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The page below is a sample from the LabCE course Drug Testing Methods in the Clinical Toxicology Laboratory. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Immunoassay Cross-Reactivity, continued

False negatives due to low cross-reactivity can also be a problem. In the screening of certain classes of drugs, each drug in that class can display varying degrees of cross-reactivity. For example, an amphetamine screen with a cutoff of 300 ng/mL may show high cross-reactivity for amphetamine but much less for methamphetamine, MDMA, MDA, and MDEA. Let’s suppose amphetamine is present in a urine specimen at 325 ng/mL for amphetamine and 975 ng/mL for MDMA. Because the cross-reactivity of MDMA in this particular immunoassay is 10 times less than that of amphetamine, the result would show that amphetamine is above the cutoff (positive) and MDMA is below the cutoff (negative). MDMA would be reported as negative but would actually be present at a concentration of 975 ng/mL.
The immunoassay for opiates is another assay that demonstrates varying levels of cross-reactivity.
Morphine and codeine elicit a strong response for the opiate immunoassay, but there is lower sensitivity for semisynthetic opioids, such as hydrocodone, hydromorphone, oxycodone, oxymorphone, heroin, and buprenorphine. Because of this cross-reactivity pattern, a negative result does not necessarily mean that the drug was not used. To illustrate further, synthetic opioids such as fentanyl and methadone may not be detected at even very high concentrations. For this reason, specific immunoassays for some of these compounds have been developed. In the screening of opiate class drugs and metabolites, there is some cross-reactivity of all opiates. However, the oxycodone screen is more specific for oxycodone, oxymorphone, and metabolites. In other words, a urine sample with a high concentration of codeine, morphine, hydrocodone, hydromorphone, or heroin in the absence of oxycodone or oxymorphone would yield a positive opiate screen and a negative oxycodone screen. However, a urine sample with a high concentration of oxycodone or oxymorphone in the absence of all other opiates could yield a positive opiate screen, as well as a positive oxycodone screen.
Reagents are being frequently refined to address cross-reactivity issues.
Examples of cross-reacting compounds for certain immunoassay classes of drugs:
  • Opiates - Quinolone antibiotics (eg, levofloxacin, ofloxacin), levorphanol, naloxone
  • Fentanyl - Trazodone
  • Phencyclidine - Venlafaxine, dextromethorphan
  • Methadone - Quetiapine
  • THC- Antiretroviral efavirenz, proton pump inhibitors (eg, pantoprazole)
  • Amphetamine - Diet pills, promethazine, l-methamphetamine, phenmetrazine, selegilene
Different assays will often have different cross-reactivities based on the detection antibodies being used.