An Article by INSTAND EQAS/PT Expert Drug Screening and Pharmaceuticals Prof. Dr. med. Werner Steimer
INSTAND has long offered a range of well-establishedEQAS/PT for drug analysis, particularly in urine. Depending on the reported results, these interlaboratory tests are evaluated qualitatively and/or quantitatively. One might think that qualitative interlaboratory tests, in particular, are easy to evaluate and interpret, following the principle that positive is positive and negative is negative. However, this is a fallacy or a dangerous oversimplification.
For simplicity—lower costs, faster turnaround, and easier handling with minimal staff involvement—many laboratories prefer immunological assays over more complex chromatographic assays. These assays, quite intentionally but also partly unavoidably, detect not only individual substances but also many different substances within entire substance groups, e.g., barbiturates or benzodiazepines, and cross-reactivity can vary among immunoassays or diagnostic antibodies. The specified cutoff values typically refer only to the substance used for calibration. In contrast, the sensitivity of other substances in the same group may differ, making the cutoff value inaccurate for those substances. Another difficulty with barbiturates is the fact that, for historical reasons, secobarbital is regularly used as the calibration substance—a substance that is no longer used as a drug today. This means that a cut-off value specified for secobarbital would not be truly accurate for any other barbiturate, unless that barbiturate were bound by the antibody in exactly the same way as secobarbital.
Conducting meaningful EQAS/PT is extremely difficult under these circumstances, as there are a large number of different assays on the market, nearly all of which have varying detection sensitivities for the various specific substances that can, in principle, be detected using these assays.
Several years ago, INSTAND and the RfB, together with two toxicological professional associations, therefore attempted to propose clinically meaningful cutoff values for RiliBäk to the German Medical Association, to use these uniformly as a basis for evaluating all assays in the interlaboratory test. The relevant committees of the German Medical Association rejected this and proposed a consensus meeting among the major suppliers, INSTAND, and the RfB. Unfortunately, no consensus could be reached at this meeting, as any agreement would have required some manufacturers to redesign, validate, and re-approve their assays.
Since, to the best of our knowledge, all assays are legally available on the market and there were and are no binding requirements regarding the necessary sensitivity of such assays, INSTAND decided at the time to focus the interlaboratory tests primarily on verifying whether each assay meets its own performance claims.
To this end, INSTAND informs all participants before the EQAS/PT which substances are added to the samples in defined concentrations that are unknown to the submitter. To assess whether a specific assay should yield a positive or negative result at a given concentration, the cut-off value of the assay used for the substance added by INSTAND is queried in addition to the result. Since the substances used by various manufacturers to calibrate the assays often do not match the substances added by INSTAND, the cutoff value for these substances, if available, must either be taken from the assay’s package insert or converted to the substance added in the interlaboratory test based on the specified cross-reactivity. Although this step is clearly described in the interlaboratory test documentation, it often causes difficulties and, if not followed, can result in failure of the interlaboratory test. Without the cut-off value, your result cannot be evaluated. In such cases, only a certificate of participation will be issued.
Of course, qualitative assays also exhibit analytical imprecision. To account for this, results may be classified as “questionable positive,” which is considered correct if the assay’s cut-off value falls within a range of +/- 45% of the selected weight in the interlaboratory test sample.
The wide variety of assays available on the market, with different cut-off values, also makes it difficult to choose appropriate concentrations of substances to add to the samples. Since imprecision is also to be expected in qualitative assays, concentrations at or near cut-off values should be avoided. This would lead to a high number of false results solely due to inherent, unavoidable variability. However, the large number of commercially available assays with different cut-off values means that, in individual cases, it can be difficult, or even impossible, to determine suitable concentrations sufficiently far removed from all cut-off values, at least when using samples capable of testing the performance of various assays.
In everyday clinical practice, therefore, a positive result may be interpreted very differently depending on the assay used. While screening assays are intended only to raise initial suspicion, the general recommendation is to order a confirmatory test if the result is positive. However, such confirmatory testing is usually only available at specialized centers and involves a significant time delay. It may therefore be clinically necessary to make decisions before the results are received. It is therefore essential to have a thorough understanding of one’s own assay and its interpretive value, including concerning different substances, which, as described above, is also required to pass the proficiency testing program.
Establishing minimum standards for drug screening assays through appropriate legal requirements, e.g., within the IVDR framework, would make sense. These could then serve as the basis for a corresponding assessment in the proficiency testing program and allow comparison of various assays available on the market. Until then, I fear that, due to economic considerations, nothing will change regarding the—in this case—rather problematic diversity of different assays.
I hope I have motivated you to become more familiar with your drug screening assay, so you are better equipped to interpret results accurately in everyday clinical practice.
If you have any questions or suggestions, please feel free to contact me at any time.
Prof.Dr. Werner SteimerINSTAND
INSTAND EQAS/PT Expert Drug Screening and Pharmaceuticals