radiocolloids and blue dye is the gold standard for correct identification and biopsy of SN with 100% of favourable results. The technique is simple, fast and effective and permits to select the patients that need other surgical and oncologic procedures.

Adult↗
PubMed1982

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green.

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3- to 5-fold reduction in the fluorescence intensity of intracellular MG-PY. Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG-PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24, 48, and 72 hr of cell culture were monitored. The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: a) actinomycin D inhibited the increases in cellular RNA and DNA; b) hydroxyurea inhibited the increases in cellular RNA were only slightly reduced; c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.

Animals↗
PubMed2004

Isolation and characterization of microorganisms capable of decolorizing various triphenylmethane dyes.

Various soil and sludge samples collected from the vicinity of textile dyeing industries and waste disposal sites were used for enrichment of microbial population in the presence of triphenylmethane (TPM) dye Acid Violet-17 (AV-17). Twenty-five (25) isolates were screened for their ability to decolorize AV-17 dye added at a rate of 10 mgl(-1) in mineral salts medium (MSM) agar plates. Five bacterial isolates belonging to Bacillus sp., Alcaligenes sp. and Aeromonas sp. were selected on the basis of their higher decolorization ability and were used to develop a bacterial consortium. The consortium was able to efficiently decolorize various TPM dyes viz. Acid Violet-17 (86%), Acid Blue-15 (85%), Crystal Violet (82%), Malachite Green (82%) and Brilliant Green (85%). The consortium will be further used for designing efficient and cost effective treatment system for effluents of textile processing industries (TPI).

Aeromonas↗
PubMed1981

Cytofluorometry on cells isolated from paraffin sections after blocking of the background fluoroscence by azocarmine G.

A technique for isolation of cells from paraffin embedded tissue is indispensable for the performance of Feulgen-DNA cytofluorometry in parallel with the definition of histological characteristics. Background fluorescence due to nonspecific dye-binding by a "pseudo-plasmal reaction" is usually found to be so intense on cells isolated from formalin-fixed tissues, that we are often forced to abandon quantitative DNA determinations. In the present work, we report the fixation of tissues with Carnoy's fixative for 12 h at 5 degrees C not only reduces nonspecific dye-binding but also facilitates the process of cell isolation. Furthermore, we find that pre-treatment of cells isolated from Carnoy-fixed tissues with acidic azocarmin G solution completely blocks nonspecific dye-binding in subsequent acriflavine Feulgen nuclear staining. This combination of techniques for specimen preparation enables us to carry out Feulgen-DNA cytofluorometry on cells isolated from histological sections with satisfactorily low coefficients of variation (less than 8%). The techniques should be widely applicable for parallel DNA determinations and histology.

Animals↗