In toxicology, puzzling out the unexpected negative

Amy Carpenter Aquino

November 2022—In cases of unexpected negative results in toxicology testing, avoid overinterpretation, know your assays and providers, and don’t put off definitive testing when it’s needed, though it’s not a panacea.

That’s some of the advice Nicholas Heger, PhD, NRCC, medical director of clinical operations and lab support and co-director of clinical chemistry at Tufts Medical Center, shared in an AACC session in July on toxicology investigations, focused on urine drug screening for compliance and pain management and using his lab’s patient cases.

“Hold yourself back from trying to suggest the patient may have diverted, sold the drug, done x, y, or z. It’s tempting sometimes to come up with scenarios to potentially explain a negative or positive result,” he said, but “that’s not what our job is. Our job is to interpret the data we have in front of us, report it objectively, and move on from there.”

For patients in medication-assisted treatment programs, if a result doesn’t match what’s expected, “we wouldn’t want that patient to be discharged from that program,” or an infant to be taken from its mother, “because the lab didn’t do what it’s supposed to do to confirm a presumptive positive result.”

Ask yourself at the start whether the assay the laboratory is using “can do what it is I want it to do,” he said.

For example, several structurally related compounds, such as codeine (at 500 µg/mL) or heroin (at 300 µg/mL), would test negative at the 100 ng/mL cutoff of a particular oxycodone urine immunoassay. “There are several other naturally occurring opiates that are not picked up well by the oxycodone assay, and that’s perfectly expected,” said Dr. Heger, who is also assistant professor of anatomic and clinical pathology at Tufts University School of Medicine. The concentrations listed for this assay’s package insert are micrograms per mL, not nanograms per mL, so the oxycodone immunoassay wouldn’t be expected to pick up compounds like morphine, codeine, heroin, hydromorphone, and others.

For Dr. Heger, “package inserts are gold” and the first place he goes when working up a case. “And even after having looked up the same package inserts year after year, I find myself gravitating back to them to answer a lot of those questions,” he said.

If the laboratory is performing confirmatory testing in-house—generally with gas chromatography mass spectrometry or tandem mass spectrometry—“look at your in-house standard operating procedures, perhaps some of your validation data where you worked up cross-reactivities with other structurally related drugs,” he said, noting, “That can be helpful as well.”

As can personal experience. “As you start to review more urine drug screens, you will start to come across some interesting things at your own facility that you may not have realized aren’t picked up by your assays,” he said.

Review drug metabolism pathways, he advises. Residents and physicians may be unaware of drug interrelatedness, especially with opiates and benzodiazepines, for which there are many common pathways. “There are lots of common drugs and common metabolites, and reviewing this can be important.” Because many screening assays are class assays—benzodiazepines, barbiturates, opiates, amphetamines—“it’s important to review the drug metabolism pathways to determine whether the assay you’re running will or will not pick up what you want.”

Next month:
Unexpected positive results

Detection varies by method and manufacturer. For example, one manufacturer of a commercially available immunoassay for fentanyl designed its assay to pick up fentanyl at 2 ng/mL and claims it doesn’t pick up norfentanyl at all; the other manufacturer’s assay detects fentanyl at 2 ng/mL and norfentanyl at 5 ng/mL. If the laboratory were using the first manufacturer’s assay and had a patient who was on a waning dose of fentanyl, the assay “might not pick them up as having used fentanyl recently because it wouldn’t pick up norfentanyl.”

Stay updated on assay versions, he said. “It has happened more than once where the manufacturer has released a new version of the assay parameters, and it was not updated on the instrument itself. Same thing with formulation.” Some third-party assays have “fairly elaborate” preparation steps—bringing the assay to room temperature or mixing part A with part B and letting it sit in the refrigerator for 24 hours, for example. “Follow all of that carefully and make sure your assay’s performing the way you expect.”

Lot-to-lot variability is not as common of an issue, but does happen occasionally. “There may be different lots of antibodies, and that could potentially explain an unexpected negative.”

Run the right assay for the right drug. A person can hear or transcribe the wrong test, write the wrong test on the requisition, or choose the wrong test from the medical record.

Dr. Heger presented the case of a 30-year-old female who was seen for chronic pain post-cesarean section. The patient was prescribed 2 mg of Dilaudid (hydromorphone, a semi-synthetic opiate) every six hours PRN. The urine drug screen was negative for opiates, and the provider didn’t understand why.

A pathology resident who looked into the case found no problems with calibration or quality control, no recent lot changes of the assay, and no labeling problem. The specimen had been automatically aliquoted by the track system.

The laboratory sent the specimen out for confirmatory testing by liquid chromatography–tandem mass spectrometry. The result: 900 ng/mL of hydromorphone. “So clearly there’s hydromorphone in the sample, which we would expect,” Dr. Heger said.

The resident then pulled the package insert: “This particular assay for opiates does not strongly cross-react with hydromorphone,” Dr. Heger said. “We need concentrations of 1,400 ng/mL at least or higher to report a positive result for the opiates assay.”

The laboratory explained to the provider that the assay was best designed to pick up naturally occurring opiates, such as morphine and codeine, and less so for hydrocodone and hydromorphone. The laboratory decided to improve its communication with and education of providers by using links on its intranet site about common cross-reactants and at what concentrations for some assays.