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PubMed1961

Studies on fluorescent antibody staining. I. Non-specific fluorescence with fluorescein-coupled sheep anti-rabbit globulins.

1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules.

Animals↗

PubMed1991

Effect of intracanalicular collagen implants on the absorption of topically applied sodium fluorescein.

Absorbable intracanalicular collagen implants were placed in both canaliculi of one eye of nine human volunteers. The other eye served as a control. Twenty-four hours later 2% sodium fluorescein was placed into both conjunctival sacs. Serial corneal fluorescein concentrations were measured with a scanning ocular fluorophotometer from 2 to 90 min after fluorescein administration. The mean corneal fluorescein concentration averaged over all time points was greater in 7 of 9 eyes with collagen implants compared to unimplanted controls. When the data from each subject were analyzed collectively, the mean corneal fluorescein concentration in the implanted eyes (1,218 micrograms/ml +/- SEM 83) was significantly greater (p less than 0.001) than the mean concentration in the control eyes (823 micrograms/ml +/- SEM 83). The use of absorbable intracanalicular collagen implants may increase the bioavailability of topically applied ocular solutions.

Absorption↗

PubMed1992

Corneal staining characteristics after sequential instillations of fluorescein.

Twenty-one healthy noncontact lens-wearing subjects were screened for the presence of corneal staining with fluorescein after sequential instillations of the vital stain (Phase I). These staining characteristics were further explored by monitoring the results of sequential instillations of fluorescein at 2-h intervals over a 12-h period (Phase II) and by patching 1 eye overnight and observing corneal staining after sequential instillations of fluorescein in the morning after patch removal (Phase III). In Phase II more eyes stained in the early morning. Throughout the day the most common staining areas were inferior and nasal. In Phase III, 9 of 12 patched eyes presented with fluorescein staining, as did 7 of the 12 unpatched eyes. Corneal staining after sequential instillations of fluorescein is more likely to be observed early in the day and is not reduced by overnight patching.

Adolescent↗

PubMed2006

Validity of fluorexon disodium versus sodium fluorescein for use in Goldmann tonometry.

PURPOSE: To evaluate the safety, validity, and comfort of 0.35% fluorexon disodium and 0.4% benoxinate (Flura-Safe) compared with the gold standard of 0.25% sodium fluorescein and 0.4% benoxinate for Goldmann applanation tonometry (GAT). METHODS: This was a double-masked, randomized, crossover clinical trial. Subjects received either the standard or study formulation for GAT on visit 1 and the other formulation 1 week later. At each visit, tonometer mire quality, adequacy of fluorescence, ease of intraocular pressure (IOP) measurements, the IOP value, and anesthetizing efficacy of the formulation were assessed. Subjects graded general comfort, soreness and irritation, and burning and stinging of each formulation at 1 and 5 minutes after drop instillation. RESULTS: Sixty-seven subjects completed the study. The mean IOP was 13.9 +/- 2.7 with fluorexon and 13.9 +/- 2.8 mm Hg with fluorescein OD and 14.0 +/- 2.8 with fluorexon and 13.9 +/- 2.5 mm Hg with fluorescein OS. The measurements with the 2 formulations were highly correlated for OD and OS, and the differences between the 2 measurements were not clinically significant. There was also no significant difference between the 2 drops in mire clarity, adequacy of fluorescence, or corneal anesthesia. However, fluorexon was statistically more comfortable (P = 0.039) and caused less stinging and burning (P = 0.014) at 1 minute versus the fluorescein formulation. CONCLUSION: Not only was the new fluorexon product accurate and effective in GAT, it was also statistically more comfortable and had a less stinging and burning effect at 1 minute after drop instillation than the traditional fluorescein formulation. Because fluorexon is less likely to stain soft contact lenses, this may be the dye-anesthetic formulation of choice for practices that routinely perform GAT.

Adult↗

PubMed1987

Oxygen radicals mediate cell inactivation by acridine dyes, fluorescein, and lucifer yellow CH.

Acridine dyes, fluorescein and lucifer yellow CH are fluorescent photosensitizers used experimentally to selectively stain and photodynamically destroy eukaryotic cells and subcellular structures. We have determined that the mechanism of light- and oxygen-dependent inactivation of E. coli by these dyes involves oxygen radicals and hydrogen peroxide. All of the dyes oxidized NAD(P)H+ under illumination. Superoxide (O2), detected as the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c, was a major product of the dye sensitized photooxidation. Cationic acridine dyes penetrated the membranes of E. coli and were photoreduced intracellularly. Reduced dyes diffused back into the medium and mediated the reduction of extracellular ferricytochrome c. The anionic dyes fluorescein and lucifer yellow CH were unable to mediate extracellular cytochrome c reduction, indicating that these dyes were impermeable to the E. coli membrane. Acridine dyes, when illuminated, inhibited the growth of E. coli in a rich medium, and induced the synthesis of SOD. Fluorescein and lucifer yellow CH did not inhibit growth or induce SOD synthesis because they were unable to enter the cells. Superoxide (O2) and hydrogen peroxide (H2O2), generated by the enzyme xanthine oxidase were toxic to E. coli B. Inactivation by xanthine oxidase was partially inhibited by exogenous SOD and completely inhibited by exogenous catalase or SOD plus catalase. Similarly, exogenous SOD plus catalase protected against inactivation by acridines and fluorescein-NADH or lucifer yellow CH-NADH mixtures. Prior induction of superoxide dismutase and catalase in E. coli B significantly protected cells against a subsequent challenge by illuminated acridine dyes. SOD and catalases preinduction combined with additions of exogenous SOD and catalase completely protected E. coli B against photodynamic inactivation by acridine yellow. The hydroxyl radical scavengers, dimethyl sulfoxide, sodium benzoate and thiourea, protected E. coli B against photodynamic inactivation by acridine orange. The results implicate O2, H2O2, and the hydroxyl radical (OH) as underlying molecular agents of the phototoxicity mediated by acridine orange, acridine yellow, fluorescein and lucifer yellow CH.

Acridine Orange↗

PubMed2002

Comparison of fluorescence of sodium fluorescein in retinal angiography with measurements in vitro.

To quantify dye leakage in ocular fluorescein angiography, the arterial concentration of sodium fluorescein has to be determined. We investigated whether the nonlinear relationship between the fluorescein concentration and the fluorescence intensity obtained by in vitro measurements corresponds with that measured in vivo in a retinal artery. The time series of fluorescence in a retinal artery were recorded using an in-house-designed and -built confocal scanning laser ophthalmoscope in 11 healthy volunteers. Three different doses of sodium fluorescein were injected successively. About 10 min after the last injection a venous blood sample was drawn. The three in vivo peak intensities were fitted by least squares on the in vitro calibration curve using the first peak concentration and an intensity scaling factor as the two unknown parameters. The fit showed that the saturation of the three in vivo peak intensities corresponded well with the in vitro data. Calculation of the intensity scaling factor from the blood sampling data confirmed the result of the fit. The fitted concentration was verified by showing that the cardiac output necessary to obtain this concentration was within the physiological range. The fluorescence measured in our in vitro experimental setup corresponded well with the in vivo measurements. Therefore, the results from in vitro measurements can be applied in the analysis of fluorescein angiograms.

Adult↗

PubMed1985

Outward transport of fluorescein from the vitreous in aphakic eyes.

By administering fluorescein intravenously to 95 patients we calculated the ratio of fluorescein concentration in the vitreous at the time of its peak level compared with the estimated unbound concentration of fluorescein in the plasma at the same time. We studied 12 normal phakic and 83 aphakic eye approximately two months, one year, and more than two years after cataract extraction. All the eyes had undergone intracapsular cataract extraction or extracapsular cataract extraction, with or without posterior capsulotomy, because of senile cataract. The calculated ratio in patients with intracapsular and extracapsular lens extraction was statistically significantly reduced at two months and one year after cataract extraction and was normalised at more than two years after the operation in comparison with normal subjects. The ratio was statistically extracapsular extraction at two months and one year after surgery. Posterior capsulotomy had no effect on the ratio. The ratio, we considered, at least partially reflects the outward transport of fluorescein from the vitreous cavity. Although the findings reflect subclinical phenomena, they are of importance when considering postoperative sequelae. The posterior lens capsule, zonule, and intact anterior vitreous face may be essential for the anterior uvea to function in the outward transport of fluorescein from the vitreous cavity.

Aged↗

PubMed1990

Fluorescein transport in isolated proximal tubules in vitro: epifluorometric analysis.

An epifluorometric method was used to quantify the bidirectional fluxes of fluorescein across the basolateral surfaces of nonperfused rabbit tubule segments in vitro. Proximal S2 segments, but not cortical collecting tubules or cortical thick ascending limbs, accumulated fluorescein to levels in cytoplasm over 100-fold greater than in the external medium. The rate of intracellular fluorescein accumulation was dependent on the concentration of the ligand in the external bath. The apparent Km was 10 microM and the Vmax was 623 x 10(-6) mol.min-1.l-1. Probenecid and ouabain inhibited fluorescein accumulation. We conclude that fluorescein is transported into the cytoplasm of proximal tubules by basolateral mechanisms that share features in common with the classical organic anion system. This fluorescent compound offers some unique advantages for the study of the organic anion transport system in intact cells.

Animals↗

PubMed1991

Astrocytes within the hypothalamic arcuate nucleus contain estrogen-sensitive peroxidase, bind fluorescein-conjugated estradiol, and may mediate synaptic plasticity in the rat.

Estrogen treatment induces synaptic plasticity accompanied by damaged structures and aggregates of peroxidase in astrocytes in the hypothalamic arcuate nucleus of the rat. Synaptic plasticity also occurs within the arcuate nucleus after physiologic surges of estrogen. Although the function of estrogen-induced peroxidase is unclear at present, in other systems peroxidase can generate free radicals by catalyzing the oxidation of some molecules, including estrogen. Because free radicals underlie remodeling in a number of tissues, estrogen-induced free radicals could mediate synaptic remodeling within the arcuate nucleus. Although they contain estrogen-inducible peroxidase, astrocytes do not contain estrogen receptors as measured by conventional techniques, suggesting that estrogen-inducible peroxidase arises from some novel mechanism. Estrogen could induce peroxidase within receptor-deficient astrocytes by binding to receptors in neurons and stimulating the release of some factor that interacts with astrocytes. Alternatively, estrogen could act directly on astrocytes in the absence of estrogen receptors. Although astrocytes in the hypothalamus of the rat do not contain classical nuclear estrogen receptors, they do bind fluorescein-conjugated estradiol in extranuclear sites. The distribution of fluorescein-conjugated estradiol binding within the hypothalamus overlaps that of peroxidase-rich astrocytes, and double labeling reveals many cells with the stellate morphology of astrocytes, containing both peroxidase and fluorescein-conjugated estradiol binding. However, because peroxidase and fluorescein-conjugated estradiol always occupy different compartments of the cell, the fluorescein-conjugated estradiol is not binding to peroxidase.

Animals↗

PubMed1989

Comparison of variable region primary structures within an anti-fluorescein idiotype family.

Previous reports described the properties of a high affinity (Ka = 1.7 X 10(10) M-1) prototype anti-fluorescein monoclonal antibody 4-4-20, an intermediate affinity (Ka = 3.7 X 10(7) M-1) prototype 9-40, and Ig members of the 9-40 idiotype family (comprised of 3-24, 5-14, 5-27, 10-25 and 12-40). Although the seven monoclonal anti-fluorescein antibodies expressed similar active site structural determinants (idiotypes) as determined serologically, each was characterized by different affinities for fluorescein and fine specificity binding patterns. Partial heavy (H)- and light (L)-chain N-terminal amino acid sequence analyses revealed all antibodies (except 5-27) were composed of highly homologous VHIII(C) and V kappa II subgroup genes, respectively. Antibody 5-27 utilized a VHIII(B) and a V kappa V subgroup genes and shared low V-region sequence homology with 4-4-20, 9-40 and the remaining 9-40 idiotype family. In addition, complete 4-4-20, VH- and VL-region primary structures were determined to better understand antibody-antigen interactions. Antibody 4-4-20 utilized a VHIII(C) subgroup VH-gene, a truncated Sp2 D gene segment, JH4, a V kappa II subgroup VL-gene, and J kappa 1. Antibody 4-4-20 VH and VL complementarity-determining regions contained many basic and aromatic amino acid residues capable of interaction with fluorescein. Results are discussed in terms of idiotypic and fluorescein-binding characteristics as well as antibody structural and functional diversity in the immune response.

Amino Acid Sequence↗

PubMed1984

The value of fluorescein for predicting viability muscle tissue after high-energy trauma.

Skeletal muscle subjected to high-energy trauma shows discolouration, which in earlier studies was found to imply devitalization. Fluorescein staining has been used to assess the viability of many tissues, and in the present study discolouration was compared with fluorescein staining. Trauma was caused to one thigh of anaesthetized pigs with a high-velocity spherical missile, after which the distribution of discolouration in the traumatized muscle was outlined and photographically recorded, as also were the areas stained with intravenously injected fluorescein. Recordings were made two hours and five hours after the trauma. In distinctly discoloured muscle there was no change in colour with time and no fluorescein staining. In slightly discoloured areas, presenting reduced fluorescence, there was normalization of colour with time as well as increased fluorescence. The observations imply that slightly discoloured traumatized muscle may recover, in contrast to distinctly discoloured muscle. Further, the simple and safe fluorescein staining procedure can be a useful aid in the clinical assessment of viability in traumatized muscle.

Animals↗

PubMed1978

[Limited diffusion of dye between choroid and retina during animal experiments after injection of fluorescein and rhodamin (author's transl)].

For contributing to the question of an existence of a diffusion of the unbound part of the fluorescein between choroid and retina in fluorescein angiography, on pigmented rats, after intravenous injection of Fluorescein sodium and Rhodamine B respectively, comparing observations were effectuated. Rhodamine B in its unbound form showed a distinct diffusion between choroid and retina and coloured all retinal layers in the late phases whereas in the case of the Fluorescein sodium, only a poor diffusion was observable. The similar spectral bands of the primary and secondary fluorescences render difficult the observation of the behaviour of the fluorescein dye.

Animals↗

PubMed1996

Fluorescein as an easy, low-cost, indirect, or reverse intraoperative marker to rule out perilymph versus local injection.

Clear fluid found in the dependent portions of the middle ear (round and oval windows) is generally assumed to be perilymph (PL). However, all clear fluid observed at middle ear exploration is not necessarily PL. PL should be distinguished from local injection. An accumulation of the local injection could be confused with perilymph fistula (PLF) diagnosis. There is no standard way of distinguishing perilymph from local injection, as both are clear, watery fluids. Fluorescein, tagged with a mixture of xylocaine and epinephrine (FLOTAX) was used as the local injection in 10 patients undergoing middle ear exploration for possible PLF.FLOTAX was injected into the vascular strip and ear canal skin in routine fashion. Transtympanic endoscopy and special Zeiss custom fluorescein filters were used to document any accumulation of fluid in the dependent portions of the middle ear and confirm whether any fluorescein (from the FLOTAX) was present. The middle ear was checked both before (endoscopically) and after raising the tympanomeatal flap. After the "local" injection, FLOTAX was observed to accumulate in dependent portions of the middle ear before raising the tympanomeatal flap in six of 10 ears. In the other four ears, FLOTAX slowly seeped into the middle ear cleft after the tympanomeatal flap was elevated and manipulated. Fluorescein can be used as an inexpensive, indirect intraoperative marker or "reverse test" for possible PLF. By eliminating a likely and common offender (local injection), any accumulation or reaccumulation of clear fluid that is not fluorescein tagged may more confidently be identified as possibly perilymph. The use of FLOTAX helps eliminate the confusion between the local injection and potential PLF. It does not in any way eliminate the possibility of other transudates from incisions, allergic reactions, or other manipulation of the tympanomeatal flap or middle ear mucosa.

Anesthetics, Local↗

PubMed1976

Fluorescein angiography of the normal and diseased ocular fundi of the laboratory dog.

A method was determined for fluorescein angiography of the ocular fundus of the laboratory Beagle, using a portable fluorescein fundus camera. A 10% solution of sodium fluorescein was injected by a syringe pump in the right cephalic vein of each dog. Acepromazine maleate (0.5 mg/kg, IV) with ketamine hydrochloride (10 mg/kg, IM) provided chemical restraint. Of the 10-, 25-, and 50-mg/kg dosages of fluorescein, 25 mg/kg permitted satisfactory fluorescence of the choroidal and retinal vasculature and the recirculation phase. High-speed color film permitted good quality photography of the tapetal and nontapetal retinal vessels. Reduced retinal blood vessel and pigment epithelium permeability was demonstrated by fluorescein angiography in dogs with chorioretinitis associated with systemic blastomycosis, retinitis associated with distemper, serous retinal detachment, and preretinal hemorrhage.

Animals↗

PubMed2003

Photodynamic therapy of subfoveal choroidal neovascularization with verteporfin: fluorescein angiographic guidelines for evaluation and treatment--TAP and VIP report No. 2.

OBJECTIVE: To describe fluorescein angiographic guidelines for the use of verteporfin therapy in patients with subfoveal choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) or other conditions based on 2-year vision outcomes from the Treatment of Age-Related Macular Degeneration With Photodynamic Therapy (TAP) Investigation and Verteporfin in Photodynamic Therapy (VIP) Trial. METHODS: Three multicenter, double-masked, placebo-controlled randomized clinical trials at 28 ophthalmology clinical centers in Europe and North America involving prospectively identified patients with best-corrected visual acuity (Snellen equivalent) of approximately 20/20 to 20/200, subfoveal CNV secondary to AMD or pathologic myopia with evidence of CNV, and a lesion greatest linear dimension of 5400 micro m or less. Fluorescein angiography was to be performed on all patients at enrollment and at regular 3-month follow-up visits through 2 years. The initial treatment laser spot size and all subsequent treatment decisions were based on the investigator's interpretation of these fluorescein angiograms. Photographic materials forwarded to the Wilmer Photograph Reading Center were reviewed by masked graders. MAIN OUTCOME MEASURES: Baseline angiographic features, including lesion composition and size, morphologic response to treatment during follow-up (eg, absence of leakage), and reliability (kappa values) of grading selected characteristics based on a 10% regrading of baseline visits. RESULTS: Terms and examples of different lesions and lesion components are provided to assist recognition of fluorescein angiographic characteristics of choroidal neovascular lesions that were important in determining when and where to apply verteporfin therapy. The kappa statistics for agreement of identification of lesion characteristics by the Wilmer Photograph Reading Center for these trials ranged from 0.70 to 0.85. CONCLUSIONS: Ophthalmologists should consider interpreting fluorescein angiographic images of subfoveal lesions with terms provided to follow recommendations regarding which patients are most likely to benefit from verteporfin therapy based on results from the TAP Investigation and VIP Trial.

Choroidal Neovascularization↗

PubMed1994

Detecting recurrent choroidal neovascularization. Comparison of clinical examination with and without fluorescein angiography.

OBJECTIVE/DESIGN: To evaluate prospectively the ability of three retina specialists to detect recurrent choroidal neovascularization (CNV) after clinical examination alone and then with fluorescein angiography at 3 and 6 weeks and at 3, 6, 9, and 12 months after laser photocoagulation. SETTING: Single tertiary retinal referral center. PATIENTS: All patients who had laser treatment for CNV within 14 months of their study visit. One hundred thirty-seven eyes of 134 patients were evaluated during 401 visits. MAIN OUTCOME MEASURES: Sensitivity, specificity, positive predictive value, and negative predictive value of clinical examination with biomicroscopy to detect recurrent CNV when defined as leakage on the periphery of the laser-treated area on the fluorescein angiogram. RESULTS: Ninety-seven definite or probable recurrences in 56 eyes were identified on the fluorescein angiogram. Clinical examination had a sensitivity of 59%, specificity of 94%, positive predictive value of 76%, and negative predictive value of 88%. These figures varied somewhat by underlying cause, age, time since treatment, and lesion location. Using either a reported or measured loss of vision with the results of biomicroscopy as an indication of recurrence increased the sensitivity to 77% but reduced the specificity to 81%. CONCLUSIONS: Clinical examination probably cannot replace fluorescein angiography in detecting all recurrent CNV after laser treatment. However, for follow-up visits in which recurrent CNV was not suspected on biomicroscopy, definite or questionable recurrent CNV was identified on the fluorescein angiogram only 12% of the time, while the absence of recurrent CNV using this method was confirmed 88% of the time.

Aged↗

PubMed1994

Fluorescein derivatization of fibrinogen for flow cytometric analysis of fibrinogen binding to platelets.

Dog and human fibrinogen were derivatized with N-hydroxysuccinimido-fluorescein and utilized for flow cytometric estimation of fibrinogen binding to activated platelets. Fluorescein-fibrinogen binding fulfilled the criteria for specific binding to platelets; the binding was saturable, dependent on agonist activation, and inhibited by unlabeled fibrinogen. In addition, EDTA and barbourin, a KGD-containing peptide, were found to inhibit the binding of fluorescein-fibrinogen. Fluorescein-fibrinogen bound to dog platelets with an apparent affinity of 0.31 microM after stimulation with either adenosine-5'-diphosphate (ADP) or plateletactivating factor. The labeled fibrinogen was also used to study the fibrinogen binding capacity of aged, biotinylated platelets. Aged platelets were indistinguishable from young platelets with regard to fibrinogen binding in response to ADP. These studies document that direct derivatization of fibrinogen with fluorescein generates a useful probe for analyzing fibrinogen binding to platelets with flow cytometry.

Adenosine Diphosphate↗

PubMed1988

Comparison of skin surface fluorescence and fluorescein tissue concentration with the dermatofluorometer.

Recently sodium fluorescein has been used to determine viability of tissue and assess blood flow. No study, however, has documented the correlation of surface fluorescence as measured by the dermatofluorometer with the actual distribution of fluorescein in tissue. Abdominal skin flaps were raised on Sprague-Dawley rats. Following intravenous injection of sodium fluorescein, skin surface fluorescence was measured and compared with tissue fluorescein concentration. This study confirms that skin surface fluorescence correlates well with actual fluorescein distribution in tissue. Furthermore, the study clearly illustrates the statistically significant variation of surface fluorescence as a function of distance from the vascular pedicle.

Animals↗