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PubMed2001

Histological techniques and microscopic analysis of biological agents for preservation of human bone remains.

Microscopy and histology techniques can be applied to morphological study of fungi and bacteria contaminating ancient human osteological remains. Undecalcified samples are cut with a diamond rotary blade microtome and an original technique was applied using adhesive tape to prevent damage to the bone surface during sectioning. We used light microscopy, polarized light microscopy, and epi-illumination fluorescence systems. Common dyes can be applied to 80 microm sections using classic staining techniques to reveal the architecture of bone and the presence of infecting biological agents.

Bone and Bones↗
PubMed2003

Technical limitations of sentinel node biopsy in breast cancer: a single surgeon's experience.

Few studies have attempted to critically identify patient- and tumor-related factors that limit sentinel node biopsy (SNB). These studies have been limited by sample size and surgeon variability. The present study attempts to enumerate these limitations in a unique group of patients. One hundred twenty-five SNBs performed by a single surgeon between May 1997 and June 2001 were reviewed. Overall SNB was successful in 96 per cent of patients with a 97 per cent correlation with the axillary node dissection. Sentinel node identification was not affected by age, tumor size, tumor location, prior segmental resection, or neoadjuvant therapy. No false negatives were noted in the neoadjuvant group. The use of blue dye alone significantly understaged patients when compared with isotope alone (P = 0.02). SNB is a highly accurate method to identify axillary metastases and its limitations are not affected by patient or tumor related factors. In the present study SNB detection by both isotope and blue dye has been shown to be superior to blue dye alone. This finding demonstrates that these limitations may be overcome with the standardization of the technique used.

Age Factors↗
PubMed1992

Methyl green and its analogues bind selectively to AT-rich regions of native DNA.

Methyl green has long been used as a DNA stain in histochemistry. The sequence selective binding of the cationic triphenylmethane dyes methyl green, crystal violet and Malachite green to DNA was investigated by DNAase 1 and micrococcal nuclease footprinting. At low concentrations the ligands showed similar footprinting patterns which centred around AT-rich regions with a mild preference for hompolymeric A and T. At higher concentrations the dyes bound to almost all available DNA sites. Models, with and without intercalation are discussed to account for the specific binding.

Base Sequence↗
PubMed1989

Refinement of the Coomassie brilliant blue G assay for quantitative protein determination.

Minor modifications of the Bradford method [1976) Anal. Biochem. 72, 248-254) increase the sensitivity of the Coomassie brilliant blue G microassay for protein quantitation. This is reached by selecting the proper dye source, by alterations in the dye, ethanol, and phosphoric acid concentrations, and by including Triton X-100 in the reaction mixture. The modified assay conditions allow proteins to be detected above 0.2 micrograms and from a solution above 0.4 micrograms/ml. The extinction coefficient of the dye-protein complex was 0.144 +/- 0.010 for 1 microgram bovine serum albumin in the final reaction mixture of 1 ml. In general, the modified procedure exhibits the same specificity, advantages, and drawbacks as described for the original Bradford assay.

Octoxynol↗
PubMed2000

Dye removal, catalytic activity and 2D crystallization of chloroplast H(+)-ATP synthase purified by blue native electrophoresis.

The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F(1)) and its subcomplexes CF(O) and CF(1) were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization.

Catalysis↗
PubMed2000

Standardized probe-directed sentinel node dissection in melanoma.

BACKGROUND: Radiopharmaceutical agents appear to improve the accuracy of sentinel node (SN) identification in patients with early-stage melanoma, but the optimal radiopharmaceutical agent and its timing from injection to surgery remain controversial. We undertook this investigation to examine the utility of 3 methods of intraoperative lymphatic mapping with radiopharmaceutical-directed sentinel lymphadenectomy (LM/SL). We suspected that concurrent injection of radiopharmaceutical and blue-dye would lead to the greatest success of SN identification. METHODS: The study was composed of 247 consecutive patients who had American Joint Committee on Cancer stage I or II melanoma. Before LM/SL, all patients underwent cutaneous lymphoscintigraphy by 1 of 3 techniques: technetium 99m (Tc 99m) human serum albumin (HSA) injected at least 24 hours before LM/SL (124 patients), Tc 99m filtered sulfur colloid (SC) injected no more than 4 hours before LM/SL (same-day SC, 95 patients), or Tc 99m SC injected at least 18 hours before LM/SL (prior-day SC, 28 patients). At the time of LM/SL, isosulfan blue dye was injected alone (SC groups) or with a second dose of HSA (HSA group). A hand-held gamma probe was used to determine the radioactive (hot) counts of blue-stained and nonstained nodes, and the in vivo and ex vivo node-to-background count ratios of the nodes were compared. RESULTS: Preoperative LS identified 299 drainage basins; LM/SL identified at least 1 SN in 119 (98%) of 121 basins using same-day SC, 142 (97%) of 146 basins using HSA, and 32 (100%) of 32 basins using prior-day SC. There was no difference (P = .62) in the accuracy rate between the 3 techniques. The total number of SNs was 463. Same-day SC yielded higher intraoperative node-to-background count ratios than did either of the other techniques (P < .0001). Same-day SC also had the greatest relative change in radioactivity between the blue sentinel node and the post-excision basin (P < .0001), and the highest rate of SNs that were both blue and hot (in vivo or ex vivo ratio > or = 2, P = .05). CONCLUSIONS: LS and LM/SL performed on the same day with a single injection of filtered Tc 99m SC serves as the most useful method for probe-directed LM/SL. This technique demonstrated the highest in vivo and ex vivo count ratios, fall-off of radioactivity between the excised nodes and post-excision basin, and concordance between blue dye and hot nodes. It should be recommended as the method of choice for probe-directed LM/SL.

Humans↗
PubMed2006

Lymphography prior to laparoscopic Palomo varicocelectomy to prevent postoperative hydrocele.

BACKGROUND: We report our experience with preoperative lymphography to identify and perioperatively preserve the ligature of the lymphatic vessels to reduce the incidence of postoperative testicular hydrocele in patients undergoing laparoscopic Palomo varicocelectomy for adolescent varicocele. MATERIALS AND METHODS: Twenty-seven consecutive patients with varicocele had preoperative lymphography. The mean age was 13.5 years (range, 8-18 years) and the mean grade of varicocele was III. We performed lymphography with intrascrotal isosulfan blue. The laparoscopic Palomo procedure was successfully carried out in all patients. RESULTS: In 17 patients (63%) we were able to identify and conserve the lymphatic vessels by lymphography. Mean follow-up was 9.5 months (range, 6-24 months). None of the 27 patients had a recurrence. None of the 17 patients with positive lymphography had a testicular hydrocele. One of the 10 remaining patients developed a sizable hydrocele. CONCLUSION: Preoperative lymphography prior to laparoscopic Palomo varicocelectomy is a simple and feasible method for preventing testicular hydrocele. However, the method should be standardized to identify the exact site, the correct level of injection of blue dye, and to determine the optimal time to perform lymphography prior to the procedure.

Adolescent↗
PubMed1985

Effect of estrogen on the affinity of malachite green for staining cardiac lipid inclusions in mice.

The effect of estradiol-17-beta on lipids of the ventricular myocardium of mice has been studied with a cytochemical technique in which malachite green was added to glutaraldehyde. This malachite green-glutaraldehyde fixative enhances the visualization of certain phospholipid-related elements. Estrogen induces an affinity of ventricular cardiac lipid inclusions for the cationic dye malachite green. The staining affinity is evidenced only in the estrous female, not in diestrus. In oophorectomized animals, malachite green staining is seen only following estradiol injection, but this effect is blocked by progesterone. In the male, ventricular lipids do not stain, nor do they develop malachite green affinity with estrogen stimulation. These results imply a blockade of the estradiol-mediated dye affinity by progesterone and testosterone. This reinforces the concept of the heart as a target organ for sex steroids and expands the previously described estrogen effects on myocardium.

Animals↗
PubMed2004

Sentinel node evaluation in gynecologic cancer.

The sentinel node evaluation has revolutionized the modern surgical management of cutaneous melanoma and breast cancer. In gynecologic oncology, sentinel node mapping has been mainly studied in vulvar and cervical cancer. In vulvar cancer, data from 12 studies including 353 cases indicate that the sentinel node detection rate is 92% and the negative-predictive value is 99%. Three groin recurrences have been documented so far (< 1%). The technique has more recently been studied in cervical cancer. Data from 12 studies including 323 cases indicate a lower sentinel node detection rate of 80% to 86% and a negative-predictive value of 99%. Three false-negative cases have been reported so far (< 1%). Review of the literature suggests that the combined approach with blue dye and lymphoscintigraphy is superior to the blue dye alone for sentinel node detection. It also suggests that the sentinel node mapping technique is feasible in vulvar and cervical cancer and that it may become a valuable alternative to the traditional groin and pelvic lymphadenectomy. However, results have not been duplicated in large multi-institutional trials, and the technique should still be performed in the context of clinical trials. Complications of the sentinel node mapping technique are rare and usually benign but physicians should be aware of the serious risk of anaphylactic reaction to the blue dye (1% to 2%). Before this technique becomes a standard approach in the management of gynecologic malignancies, more data will be needed to clarify some of the related controversies.

Female↗
PubMed1990

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.

Cell Nucleus↗
PubMed2005

Randomized clinical trial comparing blue dye with combined dye and isotope for sentinel lymph node biopsy in breast cancer.

BACKGROUND: Use of blue dye alone as a marker for sentinel lymph node (SLN) biopsy is effective, but combining it with isotope marking can improve the success rate. Use of the isotope adds extra cost and there are potential radiation hazards. The two techniques were compared in a randomized trial. METHODS: Women with early breast cancer (less than 3 cm) and no palpable axillary nodes were recruited. Women older than 70 years with multicentric cancers or previous surgery to the breast or axilla were excluded. Patients were randomized to either blue dye alone or combined mapping for SLN biopsy. All women had a level I and II axillary dissection after the SLN biopsy. RESULTS: A total of 123 patients were recruited, of whom five were excluded from analysis. Blue dye alone was used in 57 women and 61 had combined mapping. Baseline demographic data were similar in the two cohorts. The success rate of SLN biopsy was higher with combined mapping than with blue dye alone (100 versus 86 per cent; P = 0.002). The accuracy and false-negative rate were similar (accuracy 100 per cent for combined mapping versus 98 per cent for blue dye; false-negative rate 0 versus 5 per cent). CONCLUSION: Combined mapping was superior to blue dye alone in identification of the SLN, but accuracy and false-negative rates were similar.

Breast Neoplasms↗
PubMed1981

"In situ"--measurements of protein contents in the brush border region along rat jejunal villi and their correlations with four enzyme activities.

In order to quantify the amount of protein in the small intestinal brush border region at different villus sites, cryostat sections of adult rat jejunum were stained with Naphthol Yellow S, Dinitrofluorobenzene and Coomassie Brilliant Blue and the dye deposits were evaluated cytophotometrically. Judged by the absorbance spectra in the tissue sections and the increase in absorbance as a function of the optical pathway (section thickness), Naphthol Yellow S proved to be the most suitable quantitative protein stain. By continuously measuring the absorbance of this dye at lambda 440 nm rectangular to the villus in the longitudinal axis of the enterocytes, a peak was registered in the brush border region which clearly could be differentiated from the apical cytoplasm. The amount of protein in the brush border region was determined at six different positions equally distributed along the villus. In parallel four brush border enzymes (neutral alpha and beta-glucosidase, unspecific alkaline phosphatase and dipeptidylpeptidase IV) were quantified by the same measuring technique in the Vmax-range of their substrate hydrolysis at equivalent villus positions. Their activities were correlated to the amount of protein. The absorbance data both for protein and for enzyme activities were significantly influenced by the villus position. They revealed an increasing gradient from the basal to the apical villus. In an additional analysis of the breadth of the dye deposits at the different measuring positions on the villus, it was shown that this parameter also ran parallel with the absorbance values.

Alkaline Phosphatase↗
PubMed1989

Nonlinearity in protein assays by the Coomassie blue dye-binding method.

The Coomassie brilliant blue (CBB) method for protein determination takes advantage of the fact that a low-pH red form of CBB reverts to a blue form when CBB binds to protein. The increase in absorbance at 595 nm of a protein-dye mixture compared to a blank containing only the dye reagent has been used to estimate the total amount of protein present in the sample mixture. A disadvantage of this method of protein determination is that the assay plot of absorbance at 595 nm versus total protein is not linear.

Hydrogen-Ion Concentration↗
PubMed2000

The role of sentinel lymph nodes in malignant melanoma.

Selective sentinel lymph node dissection should be considered a standard approach in the treatment of primary malignant melanoma. With the combination of blue dye and radioisotope mapping, the sentinel lymph nodes (SLNs) can be harvested with pinpoint accuracy. This article compares blue dye and radioisotope mapping techniques. Based on the clinical outcome data of selective sentinel lymph node dissection, micrometastasis to the SLNs carries a poor prognosis for patients with primary invasive melanoma.

Coloring Agents↗
PubMed2001

The effects of postinjection massage on the sensitivity of lymphatic mapping in breast cancer.

BACKGROUND: The technique of lymphatic mapping and sentinel lymph node (SLN) biopsy is rapidly becoming the preferred method of staging the axilla of the breast cancer patient. This report describes the impact of postinjection massage on the sensitivity of this surgical technique. STUDY DESIGN: Lymphatic mapping at the H Lee Moffitt Cancer Center is performed using a combination of isosulfan blue dye and Tc99m labeled sulfur colloid. Data describing the rate of SLN identification and the node characteristics from 594 consecutive patients were calculated. Patients who received a 5-minute massage after injection of blue dye and radiocolloid were compared with a control group in which the patients did not receive a postinjection massage. RESULTS: When compared with controls, the proportion of patients who had their SLN identified using blue dye after massage increased from 73.0% to 88.3%, and the proportion of patients who had their SLN identified using radiocolloid after massage increased from 81.7% to 91.3%. The overall rate of SLN identification increased from 93.5% to 97.8%. The proportion of nodes that were stained blue among those removed increased from 73.4% to 79.7% after massage. CONCLUSIONS: As experience increases with this new procedure, the surgical technique of lymphatic mapping continues to evolve. The addition of a postinjection massage significantly improves the uptake of blue dye by SLNs and may also aid in the accumulation of radioactivity in the SLNs, further increasing the sensitivity of this procedure.

Breast Neoplasms↗
PubMedDate not supplied

Blue-dye sentinel node mapping in thyroid carcinoma: debatable results of feasibility.

The present study aims to investigate the feasibility and influence of the lymphatic mapping and sentinel node biopsy on determination of the nodal status in thyroid carcinoma using blue-dye method. Nine consecutive patients with cytological diagnosis of papillary carcinoma were included in this study. To detect the sentinel lymphnode, intra- or perinodular injection of an average quantity of 0.5 ml (range : 0.1-1.2) of Ble Patenté V was performed intraoperatively in 8 cases only, as in one case a solitary cystic nodule occupied the entire lobe and thus any injection was impossible. After an average time of 16 minutes (range : 5-25) before dissection of the thyroid , no lymphnodes and no lymphatic afferent thereto visibly coloured were evidenced, except for spread of the vital dye into adjacent tissue and disrupted blood and lymphatic vessels at the injection site. Our results evidence that : intranodular injection, does not allow proper diffusion of the dye in the adjacent parenchyma, and in nodules smaller than 1 cm it may be difficult ; and that it is hazardous in cystic nodule because of the rupture risk; perinodular injection, at the four cardinal points, is impossible when the nodule occupies the entire lobe or the isthmus; multinodular goiter complicates the identification by palpation of the neoplastic nodule in which the dye should be injected or, if perinodular injection is given, to detect the parenchyma surrounding the nodule.

Adult↗
PubMed2002

Isosulfan blue dye reactions during sentinel lymph node mapping for breast cancer.

UNLABELLED: In the United States, identification of the sentinel lymph node (SLN) requires the use of (99m)Tc-labeled colloid, 1% isosulfan blue dye, or both to trace the lymphatic drainage of a given neoplasm. We report our experience with adverse reactions to isosulfan blue dye during SLN mapping in breast cancer. A chart review of the breast cancer SLN database was performed; it included 2392 sequential patients who underwent SLN biopsy involving isosulfan blue dye at Memorial Sloan-Kettering Cancer Center from September 12, 1996, to August 17, 2000. Thirty-nine of 2392 patients (1.6%) had a documented allergic reaction during the mapping procedure. Most reactions (69%) produced urticaria, blue hives, a generalized rash, or pruritus. The incidence of hypotensive reactions was 0.5%. Although anaphylaxis after the injection of isosulfan blue dye is rare, this article highlights the need to suspect anaphylaxis when hemodynamic instability occurs after the injection of this compound. Our experience indicates that bronchospasm and respiratory compromise are unusual and that most patients do not require emergent intubation and can be managed with short-term pressor support. In addition, our data indicate that patients with a sulfa allergy do not display a cross-sensitivity to isosulfan blue dye. IMPLICATIONS: We report the largest single-institution review of adverse reactions to injection of isosulfan blue dye during sentinel lymph node mapping in breast cancer. Bronchospasm and respiratory compromise are unusual, and most patients can be treated with short-term pressor support. Patients with a sulfa allergy do not display a cross-sensitivity to isosulfan blue dye.

Anaphylaxis↗
PubMed1975

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.

Bacteria↗