In toxicology, unraveling the unexpected positives

The immunoassay package insert revealed that oxycodone cross-reacts with the opioid test in and around 10,500 ng/mL, and the high concentration triggered suspicion about the patient spiking his urine to trigger the positive result. “Our mass spectrometry screen is geared to calculate ratios between the oxycodone and noroxycodone present” and indicate a problem: insufficient metabolite. The patient admitted to diverting and selling his oxycodone pills, saving one pill to drop in his urine at the pain clinic to get a positive result and continue receiving his prescriptions. He was referred to the behavioral medicine substance use disorder clinic for an evaluation and to start a compliance regimen. “Instead of barring him from the pain clinic, we said, ‘We’re going to give you another avenue. Let’s have a talk about compliance and how to keep you on it,’” Dr. Tacker said.

In a toxicology investigation, it helps to go into a clinic and observe the specimen flow, Dr. Tacker said. “If you get an unexpected positive, was it really that patient’s urine? Potentially no.” Limited numbers of clinical assistants handle multiple urine cups. “They’re lining them up, trying to get them labeled, trying to read results. They can make mistakes. Things need to be asked at a fundamental level all the time.”

Ask too about the local laboratory’s manual aliquoting steps. “Did someone in the lab aliquot this, send it down the track, or send it to confirmation? Or did they do it in the clinic setting and then send us a labeled tube for confirmatory testing and keep the urine cup?” Know how a specimen moves, where it goes, and who touches it, “so we’re not pegging people with blame or discussions with the substance use disorder clinic because we didn’t put the label on the right tube,” Dr. Tacker said.

Assay-related issues are less likely causes of false-positive results, she said, though “things might switch on you overnight if a calibrator has drifted and it’s detected later.” Calibrator drift resulting in unexpected weak positives is more common for lower-cutoff screens, such as for fentanyl and buprenorphine, she said, and potentially if there’s an interference on top of a low cutoff screen. “So look at your raw signals from your analyzer. It could give you good information” when investigating.

With a definitive-method false-positive, carryover is the most common issue. Also possible: the presence of an isobaric ion or ion ratio settings that aren’t sufficiently stringent. “If something new comes on the scene and your ion ratios aren’t tuned exactly right, you might get a false-positive.” You find out you have an isobar, and now it needs to be differentiated from the target compound. “There’s always a learning curve in definitive testing,” Dr. Tacker said. “It’s ever-evolving.”

She cited dihydrocodeine and noroxycodone as an example. They share an ion at 302 m/z and have a close retention time on her laboratory’s assay. “We have implemented two to three levels of ion ratio checking to differentiate them so that when technologists are looking at them, they can see it looks strange and that it failed the ion ratio check.”

Dr. Tacker shared a case in which evening shift technical maintenance and quality control led to a buprenorphine urine screening failure. Her laboratory’s buprenorphine urine screening test has a cutoff of 5 ng/mL. The technologist did a 24-hour look-back and found eight weak-positive raw results to review. All eight were negative on retests performed after the calibration was set up and rechecked and QC was in place. “It happens,” Dr. Tacker said. “You get drift.”

The laboratory corrected the results, made calls to explain, and offered definitive testing at no charge and credits for any unnecessary definitive reflexes. And, of course, it examined its QC and calibration intervals (for example, perhaps calibration is set at 30 days in the instructions for use, but a more stringent calibration interval of 21 to 28 days could be sufficient to prevent drift).

Validation is another but less likely explanation for a definitive false-positive. “You cannot cross-check every single drug in every single matrix at every concentration and see where all your cross-reactivities lie. It’s something you acquire over time,” she said. And while mass spectrometry is good, it’s not perfect. “I love mass spec. I run that part of the laboratory. But we all realize what the limitations can be and try to be hyperaware of them and always course-correct as we move through our work.” And sometimes unexpected positives stem from combinations of things—“a twisted mess,” as Dr. Tacker puts it.

In another case from her laboratory, a patient with diabetes and a urinary tract infection tested ethyl glucuronide (EtG) positive and ethyl sulfate negative (alcohol metabolites). The patient denied a relapse. Her urine specimen, which had post-collection fermentation, had been stored at the clinic overnight before it reached the laboratory. There was no ethyl sulfate present (“no evidence that alcohol went through her liver,” Dr. Tacker said) but the low EtG-positive signal was unusual in this scenario, so the laboratory confirmed the result: false-positive.

Another case involved a patient with urinary retention in a prolonged clearance of prescribed oxycodone by about two days. The window for an oxycodone positive result is usually up to three to five days, but there was trace positivity in the specimen one week later. The laboratory traced the decreasing oxycodone level, but the patient said they had urinary retention. “We thought, ‘No way,’” Dr. Tacker said, “but it was actually enough to concentrate it and give us enough signal to give us that false-positive.”

Keep lists and logs of investigations and confirmations, Dr. Tacker said in closing. Her own book of investigations is always at arm’s reach, with notes in it about what test results were confirmed. “So when the phone rings, I open it up, ask for the medical number, and [say] let’s look at the meds. And I record those things because it helps me retrace my steps and find patterns.”

Amy Carpenter Aquino is CAP TODAY senior editor.