False Positive Immunoassay Results

Prescription and OTC medications (or metabolites of these medications) reported to interfere with amphetamine immunoassays include buflomedil, brompheniramine, chloroquine, chlorpromazine, ephedrine, fenfluramine, isometheptene, isoxsuprine, labetalol, mephentermine, mexiletine, N-acetylprocainamide, nylidrin, perazine, phenmetrazine, phentermine, phenylpropanolamine, promethazine, propylhexedrine, pseudoephedrine, quinacrine, ranitidine, ritodrine, tolmetin, trimethobenzamide, and tyramine (1,5,8-18).

The most common cause of false-positive results with amphetamine immunoassays is cross-reactivity with a-hydroxy amine compounds and other sympathomimetic amines found in many OTC drugs. In order to decrease these false-positive incidents and thus decrease the expense and time of confirmation testing, some laboratories adopt a policy of performing a secondary screen of all positive samples following an initial immunoassay test. This secondary screen may consist of utilizing an immunoassay with different cross-reactivity to these compounds produced by a different manufacturer or reanalysis using the same immunoassay following chemical reaction or modification of the assay in some manner. Pretreatment of samples with sodium periodate in a basic solution eliminates interference from ephedrine, pseudoephedrine, and phenyl-propanolamine by oxidative cleavage of the hydroxyl group, and this reaction has been utilized in the EMIT® amphetamine confirmation kit (Dade Behring, San Jose, CA). Another strategy for elimination of false-positive results is the addition of antibody to the target analyte resulting in neutralization of the signal in a true-positive sample but having no effect on the signal from a sample containing high concentrations of cross-reactive substances. In this situation, true positives are distinguished from false positives by the difference in signal before and after addition of the neutralizing antibody. This method was applied to a dual-channel neutralization procedure for amphetamines and used as a secondary screen that was effective in reducing false positives (19). Recently, Woodworth et al. (20) described a procedure utilizing serial dilution testing to distinguish amphetamine/methamphetamine-containing samples from samples containing cross-reacting sympathomimetic amines. Samples diluted 1:1, 1:10, and 1:20 were analyzed, and maximum slope estimates (maximum change in rate over the fractional change in concentration) were determined for each compound using the EMIT® II amphetamine/methamphetamine immunoassay. The authors were able to increase the positive predictive value of the immunoassay using optimal slope cutoffs (determined by receiver operating characteristic (ROC) analysis) to differentiate samples containing amphetamine/methamphetamine from those containing cross-reacting compounds.

The existence of d and l isomers can cause false-positive results when medication containing the l isomer is ingested. The most often mentioned example is that of Vicks® inhaler in which the active ingredient is l-methamphetamine (21). Extensive use of this product could cause false-positive results for immunoassay screening and subsequent confirmation by gas chromatography/mass spectrometry (GC/MS). Tests to differentiate the isomers are discussed in Section 5.2. Although isomer resolution is required to definitively determine isomer composition, several studies have shown no false-positive results when the inhaler was used as directed and only a few false-positive results when the inhaler was used at twice the recommended frequency (22,23). Conversely, other studies have shown that heavy use of the inhaler can result in methamphetamine concentrations as high as 6000ng/mL and concentrations of the metabolite amphetamine 200ng/mL cutoff (24,25).

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