To the Editor.—Immunoassays for prescribed or abused drugs contain reagent antibodies in concentrations designed to demonstrate their antithetical antigens by forming immune complexes. Optimal reactions occur in a “zone of equivalence,” whereas reactions in the “zone of antigen excess” can produce results that indicate less antigen than is actually present. In quantitative immunoassays, a “high dose hook effect” can be demonstrated by showing that increasing drug concentrations produce measured concentrations that first rise and then decline.I encountered a patient whose attempted suicide by benzodiazepine overdose produced such high urine concentrations of the drug and its metabolites that there was a “prozone effect” causing a false-negative qualitative immunoassay.A 58-year-old divorced white woman with familial manic-depression was socially isolated, chronically anxious, anhedonic, and unable to cope with her problems. The patient did not take her prescribed lorazepam regularly, but, at times, ingested it with alcohol despite physician warnings that respiratory arrest could result. Other prescribed medications included lansoprazole for duodenal ulcer disease and olmesartan medoxomil for hypertension.On the night she was brought to the hospital emergency department by her sister, the patient appeared depressed and said that she had ingested twelve 2-mg lorazepam tablets in the past 24 hours. Surprisingly, a screening test for urinary benzodiazepine was negative, as were tests for phencyclidine, cocaine, amphetamines, cannabinoids, opiates, barbiturates, tricyclic antidepressants, and ethanol. The test results were doubted and urine was recollected 4 hours later, but all screening test results remained negative.Serum abnormalities were limited to serum urea nitrogen of 7 mg/dL and potassium of 3.4 mEq/L. Urine specific gravity was 1.030, pH 5.0, glucose negative, ketones trace, and leukocyte esterase 1+. Microscopic examination of urine showed occasional bacteria.In view of the discrepancy between the history of benzodiazepine ingestion and the apparent lack of its urinary excretion, both urine samples were retested by gas chromatography/mass spectrometry. Lorazepam was found positive at a level of more than 20 000 ng/mL in both samples. The initial urine specimen was recovered, diluted, and analyzed in duplicate by the original automated immunoassay (Kinetic Interactive Microparticles in Solution [KIMS], COBAS/INTEGRA, Roche Diagnostics, Indianapolis, Ind). Only the 2 highest dilutions showed positive results (>1000 units) (Table).False-negative results of the KIMS test have not been reported at high drug concentrations. A recent evaluation of the assay (without glucuronidase hydrolysis) produced positive results with standards and patient samples containing benzodiazepine concentrations of up to 5660 ng/mL.1 (Because the glucuronide metabolite is a major component of the benzodiazepines in urine, a modified assay containing glucuronidase has been introduced to increase immunoreactivity. In the past, some false-negatives may have been caused by insufficient native drug.)In a true-negative KIMS test, drug-specific reagent antibodies cause rapid aggregation of drug-bound microparticles. The aggregation is monitored by rapid change in light absorbance. In a true-positive KIMS test,2 antibodies rapidly react with free (patient) drug, but they react more slowly with drug-bound microparticles. The rate of change in light absorbance is decreased in proportion to the drug concentration.False-negative KIMS results in samples containing very high drug levels could occur in several hypothetical ways. Direct, rapid precipitation of immune complexes is possible; the complexes might consist of reagent antibodies and abundant drug or complexes might consist of free drug, reagent antibodies, and microparticles. Another mechanism might involve an undetected “inhibitor” (eg, patient antibody to drug). An inhibitor could change either the expected microparticle aggregation time or its rate, and its effect would be mitigated by urine dilution. Inaccessibility of the patient and insufficient urine samples precluded testing these hypotheses.