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PubMed1988

Automated contrast fluorometry. I. Lymphocytotoxicity testing.

Automated contrast fluorometry has been carried out in lymphocytotoxicity testing using a computer-controlled microscope and direct comparison of the data with controls. The method uses heparinized lymphocytes and permits simultaneous evaluation of both fluorescein and ethidium bromide fluorescence by calculating the quotient between the green/red measurements. After comparison of the quotient with that of control samples, the raw data are automatically transformed into cytotoxicity scores. Fast and reliable measurements with minimum background fluorescence can be obtained for up to 24 hours after performance of the assay by controlling complement reactivity and the exposure time of the cells to the fluorescein. The evaluation of 15,204 cytotoxic reactions showed excellent correlation between the automated and visual results of 125 tissue typings using 120 different antibodies and of 17 HLA antibody titers using case-by-case analysis of variance. The values for the automated cytotoxicity readings were slightly higher and showed more intermediate scores than those of the visual readings suggesting a high sensitivity and objectivity of the automated system. In conclusion, this is a methods paper comparing parallel lymphocytotoxicity testing using automated and manual methods. The automated method was found to be more sensitive and faster than the manual method.

Automation↗
PubMed1986

Fluorometry of nanogram amounts of selenium in biological samples.

For fluorometry of selenium in human blood, hair, and liver and in leaves, we wet-ashed the samples with conventional nitric and perchloric acids, and then extracted piazselenol (complex of Se and 2,3-diaminonaphthalene) in cyclohexane. Selenium was back-extracted from the cyclohexane into nitric acid to remove the fluorometric interferences of trace amounts of organic compounds. This fluorometric method is rapid and suitable for routine analysis. We applied the method to human hair samples and compared it with the data for non-destructive neutron activation analysis of the hair.

2-Naphthylamine↗
PubMed1985

A pair of five-day flaps: early division of distant pedicles after serial cross-clamping and observation with oximetry and fluorometry.

In each of 2 recent patients with distant pedicles (one a groin flap and the other a cross-leg flap), we were able to perform the final division and detachment of the flap on the fifth postoperative day. Cross-clamping was used to create intermittent periods of ischemia. The periods of ischemia were progressively increased until the time of division. Fluorometry with intravenous fluorescein played a role in deciding when to divide the flap. The patients were discharged from the hospital on the sixth and seventh days, respectively. Trimming was done on an outpatient basis.

Adult↗
PubMed1979

Enzymatic fluorometry for estimating serum total bile acid concentration.

Fasting serum total bile acid (SBA) levels were estimated by enzymatic fluorometry (EF) in 36 subjects without liver disease, 28 with hepatic lesions and impaired hepatic function, and 79 with hepatic lesions and normal function. Fasting and postprandial EF-SBA levels were compared in nine normal subjects and nine patients with cholestasis, and SBA assays by EF and gas-liquid chromatography (GLC) were compared in 28 patients with hepatic lesions and impaired function. Levels of SBA were below 9 mumole/L in all but two of the 36 subjects without liver disease, and above that level in all 28 with impaired hepatic function and 17 (24%) of the 70 with hepatic lesions and normal liver function. In most subjects, EF detected notable postprandial rises in SBA. Enzymatic fluorometric and GLC-SBA values were closely correlated (coefficient of correlation, 0.869).

3-Hydroxysteroid Dehydrogenases↗
PubMed1982

Use of noninvasive fluorometry and spectrophotometry to study epithelial metabolism and transport.

Various examples illustrating the use of spectrophotometry and fluorometry in epithelia are presented. The first example uses the redox level of cytochrome aa3, measured spectrophotometrically as an index of tissue anoxia in cortical tubules and slices from the rabbit kidney. In the second example the redox level is used to measure the kinetics of aerobic energy production during transition to anoxia in the midgut of the tobacco hornworm. In the third application, the redox level of mitochondrial NADH is measured fluorometrically in a cortical tubule suspension from the rabbit kidney. Inhibition of active transport work causes reduction of NAD whereas increased work elicits oxidation of NAD, both occurring as expected from mitochondrial transitions to a lesser or more active state, respectively. Another use of NADH fluorescence is the determination of the relative effectiveness of metabolic substrates to deliver reducing equivalents to the respiratory chain in a particular tissue. Redox changes in mitochondrial NAD may be used to distinguish between primary metabolic and primary transport effects of hormones, drugs, and changes in the state of the organism. Finally, examples are provided of the use of an intracellular pH-sensitive dye and an extracellular calcium-sensitive dye in kidney tubules.

Animals↗
PubMed1995

Detection of prostate-specific antigen mRNA by reverse transcription polymerase chain reaction and time-resolved fluorometry.

We have developed a time-resolved fluorometric hybridization assay for detecting prostate-specific antigen (PSA) mRNA amplified by reverse transcription polymerase chain reaction. During PCR, digoxigenin-11-dUTP is incorporated into the amplified product. An oligonucleotide internal to the primers is used as a specific probe, being biotinylated and captured on streptavidin-coated microtiter wells. Denatured PCR product hybridizes with the probe, and the hybrids are detected with an alkaline phosphatase-labeled antidigoxigenin antibody. We used the phosphate ester of fluorosalicylic acid as the substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which we can measure by time-resolved fluorometry. A signal-to-background ratio of 10 was obtained when 160 PSA cDNA molecules were present in the preamplification sample. Also, mRNA corresponding to one LNCaP cell in the presence of 10(6) PSA-negative cells can be detected (signal-to-background ratio of 3.1). Samples containing 100, 1000, and 50,000 LNCaP cells gave CVs of 12.4%, 4.9%, and 6.8%, respectively (n = 10).

Fluorometry↗
PubMed1995

[Redox fluorometry study of corneal flavoproteins following hypoxia. Preliminary results].

PURPOSE: The cornea is frequently associated to hypoxia, whether during residence in the heights or more often when wearing contact lenses. To evaluate the corneal modifications induced by hypoxia at an infraclinical stage, we have used redox fluorometry that enables to study in vivo the metabolic response of the cells while measuring the fluorescence of the flavoproteins (FAD) of the corneal cells. METHODS: The variations of the corneal fluorescence were studied in 12 healthy subjects, before and after a topically-induced 5-minute corneal hypoxia, submitting 2 eyes to a prehumidified flow of nitrogen 100%. The results were compared to those found in the same subjects after exposure under the same conditions to an ambiant air flow (N2 = 69%; O2 = 21%). The measurements of the corneal fluorescence were carried out with the fluorophotometre Flurotron Master FM2. RESULTS: The authors did not find any statistically significant difference in the corneal fluorescence between the right and the left eyes of these 12 subjects, whether under normal conditions, under hypoxia, or under air flow (wilcoxon T-test, Friedman test). CONCLUSION: As there are no significant results, these authors suggest that the chosen exposure time, although sufficient in vitro to induce a modification of the fluorecence of the cellular flavoproteins, may be too "short" in vivo. The use of complementary filters with the FM2 system would yield more information. The study of these results led the authors to broaden their search whether by looking for conditions for general hypoxia (hypobarric box) or by increasing local hypoxia (contact lenses).

Adult↗
PubMed1995

[Diagnosis of meconium aspiration syndrome by fluorometry technique].

OBJECTIVE: To establish a new diagnostic method of neonatal meconium aspiration syndrome (MAS). METHODS: The fluorometry technique was used for examination of urine samples from three groups of neonates. The neonates were divided into 3 groups: Group I, MAS (n = 17); Group II, no MAS, but amniotic fluid with meconium stain (n = 24); Group III, normal control (n = 27). Urine fluorescence meconium index (UFMI) and urine meconium index (UMI) were calculated. RESULTS: UFMI values of group I were higher than that of group II and III. The sensitivity and specificity of UFMI were 100% and 97% respectivety. However, it were 51% and 48% respectivety in UMI. CONCLUSIONS: UFMI is an index for identification of Zn-coproporphycin, and it is a more sensitive index for diagnosis of MAS.

Coproporphyrins↗
PubMed1996

Quantitative RT-PCR combined with time-resolved fluorometry for determination of BCR-ABL mRNA.

A microtiter well-based quantitative reverse transcriptase-PCR assay for determination of BCR-ABL mRNA, which relies on coamplification of the target with an RNA internal standard (IS), was developed. The hapten digoxigenin (Dig) is incorporated during PCR. Target RNA and IS contain identical primer recognition sites and generate same-sized amplification products distinguishable by hybridization with probes specific to the molecules' central part. The hybrids are determined with an anti-Dig-alkaline phosphatase conjugate with fluorosalicylphosphate as substrate. Fluorescent complexes of fluorosalicylate-Tb(III)-EDTA are measured by time-resolved fluorometry. The ratio of fluorescence values for target and IS is linearly related to initial target RNA in the range of 1000 to 200000 molecules. Samples containing K562 total RNA amidst 1 microgram of RNA from normal cells give fluorescence ratios that are linearly related to 30-10000 K562 cells. CVs for 30, 200, and 900 K562 cells are approximately 11%.

Digoxigenin↗
PubMed1997

Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry.

We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations (delta F508, G1717-->A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are > 10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.

Alleles↗
PubMed1997

Quantification of parathyroid hormone-related protein mRNA by competitive PCR and time-resolved lanthanide fluorometry.

Using dissociation and enhancement time-resolved lanthanide fluorometry, we have developed a quantitative competitive (QC)-PCR for measuring parathyroid hormone-related protein (PTHrP) mRNA after reverse transcription. A cloned PTHrP cDNA target was also modified by deletion of 10 bp and insertion of 21 bp in the midregion of the fragment and cloned for use as a competitor (i.e., internal standard). Two primers spanning 362 bp of target and 373 bp of competitor were designed and one of the primers was biotinylated. Two oligonucleotide probes, one recognizing the target and the other hybridizing to the competitor, were labeled with Eu chelate. Two equal aliquots of PCR products were assayed with each probe separately in streptavidin-coated wells. After 35 PCR cycles, the competitor signal decreased exponentially (y = e(3.74 - 0.624x); r2 = 0.965) and the target signal increased exponentially (y = e(1.14 + 0.497x); r2 = 0.984) when 1000 copies/tube of the competitor and 0-100,000 copies/tube of the target DNA were added. Log-transformed data for the ratio of target to competitor signals (y) and the copies of the target DNA added (x) were used for plotting the linear calibration curve (y = 2.79 + 2.76x; r2 = 0.976). This QC-PCR enables analysis of multiple samples simultaneously and can be used to study PTHrP gene expression in malignancy and physiology.

Europium↗
PubMedDate not supplied

Time-resolved fluorometry (TRF)-based immunoassay concept for rapid and quantitative determination of biochemical myocardial infarction markers from whole blood, serum and plasma.

We report the development of a time-resolved fluorometry-based immunoassay concept for the rapid measurement of three cardiac markers from whole blood, serum or plasma. Using a universal all-in-one (AIO) dry reagent concept, all the analyte specific reagents are built into a single microtire well, to which an identical assay protocol is applied. Addition of 5-20 microL sample (whole blood, serum or plasma) together with a universal buffer initiates the reaction, which is brought close to equilibrium in 15 min. After the wash step the Eu chelate-derived signal is measured directly from the dried surface. Application of this concept to the three cardiac markers illustrates its ability to provide rapid, highly sensitive and fully quantitative results over a large dynamic range with good reproducibility. Such a performance, especially when using whole blood specimens, is largely a consequence of the inherently fluorescent and stable Eu-chelate employed in the system. Correlation to commercial assays was excellent for all three analytes, as was between-sample matrix correlation using the AIO assays. The presented assay concept enabling a simple automation is particularly suited for point-of-care applications, where the performance characteristics are fully comparable to state-of-the-art central laboratory immunoassays.

Biomarkers↗
PubMed1989

Digoxin immunoassay with monoclonal and polyclonal antibodies using time-resolved fluorometry.

We describe a nonisotopic heterogeneous competitive immunoassay of digoxin in serum using either Fab fragments of a polyclonal antibody or a high-affinity monoclonal antibody. In the assay, digoxin competes with immobilized digoxin (digoxin:thyroglobulin conjugate) for binding to a biotinylated immunoreactant (Fab or monoclonal). The amount of biotinylated moiety bound to the solid phase (white polystyrene microtiter wells), which is inversely related to the amount of digoxin in the sample, is then quantified by adding streptavidin labeled with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10- phenanthroline-2,9-dicarboxylic acid (BCPDA) in the presence of excess Eu3+. The fluorescent immunocomplex formed is measured directly on the dry solid phase by time-resolved fluorometry. The assay is simple to perform and its characteristics are similar to those of other currently used immunoassay techniques. The Fab fragments and the monoclonal antibody procedure performed equally well on the system. Our results suggest that monoclonal antibodies with high affinity for digoxin can be used for the routine determination of the drug in serum.

Antibodies, Monoclonal↗
PubMed1991

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching.

We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T1, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40 degrees C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T1, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.

Acrylamide↗
PubMed1986

Assessment of the in vitro growth of Plasmodium falciparum using fluorometry.

The growth of Plasmodium falciparum in vitro was quantitatively assessed by applying fluorometry using ethidium bromide. The fluorescence intensity of parasites stained with this dye was found to parallel the uptake of 3H-hypoxanthine into nucleic acids during one growth cycle of development. The assay system can be used as a substitute of morphological and radiometric methods in drug-sensitivity tests and for the screening of antimalarials.

Animals↗
PubMed2005

Traceability in fluorometry--Part I: Physical standards.

The inter-instrument, inter-laboratory, and long-term comparability of fluorescence data requires the correction of the measured emission and excitation spectra for the wavelength- and polarization-dependent spectral irradiance of the excitation channel at the sample position and the spectral responsivity of the emission channel employing procedures that guarantee traceability to the respective primary standards. In this respect the traceability chain of fluorometry is discussed from a radiometrist's point of view. This involves, in a first step, the realization of the spectral radiance scale, based on the blackbody radiator and electron storage ring, and the spectral responsivity scale, based on the cryogenic radiometer and their control via key comparisons of the national metrology institutes. In a second step, the characterization including state-of-the art uncertainties of the respective source and detector transfer standards such as tungsten strip lamps, integrating sphere radiators, and trap detectors used to disseminate these radiometric quantities to users of spectroscopic techniques is presented.

Journal Article↗
PubMed2005

How to improve quality assurance in fluorometry: fluorescence-inherent sources of error and suited fluorescence standards.

The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards.

Journal Article↗
PubMed2004

Estimating photosynthetic electron transport via chlorophyll fluorometry without Photosystem II light saturation.

Estimates of thylakoid electron transport rates (J(e)) from chlorophyll fluorometry are often used in combination with leaf gas exchange measurements to provide detailed information about photosynthetic activity of leaves in situ. Estimating J(e) requires accurate determination of the quantum efficiency of Photosystem II (Phi(P)), which in turn requires momentary light saturation of the Photosystem II light harvesting complex to induce the maximum fluorescence signal (F(M)'). In practice, full saturation is often difficult to achieve, especially when incident photosynthetic photon flux density (Q) is high and energy is effectively dissipated by non-photochemical quenching. In the present work, a method for estimating the true F(M)' under high Q was developed, using multiple light pulses of varying intensity (Q'). The form of the expected relationship between the apparent F(M)' and Q' was derived from theoretical considerations. This allowed the true F(M)' at infinite Q' to be estimated from linear regression. Using a commercially available leaf gas exchange/ chlorophyll fluorescence measurement system, J(e) was compared to gross photosynthetic CO(2) assimilation (A(G)) under conditions where the relationship between J(e) and A(G) was expected to be linear. Both in C(4) leaves (Zea mays) in ambient air and also in C(3) leaves (Gossypium hirsutum) under non-photorespiratory conditions the apparent ratio between J(e) and A(G) declined at high Q when Phi(P) was calculated from F(M)' measured simply using the highest available saturating pulse intensity. When F(M)' was determined using the multiple pulse / linear regression technique, the expected relationship between J(e) and A(G) at high Q was restored, indicating that the Phi(P) estimate was improved. This method of determining F(M)' should prove useful for verifying when saturating pulse intensities are sufficient, and for accurately determining Phi(P) when they are not.

Journal Article↗