Moving beyond immunoassays for poisoned patients

Amy Carpenter Aquino

April 2020—Bypassing immunoassays as the first step in toxicology testing can minimize clinician calls to the laboratory about negative toxicology reports for apparently overdosed patients and save diagnostic time.

“Immunoassay is not the right approach to monitor the compliance of the patient with benzodiazepines and opioids,” because it may not detect all drugs in a class due to poor cross-reactivity, Amitava Dasgupta, PhD, DABCC, professor of pathology and laboratory medicine, University of Texas McGovern Medical School, said in a session at last year’s AACC annual meeting. He presented several cases illustrating the limitations of immunoassays in toxicology screening.

Amphetamine immunoassays suffer from interferences with over-the-counter cold medications and poor cross-reactivity with amphetamine-like designer drugs, which cannot be detected until the concentration reaches a level of 20,000 ng/mL—“a severe overdose,” he said. And patients poisoned with novel psychoactive substances also show negative toxicology reports.

Dr. Dasgupta’s co-presenter was Kara Lynch, PhD, DABCC, co-chief of the core laboratory at Zuckerberg San Francisco General Hospital and associate professor of laboratory medicine at the University of California, San Francisco. Dr. Lynch talked about testing strategies beyond immunoassays and how her laboratory uses high-resolution mass spectrometry with a quadrupole time-of-flight instrument to crack its toughest toxicology cases.

In one case Dr. Dasgupta presented, a physician called the lab because an initial drug screen for a possible overdose patient was negative for opiates but a second screen performed hours later was positive. The clinician had prescribed naloxone after the initial screen, but it did not reverse the patient’s overdose symptoms, indicating they were not opioid-related. The patient was a known marijuana abuser, so the clinician was also surprised to see a negative THC result.

“A sample was sent to a reference laboratory, which confirmed the presence of naloxone and JWH-073, a common designer synthetic cannabinoid,” he said. The positive result in the second urine screen was due to the naloxone, which is an opioid and cross-reacts with opiate immunoassays at high concentrations. “Any patient who gets naloxone will test positive.”

Another patient known to take tramadol was admitted to the emergency department in November with a drug overdose. The patient’s initial drug screen result was negative for opiates but positive for phencyclidine (PCP). “That confused the clinician because the patient said he took too much tramadol,” for which the patient was given and responded to naloxone therapy. Confirmation tests were negative for PCP and positive for tramadol. Why was the PCP positive?

“I usually see PCP-positive results in January, February, and March because of dextromethorphan,” a cough suppressant, Dr. Dasgupta said. But this case was in November. “We learned that tramadol can also cause a false-positive PCP result.”

He referred to a case in the literature in which a 42-year-old man died at home (Hull MJ, et al. Am J Forensic Med Pathol. 2006;27[4]:359–362). The man’s postmortem urine drug screen was positive for PCP with the Syva Emit II Plus phencyclidine assay, and toxicology analysis showed a tramadol level of 14 mg/L, “which is very high because therapeutic level is 100 to 300 ng/L or 0.1 to 0.3 mg/L,” Dr. Dasgupta said. “But there is no PCP confirmed, so the authors speculated that tramadol was causing this false-positive PCP level.”

Dr. Dasgupta shared the finding of an Australian study on cross-reactivity in the CEDIA buprenorphine immunoassay by opiates (Saleem M, et al. Ann Clin Biochem. 2017;54[6]:707–711). “Is the opioid buprenorphine cross-reacting with the opioid immunoassay?” he said. “When you use this assay at a 5 µg/L cutoff, do you need to do any opiate testing?”

The authors found that cross-reactivity in the CEDIA buprenorphine immunoassay by opiates at concentrations less than 2,000 µg/L will not cause a false-positive buprenorphine result. “The HHS increased the cutoff level from 300 to 2,000 in 1998,” he said. “However, some private employers still use 300.”

In another case, a patient who presented to the ED in an overdosed state was given naloxone and later died. “When he was still alive, we did the opiate screen using ELISA mass spec and we didn’t find anything,” Dr. Dasgupta said. “But it was an opiate-related death, so the case went to the medical examiner’s office where they have a much broader screen.” The forensic test results showed that the patient had taken the over-the-counter antidiarrheal drug loperamide. The suggested maximum dose is four tablets. “When the family members showed up, we found that he probably took 30 to 50 tablets to get high.”

Loperamide is an opioid with poor bioavailability but when taken in excess, it crosses the blood-brain barrier and produces euphoria (Eggleston W, et al. Ann Emerg Med. 2017;69[1]:83–86). “If it goes to the bloodstream, it’s very potent, as potent as fentanyl,” Dr. Dasgupta said, calling the drug “the poor man’s methadone.” The therapeutic level is 0.24 to 3.1 ng/mL. Death from cardiac dysrhythmia has been reported in patients with loperamide levels as low as 77 ng/mL and as high as 140 ng/mL.

In one of two cases that illustrate the problems in detecting benzodiazepine by immunoassay, a clinician complained to the laboratory when a patient on lorazepam had a negative toxicology screen. GC-MS confirmed lorazepam at a level of 1,566 ng/mL; the EMIT (enzyme multiplied immunoassay technique) cutoff for lorazepam is 600 ng/mL. What went wrong?

“We thought that most of the lorazepam was conjugated with glucuronic acid, and when a benzodiazepine is conjugated, it has a much lower cross-reactivity” with the immunoassay, Dr. Dasgupta said.

He and his colleagues tested a theory that the EMIT benzodiazepine immunoassay sensitivity could be increased. “We incubated urine with beta-glucuronidase to break up the glucuronic acid conjugate,” he said (Dixon RB, et al. Ther Drug Monit. 2015;37[1]:137–139).

Thirty-one urine specimens collected from patients taking benzodiazepines were treated with beta-glucuronidase for two hours at 47°C, and then tested with the EMIT benzodiazepine immunoassay on the Vista 1500 analyzer (Siemens Healthineers) and with an LC-MS/MS assay. All 31 specimens showed benzodiazepine levels above the 200 ng/mL cutoff concentration by LC-MS/MS, but 11 of the 31 specimens showed a negative response by the EMIT, for a false-negative test result rate of 35.5 percent.

“Even if you break the conjugated compound and you are using the benzodiazepine immunoassay, it’s not adequate for monitoring compliance,” Dr. Dasgupta said. He advises labs to tell clinicians who want to test for compliance to skip the benzodiazepine screen and go right to the confirmation assay.

In another case, a 58-year-old woman was brought to the ED after she attempted suicide by taking 12 2-mg lorazepam tablets. Her urine benzodiazepine test result was negative using an immunoassay with a cutoff of 200 ng/mL. The clinician doubted the result and ordered a second urine benzodiazepine screen 14 hours later, which was also negative. Both urine samples were then tested by GC-MS, and those results showed a benzodiazepine level of more than 20,000 ng/mL (Wenk RE. Arch Pathol Lab Med. 2006;130[11]:1600–1601).

“This is a prozone or hook effect,” Dr. Dasgupta said. “There was so much benzodiazepine that it oversaturated the binding site, causing a false-negative result. We know that the hook effect causes falsely lower values with sandwich assays for big molecules such as hCG. This is the only case I know where a small molecule like benzodiazepine gave a falsely low value due to hook effect.”

Another case highlighted the challenges presented by synthetic cannabinoids, which standard drug screens don’t detect (Bassir Nia A, et al. J Psychopharmacol. 2016;30[12]:1321–1330). The patient was admitted to the ED with a suspected overdose, but the toxicology report was negative for all drugs except the inactive cannabis metabolite THC-COOH. “The clinical picture indicated a life-threatening overdose not consistent with THC-COOH because the THC value [55 ng/mL] was very low,” Dr. Dasgupta said. “We thought it was maybe a synthetic cannabinoid.”

The clinician ordered a synthetic cannabinoid panel (sent to a reference laboratory), and the patient had a positive result for JWH-018, one of several types of synthetic cannabinoid compounds (Yeruva RR, et al. Innov Clin Neurosci. 2019;16[1–2]:31–32).

“Synthetic cannabinoids account for 39 percent of all psychoactive drugs, and there are more than 177 compounds reported,” Dr. Dasgupta said, noting they’re far more dangerous than marijuana. “It’s a nightmare.”

Several companies offer synthetic cannabinoid immunoassays, Dr. Dasgupta said, but the problem is that they detect the most common synthetic cannabinoids. “They cannot detect everything, so you need an LC-MS/MS assay. And the limitation of developing an LC-MS/MS assay is the lack of a pure standard.”

Bath salts, too, present a problem. Many fatalities have been associated with overdoses of synthetic cathinone, of which bath salts are a synthetic derivative. They cannot be detected with amphetamine/methamphetamine immunoassays, Dr. Dasgupta said.

Randox Toxicology was the first company to offer an immunoassay to detect bath salts in urine. Its Drugs of Abuse V Biochip Array assay has two synthetic cathinone antibodies: Bath Salt I targets mephedrone/methcathinone, and Bath Salt II targets 3’,4’-methylenedioxypyrovalerone (MDPV)/3’,4’-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP).

“You cannot detect everything,” Dr. Dasgupta said, “but at least you can detect the most commonly abused bath salts and synthetic cannabinoids.”

At Zuckerberg San Francisco General Hospital, to solve the most challenging toxicology cases, the laboratory uses liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) on a quadrupole time-of-flight mass spectrometry (QTOF-MS) system. The laboratory serves as a regional toxicology laboratory, taking on difficult cases from other Bay area hospitals referred through the Northern California Poison Control Center.