Cutoff Concentrations and Immunoassay Evaluations

Because a cutoff is the concentration of drug below which all specimens are considered to be negative, the cutoff decision has a direct impact on the detection time window and the positive rate. The most commonly used method for immunoassay performance comparisons is to evaluate the so-called true-positive (TP), true-negative (TN), false-positive (FP), and false-negative (FN) of the assay. These results can then be used to calculate the specificity [TN / (TN + FP)] x 100%, sensitivity [TP / (TP + FN)] x 100%, efficiency [(TP + TN) / (TN + FP + TP + FN)] x 100%, or positive or negative predictive values of the assay. Because the criteria for either true or false are based on the comparison of immunoassay and GC/MS interpretation at their respective screening and confirmation cutoff levels, the goals and strategies for balancing the relative performance around the selected cutoff concentrations are among the important considerations for designing an immunoassay for cannabinoid testing.

Traditionally, the cutoff decision can be made by considering the assay limit of detection or a predefined, higher concentration. Although not generally inferred in the context of drug testing, cutoff sometimes is used to refer to the analyte concentration at which repeated tests on the same sample yield positive results 50% of the time and negative results for the other 50%. In a near-cutoff zone as concentrations close to the cutoff value, some results may be positive or negative for different analytical methodologies or for repeated testings using the same method. For most drug-testing programs, the "administrative cutoffs" were chosen with the consideration that the cutoff is sufficiently above the assay limit of detection, yet low enough to allow the detection of drug use within a reasonable time frame (90,91). One of the earlier concerns in setting the immunoassay cutoff for cannabinoids was the risk of falsely identifying urine samples as positive for individuals exposed to passive marijuana smoke. Nonetheless, further studies on passive inhalation have led to the conclusion that the levels of cannabinoids in the body from passive inhalation would not be enough to cause urine specimens from a non-marijuana user to test positive using a screen cutoff concentration of 50 ng/mL (72,106,107).

Several studies have since demonstrated that higher positive rates for marijuana detection were achieved by lowering the initial testing cutoff in urine (100-105). The sensitivity vs specificity tradeoff also reflects the fact that the target analyte specificity is related to the detection rate of cannabinoid immunoassays, especially for samples that contain THC-COOH concentrations between the mandated GC/MS cutoff and the mandated (or chosen) immunoassay cutoff levels (100-105,108-110).

Luzzi et al. (111) investigated analytical performance of drug detection below the SAMHSA cutoffs and showed that the accuracy of urine drug-screening results between the SAMHSA-specified cutoffs and the precision-based cutoffs was less than the accuracy for specimens above the SAMHSA cutoffs. The use of the precision-based cutoff for clinical drug testing increased both the number of screen-positive specimens and the detection of specimens that yielded positive results on confirmatory testing. However, the confirmatory rates for subcutoff-positive specimens were lower than for specimens screened positive at cutoff. When choosing 35 ng/mL as the subcutoff for EMIT screening, 90% of the subcutoff-positive THC specimens contained THC-COOH by GC/MS analysis. Similarly, Hattab et al. (112) stated that the immunoassay cutoff could be further lowered for detecting maternal and neonatal drug exposure. Using the lower thresholds, drugs were detected in 4-5% of the subjects that had screened negative at the conventional threshold concentrations. GC/MS analysis confirmed the presence of cannabinoids in 74% of urine specimens that rescreened positive at a lower cutoff.

The target ranges of cutoff concentrations for alternative specimen testing are significantly lower than those for urine drug testing. The application of alternative specimens for drug testing is still an evolving field, and there have been ongoing discussions and studies over recent years (23,27-29,42,45,113-122). In a prevalence study that compared positivity rates of oral fluid test results with urine test results for different drugs, the screening and confirmation cutoff concentrations selected for oral fluid cannabinoids testing were 3 and 1.5 ng/mL, respectively (27). The overall confirmed-positive prevalence rate for oral fluid testing at these cutoff concentrations was 3.2%. In comparison, the confirmed-positive prevalence rates for urine testing using 50 and 15 ng/mL as the respective screening and confirmation cutoffs were 1.7% for federally mandated urine testing and 3.2% for private sector workplace testing.

With the low cutoff concentrations for oral fluid cannabinoid screening and confirmation, oral fluid testing also has the potential to produce positive results from passive cannabis smoke exposure. In a controlled dosing study, Niedbala et al. reported that two individuals who were passively exposed to the smoke from 10 cannabis cigarettes produced positive screening results, which failed to test positive by GC/MS/MS

(27). In a subsequent study with five cannabis smokers and four passive subjects, the authors observed a biphasic pattern of decline for THC in oral fluid specimens collected from active smokers, whereas the pattern of THC decline was linear in specimens collected from passive subjects (28). The authors concluded that the risk of positive oral fluid tests from passive inhalation is limited to a period of approx 30 minutes following smoke exposure.

In the latest version of the Proposed SAMHSA Guidelines (91), the following cutoff concentrations are recommended for detecting cannabis abuse:

1. Initial tests:

a. 1 pg marijuana metabolite/mg hair sample.

b. 4 ng marijuana metabolite/sweat patch.

c. 4 ng "THC parent drug and metabolites"/mL oral fluid specimen.

d. 50 ng "THC metabolites"/mL urine specimen.

2. Confirmation:

a. 0.05 pg THC-COOH/mg hair sample.

b. 1 ng THC parent drug/sweat patch.

c. 2 ng THC parent drug/mL oral fluid specimen.

d. 15 ng THC-COOH/ mL urine specimen.

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