Urine Fluorescence Research Articles

Novel photodynamic diagnosis of bladder cancer: Ex vivo fluorescence cytology using hypericin

In this study we have evaluated the use of hypericin ex vivo urine fluorescence cytology as a non-invasive method for detecting early bladder cancers. To date this is the first study reported using this technique with hypericin. Urine samples from patients with early bladder cancers were processed for fluorescence cytology by incubation with hypericin, a novel photosensitizer. Normal urine samples incubated with hypericin served as normal controls. Laser confocal microscopy and spectroscopy was used to detect the fluorescence in the exfoliated low-grade urothelial tumor cells. Fluorescence cytology was considered positive if hypericin fluorescence of the low-grade urothelial tumor cells was detected to be stronger (>8.5 times) compared to the baseline fluorescence established for normal urine samples. Automated analysis for an objective reproducible outcome appears possible. The possibility of detection of malignant urothelial cells in early cancer makes ex vivo fluorescence cytology promising for routine diagnostic screening.

Correlative studies of urine fluorescence and free radical indicators.

Inflammation results in the production of free radicals which damage proteins, lipids, and nucleic acids. The products of these reactions are cleared, in part, by the kidney and appear in the urine. In this study, urine fluorescence was measured in individuals with various nephropathies to determine the value of these assays for detecting the excretion of end products of radical-mediated chemical reactions. Urine fluorescence was quantified at wavelengths which correspond to the presence of advanced glycosylation endproducts (AGE) and dityrosine (di-TYR). The samples were also tested for isoprostanes, nitrite/nitrate, hydrogen peroxide, and thiobarbituric acid reactive substances (TBARS). Fluorescence values, expressed per ml urine or per mg creatinine (crt), were not normally distributed and covered a wide range. There were significant differences in fluorescence among groups of patients classified by diagnosis, but the differences did not allow sharp distinction of diagnostic categories. Fluorescence assays correlated significantly with TBARS but not with isoprostanes, nitrite/nitrate, or hydrogen peroxide. Fluorescence tended to increase with age. Gender and race did not affect the results. Because of the many factors which can affect free radical production and tissue injury, the value of urine fluorescence assays to screen for radical-mediated toxicity appears to be limited. Serial studies of patients will be needed to determine whether urine fluorescence will be useful to monitor responses to treatment or be predictive of progression vs. remission of renal disease.

Diagnostic use of physicians’ detection of urine fluorescence in a simulated ingestion of sodium fluorescein–containing antifreeze

Study Objective: We sought to assess physicians’ ability to accurately determine the presence or absence of sodium fluorescein (SF) in urine at a concentration corresponding to that present after ingestion of a toxic amount of commercial automotive antifreeze. Methods: We studied 2 different urine specimen evaluation formats—one presenting isolated specimens, and the other presenting specimens grouped for comparison—to determine whether the visual clues afforded by grouped comparison aided the accuracy of the evaluation. On each study day, 3 urine specimens (1 control specimen obtained before SF administration and 2 specimens obtained after SF administration) were obtained from each of 9 or 10 volunteers. Each of these 27 or 30 urine specimens were presented sequentially and in random order to 2 emergency physicians during separate evaluation time periods. Each physician was asked to classify each specimen as fluorescent or nonfluorescent (sequential format). After a rest period, each physician, again separately, was asked to look at the same 27 or 30 urine specimens, this time all together in a test tube rack so that grouped comparisons were possible. The physicians again classified each sample as either fluorescent or nonfluorescent (grouped format). We assessed sensitivity, specificity, and accuracy of the evaluation by each presentation format (sequential or grouped). Results: Mean examiner sensitivity, specificity, and accuracy for detecting the presence of SF in urine using the sequential presentation format were 35%, 75%, and 48%, respectively, whereas the same test performance indices were 42%, 66%, and 50%, respectively, when the grouped format was used. Conclusion: Wood’s lamp determination of urine fluorescence is of limited diagnostic utility in the detection of SF ingestion in an amount equivalent to toxic ingestion of some ethylene glycol–containing automotive antifreeze products. [Wallace KL, Suchard JR, Curry SC, Reagan C. Diagnostic use of physicians’ detection of urine fluorescence in a simulated ingestion of sodium fluorescein–containing antifreeze. Ann Emerg Med. July 2001;38:49-54.]

Does fluorescent urine indicate antifreeze ingestion by children?

Fluorescent urine has been reported to indicate antifreeze ingestion. Recently, we evaluated a child who was suspected of ethylene glycol ingestion. Although she had fluorescent urine, subsequent studies showed that she had not ingested antifreeze. We tested whether fluorescent urine indicates antifreeze ingestion by children. A convenience sample of urine specimens from 30 hospitalized children was obtained. All of the patients had been hospitalized for reasons unrelated to poisoning. The specimens were viewed with a Wood's lamp, and the samples were identified as fluorescent or not fluorescent. A second convenience sample of urine specimens from a group of 16 healthy children was obtained, and these specimens were identified as fluorescent or not fluorescent in a similar manner. The majority of urine specimens obtained from children are fluorescent. There is variation in the interpretation of urine fluorescence among observers. The type of container used may influence the finding of fluorescence. Fluorescent urine is not an indicator of ethylene glycol antifreeze ingestion by children.

Technologies of compliance: Surveillance of self‐administration of tuberculosis treatment, 1956–1966

The development of antibiotics for tuberculosis in the 1950s liberated patients from prolonged hospitalization and enabled physicians to bring treatment to underserved populations. However, outpatient treatment programs could only be as effective as the reliability with which patients self‐administered medications. This anxiety with non‐compliance quickly became ubiquitous in medicine. Several physicians, treating tuberculosis among the Navajo, attempted to use technologies of surveillance to overcome this limitation. They used fluorescent tracers, urine testing, and radioactive pill dispensers to expose non‐compliance. Although successful, these techniques threatened patient‐doctor relationships with secrecy and distrust. Physicians turned instead to personal surveillance through directly observed therapy.

Verification of the drug history given by potential blood donors: results of drug screening that combines hair and urine analysis.

Besides modern virus-screening methods, the avoidance of transfusion transmission of viral diseases is based on the best possible selection of healthy donors. Unfortunately, most of the relevant behavior-related risk factors are not accessible to objective verification. Drug screening can be used to validate a defined section of donor statements. It may be assumed that donors who conceal drug consumption may also conceal other relevant risk factors. Hair and urine samples from 186 young potential donors who denied having consumed drugs were investigated by gas chromatography with mass selective detection and a urine fluorescence polarization immunoassay for cannabinoids, amphetamine and amphetamine derivatives, cocaine, and opiates. Ten potential donors with 14 positive results on hair and urine analyses (6x cannabinoids, 4x cocaine, 1x opiates, 3x dihydrocodeine) could be identified in the population investigated. The donor history is not adequate for identifying potential donors with risk factors. Deliberately false statements concerning risk factors are a clear breach of trust between the blood bank and potential donors. These unreliable donors represent an incalculable risk for the transfusion recipient. Therefore, it is appropriate to validate donor statements about drug consumption by random hair and urine analyses and to exclude from the donor pool all persons revealed as drug users.

Immunoassay detection of benzodiazepines and benzodiazepine metabolites in blood.

We evaluated the capability of commercially available benzodiazepine immunoassays from four vendors (Abuscreen radioimmunoassay [RIA-a] from Roche Diagnostic Systems and serum RIA-d from Diagnostic Products Corporation; X-systems serum and urine fluorescence polarization immunoassays [FPIA] from Abbott Laboratories; and EMIT TOX serum and EMIT urine enzyme immunoassays [EIA] from Syva) to detect 22 benzodiazepines and 16 benzodiazepine metabolites in blood samples. Drug-free bovine blood was fortified with the different compounds at concentrations relevant to blood analysis: 7, 30, and 70 ng/mL. One-milliliter samples were extracted with n-butyl chloride, dried, and reconstituted with 0.3 ml. of buffer. Results from the two urine-specific kits (EIA-u and FPIA-u) produced either equivalent or reduced cross-reactivity compared with their serum kit counterparts; these results are presented but not discussed further. Among the 38 compounds, the following were detected by all four assays at concentrations of 70 ng/mL or less: alprazolam, alpha-hydroxy-alprazolam, norclobazam, diazepam, estazolam, flunitrazepam, norflunitrazepam, miclazolam, 4-hydroxy-midazolam, nitrazepam, noradinazolam, oxazepam, prazepam, 3-hydroxy-prazepam, temazepam, and triazolam. 7-Amino-clonazepam, 7-acetamido-clonazepam, 3-hydroxy-7-acetamidoclonazepam, haloxazepam, loprazolam, 7-amino-nitrazepam, 7-acetamido-nitrazepam, and oxazolam were not detected by any of the assays at concentrations up to 70 ng/mL. A number of benzodiazepines or their metabolites were detectable by some but not all of the six assays: bromazepam, chlordiazepoxide, demoxepam, and 4-hydroxy-alprazolam (both RIAs and EIA); adinazolam (RIA-a, EIA, and FPIA); norchlordiazepoxide (both RIAs); clonazepam (RIA-a and EIA); bentazepam, clotiaze-pam, and mexazolam (EIA and FPIA); 7-amino-flunitrazepam and quazepam (EIA); and alpha-hydroxy-triazolam and medazepam (RIA-a).

Determination of three aspirin metabolites in human urine by derivative synchronous spectrofluorimetry

The direct and simultaneous determination of three aspirin metabolites present in human urine (salicylic acid, salicyluric acid and gentisic acid) is described. The method involves the measurement of synchronous fluorescence spectra (Δλ= 100 nm) of untreated urine samples at a single pH (borate buffer giving pH 9.0). The combination of first- and second-derivative spectra and the zero-crossing technique allows calibration plots for the three metabolites to be obtained that are linear in the range 0.02–0.2 ppm. The solution of a system of three simultaneous linear equations is discussed. Better results are obtained by transferring the second-derivative spectra in the range λexc= 325–375 nm to a microcomputer and performing a least-squares fit of the sample to a combination of spectra of three standard solutions. This choice of wavelengths makes it unnecessary to eliminate normal fluorescent urine components.

Zero-crossing first and second derivative synchronous fluorescence spectroscopic determination of aspirin metabolites in urine.

Considerable interest has been devoted to the determination of acetylsalicylic acid (aspirin) metabolites: salicylic acid (SA), salicyluric acid (SU), and other minor metabolites in biologial fluids [l-13]. The subject is relevant regarding drug monitoring programs, and also in emergency determinations in cases of salicylate poisoning. The analysis of aspirin formulations regarding the content of SA has also attracted attention [14]. A variety of techniques have been applied to the determination of these compounds, including chromatography [l-3], spectrophotometry [4-61 and spectrofluorimetry 17-141. The latter method seems to be of choice due to its simplicity, sensitivity and specificity. However, the main urine metabolites of aspirin (SA and SU) have fluorescent emission spectra which not only overlap with each other, but also with fluorescent normal urine components. This problem has been previously overcome by various sample pre-

Urine fluorescence using a wood's lamp to detect the antifreeze additive sodium fluorescein: A qualitative adjunctive test in suspected ethylene glycol ingestions

Antifreeze ingestions require rapid and accurate differential diagnosis to prevent fatal outcomes. Sodium fluorescein is added to some commercial antifreeze preparations (ethylene glycol) to a final concentration of approximately 20 micrograms/mL as a colorant to aid in detection of automobile cooling-system leaks. For an adult human being, a potentially toxic volume of antifreeze is 30 mL, which contains 0.4 to 0.6 mg sodium fluorescein. Six male volunteers were given a 0.6-mg oral bolus of sodium fluorescein on an empty stomach. Urine was collected at two-hour intervals. Using a Wood's lamp, visually detectable fluorescence was seen with 100% reliability for two hours and 60% reliability for four hours. A second group of male volunteers was given the same dose of sodium fluorescein, and fluorescence was measured with a fluorometer during a six-hour period. Detectable fluorescence was present in all samples except the zero time point, including those with no fluorescence present by visual examination. We conclude that exposing urine to a Wood's lamp may be a useful adjunctive diagnostic test for early evaluation of patients with suspected antifreeze ingestion while awaiting definitive quantitative analysis of serum ethylene glycol concentration. A prospective clinical trial is needed to evaluate the frequency of false-positives and false-negatives.

Pentobarbital quantitation using immunoassays in Reye's syndrome patient serum.

Analysis of serum pentobarbital concentrations in 28 specimens from Reye's syndrome patients was conducted with modifications of three nonspecific immunoassay procedures originally designed to detect barbiturates in serum or urine. An adaptation of the urine enzyme-multiplied immunoassay technique (EMIT) Dupont aca barbiturate screen to the quantitation of serum pentobarbital is described. Replicate analysis of control specimens containing pentobarbital across a wide spectrum of concentrations revealed a between-day precision of less than 6%. Regression analysis revealed excellent agreement with a high-performance liquid chromatography (HPLC) method: (HPLC) = 0.98 (aca) - 0.07 (r = 0.97). Multiple linear regression analysis with a serum EMIT barbiturate screen and a urine fluorescence polarization immunoassay screen modified to quantitate pentobarbital in serum revealed excellent agreement among all methods, demonstrating that immunoassays offer a reliable approach to pentobarbital quantitation.