Potential for False-Positive Results by the TRIAGE ...

Published: Journal of Analytical Toxicology, Volume 20, Number 3, May/June 1996, pp. 209-210.

Potential for False-Positive Results by the TRIAGE™ Panel of Drugs-of-Abuse Immunoassay
A. Poklis and C.L. O'Neal

To the Editor:
In 1994, Biosite Diagnostics (San Diego, CA) introduced the TRIAGE panel for drugs of abuse (TPDA), a colloidal metal immunoassay device for rapid urinary drugs-of-abuse testing. The device is a test card impregnated with antibodies in separate areas to simultaneously detect seven classes of drugs: amphetamines, barbiturates, benzodiazepines, cocaine metabolite, opiates, phencyclidine, and marijuana metabolite (1–3). The widespread acceptance of TPDA as a screening assay for these drugs is evidenced by its use by 42% of the approximately 2200 participants in the 1995 American Association for Clinical Chemistry/College of American Pathologists Urine Drug Testing (Screening) Survey (4).
The TPDA is a self-contained assay performed in the following manner (5): the cap is removed from the reaction cup on the bottom of the device, and, by using a provided pipette, 140 µL of urine is transferred into the cup. The card has three different labeled areas: two areas impregnated with procedural control material, labeled “control negative” and “control positive”, and the “drug detection area”, which contains discreet labeled zones of impregnated antibodies for the drugs tested. These are incubated 10 min at room temperature. By using the pipette and a clean tip, the reaction mixture is transferred to the drug detection area of the card. The mixture is allowed to completely soak into the reaction area. Three drops of provided wash solution are added and allowed to soak in completely. A positive result produces a red colored bar in the drug detection area next to the drug name indicated. For the assay to be valid, control positive must have a red bar and control negative must be blank or unreactive.

The reaction cup in the TPDA contains three colored beads of the following composition: labeled drugs (purple), antibodies (white), and buffer (light tan). We noticed that when removing the lid of the reaction cup prior to adding the urine sample, one or more of the beads sometimes popped out or stuck to the lid of the device. The TPDA is an “on-site” testing device that may be used by individuals without laboratory training, such as parole or probation officers, employment personnel, nurses, and emergency room physicians. Therefore, we investigated the effect of removal of the buffer bead on the TPDA response to prepared urine specimens at various pH levels containing 125 and 150% of the cutoff concentrations of each of the seven analytes.

Our study protocol was as follows: all drug primary reference materials were obtained from Radian Corp. (Austin, TX), except for lorazepam–glucuronide and l-tetrahydrocannabinoic acid (Alltech, State College, PA). Drug-free urine was collected from laboratory personnel, and, with the exception of caffeine or nicotine metabolites, the samples tested negative for all drugs in this study. Neither caffeine nor nicotine metabolites interfered with any assay in the study. Duplicate urine specimens containing a single analyte drug containing 125 and 150% of the TPDA cutoff concentration were prepared in drug-free urine specimens at pH 4.0, 5.0, 6.0, 7.0, and 8.0 (Table I). The buffer bead was removed from the TPDA devices, and each urine specimen was then tested. As a control, one of the urine specimens was simultaneously tested with an intact TPDA device containing the buffer bead. Additionally, a drug-free control was prepared and analyzed as the analyte urine specimens.

We observed that, at pH 6.0, 7.0, and 8.0, the TPDA yielded appropriate responses: all drug-free urine specimens tested negative (9/9), all intact assays (21/21), as well as those with the buffer bead removed, were positive for the added analyte at 125 and 150% of the cutoff concentration (42/42), and the procedural control areas yielded appropriate positive and negative responses. However, we observed several false-positive results with the pH 4.0 and 5.0 urine specimens. At pH 4.0, one of the two drug-free urine pools tested with the intact TPDA yielded false-positive results for cocaine metabolite and opiates, whereas both of the procedural controls reacted appropriately (positive/positive, negative/negative). When the buffer bead was removed, the purple bead (labeled drug) did not properly dissolve in pH 4.0 urine specimens, forming instead a gelatinous soup. When transferred to the TPDA reaction area, controls usually failed because the negative control was positive (26/28). These pH 4.0 urine specimens yielded a positive response for all the TPDA analytes. The two instances where the controls passed were with the duplicate 150% cutoff opiate urine specimens, which yielded false-positive results for cocaine metabolite and amphetamines. However, on repeat analyses, the controls failed as with the other pH 4.0 urine specimens. The response of intact TPDA to pH 4.0 drug-added urine specimens was mixed: 8 of 14 urine specimens yielded appropriate control and analyte results, whereas controls failed in the remaining six tests. As with the pH 4.0 buffer bead-removed samples, each intact TPDA was positive for all seven analytes when controls failed.
When drug-added pH 5.0 urine specimens were tested with intact TPDA, all results were appropriate and controls passed, and the added analyte was detected. However, when these urine specimens were tested with buffer bead-removed TPDA, inconsistent results were observed. In 11 of 28 tests, the controls failed, and the assay was positive for all analytes. The remaining urine specimens yielded appropriate control results (17/28). TPDA correctly detected only the added drug in 8 of these 17 urine specimens (duplicate cocaine metabolite, amphetamines, and marijuana metabolite at 150%, plus one of the opiate urine specimens at 125%). However, 9 of 17 drug-added pH 5.0 urine specimens with appropriate control responses yielded false-positive results (Table II). Cocaine metabolite and amphetamines gave a consistent false-positive result. Obviously these two TPDA assays were greatly influenced by low urinary pH. Urinary pH levels of 4.0 or 5.0 are commonly encountered in specimens submitted for routine drugs of abuse testing. When performing some of the assays, as occurred with urine specimens at pH 4.0, the purple bead did not properly dissolve at pH 5.0. However, in most tests yielding false-positive results with appropriate control responses, the beads had dissolved, and the appearance of the reaction mixture was indiscernible from that of an intact TPDA test.

Our study demonstrated that if the TPDA buffer bead is missing, false-positive results can occur with urine specimens at pH 5.0 even though the device controls yielded acceptable results. Therefore, TPDA users must use care when removing the lid to the reaction cup in order to ensure that all the necessary beads are present. Unlike liquid immunoassays performed on autoanalyzers, which result in an error message if reagents are not added, the TPDA relies solely on the results of the device controls to indicate a problem with the assay. As individuals without laboratory training often perform on-site drug testing with TPDA, the manufacturer may consider adding a notice to their instructions that the analyst check for the presence of all beads in the reaction cup before adding the urine sample. Once urine is added, the reaction mixture may be indiscernible from that of an intact TPDA test.

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