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PubMed2000

The presumptive reagent fluorescein for detection of dilute bloodstains and subsequent STR typing of recovered DNA.

A presumptive reagent for dilute blood detection other than luminol is fluorescein. The sensitivity of fluorescein approaches the sensitivity of detection levels of luminol. The fluorescein detection method offers the advantages of working in a lighted environment, and the reaction persists longer than luminol. A series of diluted bloodstains, ranging from neat to 1:1,000,000, was placed on a variety of substrates. Three sets were made per substrate. One set was exposed to fluorescein, one set was exposed to luminol, and one set served as an uncontaminated control. The fluorescein signal persisted longer than luminol. However, background staining for fluorescein was observed on some substrates within 30 s to 1 min, and no background staining was observed for luminol. Stains on non-absorbent surfaces were detectable at 1:100,000 dilutions, and stains on absorbent surfaces were detectable usually at no more than 1:100. The sensitivity of detection of fluorescein was comparable to that of luminol in this study. In all cases, where sufficient DNA was recovered, typeable results at all 13 core CODIS STR loci were obtained from treated bloodstains and controls. The results from STR typing indicate that there was no evidence of DNA degradation.

Blood Chemical Analysis↗
PubMed2001

A new technique for the use of intrathecal fluorescein in the repair of cerebrospinal fluid rhinorrhea using a hypodense diluent.

The intrathecal injection of fluorescein is used in the diagnosis and treatment of skull base liquoric fistulas since it allows precise localization of the site of drainage. The fluorescein is always diluted in cerebrospinal fluid (CSF) resulting in a hyperdense solution in relation to the CSF. For this reason it is necessary to put the patient in the Trendelenburg position for 30 to 40 minutes so that the fluorescein reaches the cerebral cisterns and is visualized at the level of the fistulae. From October 1997 to May 1999 intrathecal fluorescein in a hypodense solution was used in the repair of 23 skull base defects associated with CSF rhinorrhea. This hypodense solution was obtained by diluting 0.5 cm3 of 5% fluorescein in 10 cm3 of distilled water. This solution density is 1001, which is hypodense when compared to CSF (density range 1004-1006) and therefore allows fluorescein to reach rapidly the cerebral cisterns when the patient is recumbent. The author discusses the advantages and the lack of complications with the use of fluorescein in a hypodense solution.

Adolescent↗
PubMed1975

[Vital fluorescent staining of microorganisms by 3',6'-diacetyl-fluoresceine for determination of their metabolic activity (author's transl)].

By fluorescent microscopy and spectroscopy studies involving micro-organisms which were either viable or devitalised by heat sterilisation or gamma irradiation and could not be cultured any more, fluorochrome binding with 3' , 6'-diacetyl fluorescein was shown to be linked with the viability of a cell and a function of its actual metabolic state. The incorporation of diacetyl fluorescein into cells, its storage and hydrolysis to fluorescein mean active processes taking place at high speed. Viable cells are capable of storing fluorescein intracellularly, bound to structural elements. If the storage capacity is surpassed, it will be eliminated from the cell. The mechanism of this process is discussed. Devitalised cells are not capable of active uptake of fluorescein nor of its storage and accelerated hydrolysis. Beyond this, they are incapable of fluorescein binding to structural elements. There will be only a minor homogenous staining of such cells by fluorescein. An express method based upon the results is providing information on viability, actual metabolic state, morphology, and motility of micro-organisms within a few minutes and without affecting onward culturing.

Bacteria↗
PubMed1981

Pharmacokinetics of fluorescein in the vitreous.

The pharmacokinetics of fluorescein in the vitreous after intravenous or oral fluorescein administration was studied in normal individuals. Vitreous and free blood fluorescein concentrations were measured over an 8 hr period by fluorophotometry and fluorescence polarization. A computer model was constructed based on the free plasma fluorescein concentrations and conformed closely to the experimental data. The concentration profiles of fluorescein appearance in the vitreous were then examined. The effect of alterations in retinal permeability and free blood fluorescein concentration are also discussed. This article illustrates that an increase in the concentration of fluorescein in the vitreous as measured by fluorophotometry can be caused by factors other than an increase in blood-retinal barrier permeability.

Administration, Oral↗
PubMed1998

Spectral properties of fluorescein in solvent-water mixtures: applications as a probe of hydrogen bonding environments in biological systems.

Although fluorescein is a widely used fluorescent probe in the biosciences, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. This application is based on the observation, originally made by Martin (Chem. Phys. Lett. 35, 105-111, 1975), that the absorption maximum of fluorescein undergoes substantial shifts in organic solvents related to the hydrogen bonding power of the solvents. We have extended this work by studying the spectral properties of the dianion form of the probe in solvent-water mixtures. We show that the magnitude of the shift correlates with the alpha and beta parameters of Kamlet and Taft (J. Am. Chem. Soc. 98, 377-383; 2886-2894, 1976), which provide a scale of the hydrogen bond donor acidities and acceptor basicities, respectively, of the solvents. In solvent-water mixtures, these shifts reflect general effects of the solvents on the hydrogen bonding environment of the fluorescein through water-solvent hydrogen bonding and specific effects due to fluorescein-solvent hydrogen bonding. Indeed, both the absorption and fluorescence properties appear to be dominated by these effects indicating that the spectral shifts of the dianion can be used as an indicator of its hydrogen bonding environment. We discuss the application of fluorescein as a probe of hydrogen bonding in the microenvironment immediately surrounding the fluorophore, and we illustrate the effect with reference to the fluorescein-antifluorescein antibody complex where it appears that antibodies selected during the immune response possess binding sites that are increasingly dehydrated and hydrophobic.

Antibodies↗
PubMed1997

Phototoxic reactions caused by sodium fluorescein.

PURPOSE: To report three cases of phototoxic reactions to intravenous fluorescein for retinal angiography and to describe provocative testing in a volunteer. METHODS: Three patients with phototoxic reactions were interviewed, and one volunteer underwent a controlled challenge test by applying a potent sunscreen and exposing skin areas to direct sunlight before and after fluorescein administration. RESULTS: All patients experienced marked cutaneous erythema, edema, and pain to sun-exposed areas within 1 hour of exposure. The reaction faded during a variable period of time, and one case resulted in mild epidermal desquamation and prolonged discomfort. We noted minimal skin changes in the volunteer who was exposed to the sun before fluorescein administration; however, marked blanching erythema and pain were noted after fluorescein administration and sunlight. CONCLUSIONS: Consistent with its in vitro properties as a photodynamic dye, fluorescein may rarely act as a phototoxic agent in humans at doses employed for fluorescein angiography.

Adult↗
PubMed1994

Foveal vision function before and after fluorescein angiography.

PURPOSE: Fluorescein angiography is routinely used in ophthalmologic practice with minimal changes in vision reported by the patient after the procedure. Recent animal work has suggested that fluorescein may be cytotoxic in the presence of light and may cause retinal damage. The authors examined whether any changes occur in foveal vision function after fluorescein angiography. METHODS: A battery of tests of vision function--including visual acuity, contrast sensitivity, color vision, and two-color increment thresholds designed to isolate individual cone pathways--was administered before and 48 hours after fluorescein angiography to a group of 10 patients with good visual acuity. RESULTS: Most of the patients showed significant abnormalities with the nonstandard tests; however, no significant changes in central vision function were found 48 hours after fluorescein angiography. CONCLUSION: Even sensitive vision tests show no measurable effect of fluorescein angiography on foveal vision function in eyes with significant disease.

Adult↗
PubMed2001

The use of prothrombin(S525C) labeled with fluorescein to directly study the inhibition of prothrombinase by antithrombin during prothrombin activation.

Serine 525 of human prothrombin was mutated to cysteine and covalently labeled with fluorescein to make II(S525C)-fluorescein. Kinetics of cleavage of this derivative by prothrombinase are identical to those of wild-type prothrombin. Cleavage is coincident with a 50% increase in fluorescence intensity and the product is catalytically inactive. Thus, it allows convenient monitoring of prothrombin activation without generating active thrombin. The kinetics of inhibition of factor Xa (FXa) by antithrombin (AT) and AT-heparin were measured by monitoring activation of II(S525C)-fluorescein and the hydrolysis of the chromogenic substrate S2222 in the presence of AT. With S2222 as the substrate the rate constant for inhibition of FXa, Ca(2+), and unilamellar vesicles of phosphatidylcholine and phosphatidylserine (75:25) (PCPS) vesicles by AT was 3.51 x 10(3) m(-1) s(-1); when factor Va (FVa) was included the rate constant was 1.55 x 10(3) m(-1) s(-1). In the absence of FVa, II(S525C)-fluorescein had no effect on inhibition. When II(S525C)-fluorescein was the substrate, however, FVa at saturating concentrations profoundly protected FXa from inhibition by AT, increasing the half-life from 3 min with FXa, Ca(2+), PCPS, and II(S525C)-fluorescein, to greater than 69 min when FVa was included. Thus, both FVa and prothrombin are necessary for this level of protection. In the absence of prothrombin, FVa decreased the second order rate constant for inhibition by the AT-heparin complex from 1.58 x 10(7) m(-1) s(-1), for FXa, Ca(2+), and PCPS, to 7.72 x 10(6) m(-1) s(-1). II(S525C)-fluorescein and factor Va together reduced the rate constant to less than 1% of that for FXa, Ca(2+), and PCPS. At a heparin concentration of 0.2 unit/ml, this corresponds to a half-life increase from 1 s to 136 s.

Antithrombins↗
PubMed1997

[Experimental study of detection of brain tumor at surgery using fluorescent imaging under a surgical microscope after fluorescein administration].

Total resection is the optimal treatment for malignant gliomas. However, we sometimes find an unexpected residual tumor mass on magnetic resonance imaging performed after an operation because of a macroscopically unclear margin of the tumor during operation. This study was designed to evaluate the effect of fluorescein sodium on imaging of glioma in combination with a surgical microscope for detection of the tumor at surgery in a rat glioma model. For this study, we produced two filters for the excitation and emission of fluorescein that can be easily fitted to and removed from a surgical microscope manually during the operation. For the in vivo study, Wistar rat brains bearing C6 glioma were removed at appropriate intervals after the intravenous administration of 10-20 mg/kg body weight of fluorescein sodium, and their surface and coronal sections through the tumor were observed using a surgical microscope with the filters. For the in vitro study, the cultured C6 glioma cells were exposed to 5 micrograms/ml of fluorescein sodium for 2 hours and then observed by confocal laser scanning microscopy. In the in vivo study, the C6 glioma itself and the brain within 2-3 mm of the gross surface of the tumor (probably indicating part of the perifocal edema) were well stained a brilliant yellowish green for a few hours. The normal brain was not stained. The intensity of the fluorescence was dose-dependent and was stronger under the xenon than under the halogen lamp. The in vitro study demonstrated almost no uptake of fluorescein by the C6 glioma cells. This fact indicates that the fluorescein in the stained tumor exists in extracellular spaces due to the disruption of the blood-brain barrier. These results suggest that this staining technique using fluorescein sodium and a surgical microscope with special filters during the operation may be the intravenous administration of 10 useful for detection of gliomas and warrants evaluation of clinical application.

Animals↗
PubMed1992

Fluorescein and acidosis. Implications for flap perfusion studies.

The visual fluorescein technique underpredicts survival of a skin flap during the first 24 hours after raising the flap. This problem limits its use as a research and clinical tool. There is no compelling explanation for this observation, but two facts are known: fluorescein is a derivative of phthalein, a pH indicator, and ischemic tissues become acidotic, with the pH falling below 7.0. These observations lead to the hypothesis that acidosis quenches fluorescence in distal skin flaps. No data could be found regarding this effect. Therefore, the effect of acidosis on sodium fluorescein was studied in vitro. A spectrophotometer was used to measure the intensity of the fluorescence of sodium fluorescein in buffered solutions of different hydrogen ion concentrations. Two studies were performed at different concentrations of fluorescein. At a concentration of 10(-5) g/mL, there is a drop of 26% in fluorescence intensity from pH 7.5 to 7.0, and 51% between pH 7.5 and 6.5. At 10(-7) g/mL, there is a 43% decrease in fluorescence between pH 7.5 and 6.5. This study supports our hypothesis that acidosis quenches the fluorescence of fluorescein. This effect must be considered when interpretating basic studies of skin flap microcirculation.

Acidosis↗
PubMed1985

Calculation of the permeability of the blood-retinal barrier to fluorescein.

A method is presented, for calculation of the permeability of the blood-retinal barrier to fluorescein which is based upon simultaneous determination of the free fluorescein concentration in plasma and the fluorescein concentration profile in the vitreous body. By aid of a simplified mathematical model of the eye the blood-retinal barrier permeability is calculated automatically on a computer from corresponding values of the fluorescein concentration in plasma and in the vitreous body. The present method eliminates some of the factors of uncertainty, which have been present in earlier applied fluorophotometric methods, thus contributing to increasing the exactness of the fluorophotometric method for the estimation of the permeability of the blood-retinal barrier to fluorescein. Apart from the permeability of the barrier, the diffusion coefficient for fluorescein in the vitreous body is also estimated by the present method.

Capillary Permeability↗
PubMed1989

Detection of low and high affinity binding sites with fluoresceinated human recombinant interleukin-2.

In this study, we describe a new methodology to detect and quantify lymphokine receptors, using interleukin-2 as a prototype. Human recombinant interleukin-2 (IL-2) was conjugated to fluorescein isothiocyanate. Binding of fluoresceinated IL-2 to different cell types was assessed by flow cytometry analysis, on a FACS 440 calibrated using fluoresceinated Sephadex G-25 beads. This calibration procedure allowed us to quantify the actual number of binding sites for IL-2. Fluoresceinated IL-2 did not bind to normal resting T cells, whereas a highly significant binding was observed on PHA-activated human T cells. The binding was inhibited by an excess of unlabeled IL-2 and by an excess of anti-IL-2 receptor p55 antibodies (anti-TAC). Dose curves of IL-2 showed a two plateau saturation, the first plateau corresponding to the saturation of high affinity binding sites, as assessed by correlation with the biological activity on IL-2-dependent T cells. Among the cell types tested, fluoresceinated IL-2 bound to IL-2-dependent mouse T cells (the binding in that case was not inhibited by anti-IL-2 receptor p55 antibodies), and to different p70 expressing cell lines or normal cells (MLA 144, normal large granular lymphocytes). Taken together, these results indicate that fluoresceinated IL-2 can be used to detect high as well as low affinity IL-2 binding sites.

Animals↗
PubMedDate not supplied

Fluorescein. Physiochemical factors affecting its fluorescence.

Fluorescein's property of fluorescence is reviewed. Of the many factors which affect its fluorescence, concentration is probably the most important and it best explains why leaking aqueous turns fluorescein bright green during Seidel's test. The intensity and pattern of fluorescein staining of corneal lesions is probably due to the concentration and distribution of fluorescein in the cornea. The concentration of fluorescein achieved in the retinal blood vessels during fluorescein angiography affects its fluorescence.

Corneal Diseases↗
PubMed1998

Simultaneous confocal scanning laser fluorescein and indocyanine green angiography.

PURPOSE: To evaluate clinically a recently developed confocal scanning laser ophthalmoscope for simultaneous fluorescein and indocyanine green angiography. METHODS: After comprehensive ophthalmic examination, including stereoscopic biomicroscopy, and after color fundus photographs, simultaneous confocal scanning laser fluorescein and indocyanine green angiography was performed after injection of 500 mg of fluorescein and 25 mg of indocyanine green mixed in one syringe using the Heidelberg Retina Angiograph. An argon laser beam (488 nm) and a diode laser beam (795 nm) from an external source were delivered by single-mode fibers. Emission was recorded between 500 and 700 nm and above 810 nm, respectively. Digital images were displayed simultaneously on a monitor during angiography. RESULTS: Two hundred twenty simultaneous fluorescein and indocyanine green angiograms were obtained in 193 consecutive patients with various diagnoses, including exudative age-related macular degeneration with occult and classic choroidal neovascularization. Simultaneous angiography with both dyes gave high-contrast images during all phases of the angiography, which allowed for accurate correlation of fluorescein and indocyanine green angiographic findings and correct comparison of the transit of both dyes through the retinal and choroidal circulation. Corresponding display of quasisimultaneous frames facilitated interpretation of the angiograms. CONCLUSIONS: Confocal laser scanning angiography allows for quasisimultaneous fluorescein and indocyanine green angiography. The investigation is possible at low retinal irradiance and offers high-contrast digital images. Compared with consecutive angiographic investigations using both dyes, simultaneous angiography is less time consuming, requires only one injection, and, because quasisimultaneous frames are obtained, facilitates interpretation of the angiograms.

Adult↗
PubMed1995

Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy.

An investigation on the photobleaching behavior of fluorescein in microscopy was carried out through a systematic analysis of photobleaching mechanisms. The individual photochemical reactions of fluorescein were incorporated into a theoretical analysis and mathematical simulation to study the photochemical processes leading to photobleaching of fluorescein in microscopy. The photobleaching behavior of free and bound fluorescein has also been investigated by experimental means. Both the theoretical simulation and experimental data show that photobleaching of fluorescein in microscopy is, in general, not a single-exponential process. The simulation suggests that the non-single-exponential behavior is caused by the oxygen-independent, proximity-induced triplet-triplet or triplet-ground state dye reactions of bound fluorescein in microscopy. The single-exponential process is a special case of photobleaching behavior when the reactions between the triplet dye and molecular oxygen are dominant.

Fluorescein↗
PubMed1995

Fluorescein angiography of the bladder: technique and relevance to bladder cancer and interstitial cystitis patients.

PURPOSE: Fluorescein angiography has been used in the study of bleeding vessels, neovascularity, tumors and ischemic tissues in a variety of disorders. This pilot study was designed to evaluate the feasibility, safety and relevance of this interesting technology for the evaluation of bladder wall vessels in patients with interstitial cystitis and bladder cancer. MATERIALS AND METHODS: Five patients with National Institutes of Health defined interstitial cystitis symptoms and 10 with bladder cancer were studied during cytoscopy while they were under general anesthesia. A yellow-green barrier filter (520 nm.) was placed over the cystoscope eyepiece and a blue exciter filter (465 nm.) was attached to the light source. Patients received a 5 ml. bolus of 10% fluorescein intravenously. RESULTS: After hydrodistension, glomerulations in interstitial cystitis patients were more prominent with fluorescein angiography and occurred in the venule phase. Areas of papillary transitional cell tumor and carcinoma in situ developed a brilliant yellow-green fluorescence. Adjacent normal urothelium was nonfluorescent and provided a contrasting dark background facilitating the detection of all lesions. No allergic reaction or other adverse effect related to the fluorescein injection was observed. CONCLUSIONS: These unique observations in a limited number of patients suggest that fluorescein angiography of the bladder is a safe and simple procedure. This preliminary report underscores the relevance of fluorescein angiography in the detection of bladder tumor and offers a new approach to the evaluation of bladder wall vessels in interstitial cystitis patients.

Arterioles↗
PubMed1992

Correlation between biochemical composition and fluorescein binding of deposits in Bruch's membrane.

PURPOSE: The fluorescence of drusen during fluorescein angiography is believed to have important prognostic and pathogenetic implications in age-related maculopathy. It is believed that deposits containing predominantly neutral lipids would be hydrophobic, resulting in hypofluorescence on fluorescein angiography, while the presence of polar phospholipids would be indicated clinically by hyperfluorescence because of its hydrophilic properties. To identify the potential determinants of fluorescence of drusen and Bruch's membrane, a series of macular specimens from human donors older than 60 years of age was examined. No clinical information was available concerning any previous eye disease. METHODS: In vitro fluorescein binding was recorded microscopically, and the presence of fibronectin was sought by immunohistochemistry. The results were correlated with the proportions of phospholipids to neutral lipids identified by histochemical and biochemical studies. RESULTS: It was found that high content of neutral fats was associated with lack of both fluorescein binding and fibronectin, and, conversely, in those specimens with high proportions of phospholipids, fluorescein binding was strong and fibronectin was present. CONCLUSIONS: These observations support the central hypothesis concerning biophysical changes in Bruch's membrane with age and the potential importance of fluorescein angiography in the characterization of Bruch's membrane deposits.

Aged↗
PubMed1994

Absorption of fluorescein given under the upper lid.

BACKGROUND: The need for a more efficacious approach to administer topical ocular medications prompted the authors to consider applying conventional eye drops under the upper lid rather than beneath the lower lid. Preliminary observations on patients with glaucoma using a beta-blocker beneath the upper lid suggested a drop in intraocular pressure into the normal range in some previously refractory patients being treated with the same medications. To test this clinical observation, the authors observed if there were any physiologic differences in topical fluorescein absorption into the anterior chamber when given beneath the upper lid versus the lower lid. METHODS: A 5-microliters drop of fluorescein solution was placed under the upper-lid fornix of one eye and under the lower-lid fornix of the other eye in human volunteers, and absorption into the anterior chamber was measured at hourly intervals, for a total of 3 hours. RESULTS: Hotelling T2 multivariate analysis for all 3 hours demonstrates that upper-lid administration of fluorescein results in significantly higher absorption of fluorescein into the anterior chamber than does lower-lid administration (P = 0.0088; for hours 2 and 3, the statistical differences is even more dramatic: P < 0.0044). CONCLUSION: Using conventional eye drops beneath the upper lid, the authors observed increased absorption of fluorescein into the anterior chamber when compared with lower-lid administration. Profuse tearing, especially by younger subjects, significantly and rapidly diminished anterior chamber absorption of fluorescein. It is reasonable to consider further clinical studies to test this new approach to drug delivery.

Absorption↗