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Researchers Develop Method to Reduce False Negatives in Benzodiazepine Immunoassay Screening

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NEW YORK (360Dx) – Researchers at the University of Virginia School of Medicine and the University of Florida College of Medicine have developed a method to minimize false-negative test results that occur during immunoassay screening for benzodiazepine, a class of psychoactive drugs.

The method, described this week in The Journal of Applied Laboratory Medicine optimizes absorbance cutoff values, which are thresholds for determining whether a sample is sent for mass spectrometry confirmatory testing.

In a two-week prospective study, the researchers dropped the cutoff value from 100 to 20, thereby decreasing the false-negative test rate of detecting benzodiazepines from 47 percent to 2 percent, Lindsay Bazydlo, one of the researchers who is also director of the University of Virginia School Health System Toxicology Laboratory, said in an interview. A greater number of samples are thus being sent for confirmatory testing by mass spectrometry but, more important, this increase in sample volume comes from samples that were positive and being missed, she added.

Bazydlo noted that the researchers have implemented the process clinically "and our retrospective analysis of that data shows that we have improved the false-negative rate in our clinical practice, also."

William Clarke, director of clinical toxicology at Johns Hopkins Hospital, who was not involved in the JALM study, said in an interview that the researchers' work is notable "because they are taking an evidence-based approach to setting a cutoff, rather than basing it on calibrators or work-based drug testing cutoffs."

However, the method also highlights a challenge related to how clinicians should do this type of drug screening and how immunoassays and mass spectrometers should be used. He noted that the decision to use an immunoassay screen and mass spectrometry as confirmation should depend on the sensitivity and specific details of the application, such as the duration of the drug within a patient and whether the clinician is looking to do pain management or handle an emergency.

Although prescription and illicit opioids continue to cause most overdose-related deaths, overdoses from benzodiazepines, such as Xanax and Valium, also account for a substantial number of prescription drug deaths.

So-called benzos were responsible for 30 percent of prescription overdose deaths in 2013, according to the US Centers for Disease Control and Prevention. They are second only to the abuse of opioids in the number of overdose deaths that they cause.

The American Association for Clinical Chemistry, which publishes JALM, devoted this month's issue to laboratory support of pain management, and said that advances in laboratory drug tests will play a crucial role in helping healthcare providers to ensure access to pain medication for patients who need it without contributing to escalating rates of addiction.

"People have been diverting or selling prescriptions or giving them to family members instead of taking them themselves," Bazyldo said. "That's really the biggest issue."

As a result, urine drug testing is an essential component of treating patients for chronic pain or anxiety and is used to monitor compliance during treatment.

However, testing algorithms for benzodiazepine drugs have tended to focus on minimizing false positives, which is a concern for patients who have been prescribed these drugs and for their physicians.

The researchers noted in their paper that a two-tiered approach for urine drug testing — involving urine immunoassay screening followed by confirmation by mass spectrometry — minimizes the clinical impact of false-positive urine drug sample results and maximizes the impact of false-negative test results, "as positive samples are confirmed and negative samples do not undergo further testing."

"The current algorithm used by many labs was historically established to ensure that someone is not labeled as taking a drug when they are actually not taking it," Bazydlo said. "It used to be that when you did a drug screen, a positive result was always bad. However, today if physicians have prescribed one of these substances, they are looking for the screen to be positive. Minimizing false negatives is becoming much more of a priority." 

Concerns over false negatives in benzodiazepine urine drug screens have been "widely reported in the scientific literature," she noted.

High false-negative rates come from the use of antibodies that being developed for immunoassays that are expected to detect many different benzodiazepine drugs and their metabolites, which is difficult because of varying antigen cross-reactivity, the researchers said. The assay antibodies are also known to have low affinity for the glucuronidated benzodiazepine metabolites that can be abundant in urine samples, they added.

Because only positive specimens generally receive confirmatory testing, false-negative test results are difficult to detect, they said, adding that "the consequences of a false-negative test result can be serious and include patients being wrongfully denied requisite medication and/or not detecting illicit or dangerous drug use."

Bazydlo said that the researchers' quest to improve false-negative rates was driven by a recognition that test results were yielding adverse outcomes for patients. "We knew that false-negative results on benzodiazepine drug screens fall through the cracks and wanted to find a patient-centered solution," she said.

In their study, the researchers investigated multiple approaches to improve the false-negative reporting of benzodiazepine testing by conducting a process called metabolite hydrolysis before screening and optimizing the absorbance cutoff required for reflex confirmatory testing by LC-MS/MS.

They found that the sensitivity and false-negative test rate of benzodiazepine urine testing can be substantially improved, with minimal impact on the number of false-positive test results, by optimizing the absorbance cutoff required for confirmatory testing.

Of the confirmed false-negative test results, 85 percent, or 17 of 20 samples, demonstrated absorbance values between 20 and 100, with 100 being the cutoff for a positive result, the researchers said. Implementing an optimized absorbance cutoff of 20, rather than 100, as part of a reflexive confirmation testing algorithm, decreases the false-negative test rate of detecting benzodiazepine from 47 percent to 2 percent.

Bazydlo noted that their method can be readily implemented in other laboratories that conduct the same kind of screening. "The test numbers for other laboratories may not be exactly the same as ours," she said, "but they may want to repeat some of the experiments we've done, whereby they send out for mass spectrometry testing some samples that were negative by immunoassay screening," and use that process to establish an optimal cutoff threshold that determines when samples need to be sent for confirmatory testing.

Whether to use an immunoassay screen upfront or go directly to using mass spectrometry is still uncertain and depends on the clinical context, Clarke said. "If you are in the pain management population, the question is whether you should be doing the immunoassay screen upfront. I don't think that you necessarily should. I think that you should probably be doing mass spectrometry-based screening first, but you still have to address the issue of what cutoffs do you use."

He said that the researchers "rightly point out in their paper," in the example they use with urine drug screening by immunoassay, that positive results are going to be sent to mass spectrometry and confirmed. In that context, "the goal for an evidence-based cutoff is to minimize the number of false negatives," he said. "They show a really good approach for that."