How does Canada's approved drug screening equipment work? Are the roadside drug testers reliable? And for what purpose?
Why We Need to Be Cautious:
Ontario 's Track Record Dealing With Immunoassay or ELISA system of detecting drugs:
The best place to start in trying to understand how Canada's new approved drug screening equipment works or doesn't work has to do with the limitations with any immunoassay system for detecting drugs. "Detecting" drugs is a lot different from quantifying drug concentration in the body. Ontario has already had a very bad experience with the use in child protection Court and criminal Court of drug screening results. Terrible miscarriages of justice resulted. The following are multiple excerpts from the Motherisk Inquiry Report. Please consider whether or not these findings of Justice Lang, apply to roadside drug screening. Do drugs and driving prosecutions have an exemption from good science? Or should good science be applied to carefully, in both our criminal law and our provincial law, separate "screening", from "quantitative analysis"? How can Courts construe and apply the new laws in such a way that "penalties" are NOT imposed for a mere screening fail? How do we ensure that Courts do not pay inordinate attention to the results of screening tests? How do we ensure that Canadians are protected against improper search and seizure by police using inadequately maintained and operated screening devices?
Please pay close attention to what Justice Lang has stated about:
18.104.22.168 Immunoassay-Based Screening Tests
43. Immunoassay is a biochemical test that detects the presence of the target compound through the use of antibodies. Radio-immunoassay (RIA) tests were initially used to screen hair samples for drugs of abuse. However, over the past decade, if not longer, most hair-testing laboratories have replaced RIA with enzyme-linked immunosorbent assay (ELISA) tests. ELISA is what MDTL used from 2005 to 2015.
44. ELISA tests are widely used to screen for drugs of abuse. The ELISA test is sensitive and, when appropriately validated, provides a tool for quickly distinguishing between negative samples that do not require any further testing and positive samples that do. The tests are commercially manufactured and can be purchased as “kits” from a variety of suppliers. There are separate ELISA kits for each drug or class of drug. However, there are currently no commercially available ELISA kits for alcohol markers.
45. ELISA kits typically come in the form of a plate containing 96 wells, and each well comes coated with specific antibodies that have binding sites for the target drug. The ELISA test works by adding both the sample being tested and a solution containing an enzyme-linked version of the target drug to one of the wells. Once added, any drugs in the sample will compete with the enzyme-linked drugs for the binding sites on the anti- bodies in the well. A substrate (a compound that reacts with the enzymes that are linked to the drugs) is subsequently added, causing a colour change in the enzyme-linked drugs that have bound to the antibodies – from clear to blue. To stop the assay after a predefined time (called the incubation period), an acid is added, which changes any blue colouring to yellow.
46. The test works by measuring the extent of the colour change (called the optical den- sity or absorbance) after the incubation period: The lower the amount of drugs present in the sample, the more intense the colour change because more enzyme-linked drugs will have bound to the antibodies (having had less competition for the binding sites). Conversely, the more drugs in the sample, the less intense the colour change because more of the drugs in the sample would have competed for and attached to the binding sites. The optical density is measured to assess if the result falls above or below a defined cut-off: If the former, it is considered positive, requiring further testing; if the latter, it is negative, and no further testing is required.
47. Because each ELISA kit comes with 96 wells, the ELISA test can be performed in batches, meaning that the test can be carried out simultaneously on multiple specimens. ELISA tests are therefore an efficient method to screen for drugs of abuse.
48. However, the antibodies in the wells can also bind with other compounds that are structurally related to the target drug, a phenomenon known as “cross-reactivity.” When the antibodies cross-react with structurally related compounds, the test may result in little or no colour change, even in the absence of the target drug or metabolite; in other words, a false positive.
49. Ultimately, the ELISA test is limited in what it can say about a particular sample because it relies on biochemical reactions: It does not separate out the target drug from the other substances contained in the sample. Nor does it identify the target drug in the sample. As a result, although ELISA can be used to measure the extent of the colour change in the solution, it cannot be used to measure the amount of the target drug contained in the sample. For these reasons, ELISA is not used to quantify drugs, but rather to distinguish quickly between samples that do not warrant further testing and those that do.
22.214.171.124 Confirmation Tests
50. Unlike the ELISA test, confirmation tests separate out the different compounds within a sample from one another using a technique called “chromatography.” Then, using a technique called “mass spectrometry,” they identify the separated compounds according to their mass spectral properties.
51. The confirmation tests relevant to the Independent Review are gas chromatography– mass spectrometry (GC-MS) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). The principles underlying both techniques are similar. The sample solution is transported through a hollow column using a gas or liquid. The inside of the column is coated or packed with a chemical material. Because the different compounds within the solution travel through the column at different rates, they can be separated from one another as they interact with the chemical coating or the material inside the column. The process of separation creates what is called a “chromatogram” of peaks and valleys, much the same way that colours of ink can be separated on blotting paper. The size of the peaks can be used to quantify the amount of the separated compounds (the larger the peak, the greater amount of the compound). At the end of the column, the mass spectrometer fragments the separated substances into ions that are further separated according to their molecular weight (and ionic charge) and their relative intensity. The spectral properties of the fragmented ions are then used to identify the substances contained in the sample (akin to a molecular fingerprint).
52. In LC-MS/MS tests, tandem mass spectrometry (MS/MS) is the detection tech- nique used at the end of the chromatographic process. When analyzed by MS/MS, the
fragmented ions are further fragmented to form secondary ions, referred to as “daughter ions” or “transitions.” The spectral properties of the transitions are then analyzed to iden- tify the substances contained in the sample.
53. Because of the manner in which the tests operate, both GC-MS and LC-MS/MS are widely accepted as reliable analytical methods for identifying as well as quantifying the amount of the target compounds present. GC-MS and LC-MS/MS have been called the gold standards for hair testing for drugs of abuse.
54. Another advantage of GC-MS and LC-MS/MS analysis is that, unlike ELISA, the instrument can be used to test for multiple drugs or metabolites at the same time. As a result, some laboratories use GC-MS and LC-MS/MS rather than ELISA to screen for drugs of abuse. Although these techniques are more expensive and less practical for screening large numbers of samples (because they cannot, for example, screen up to 96 samples at a time), they have the advantage of providing simultaneous screening and identification of many drugs in a single analysis.11 Even when chromatographic meth- ods are used as a screening test, however, the recommended best practice is that results should still be confirmed by a second test before being reported as positive. Ideally, the confirmation test will use a different method from the screening test (e.g., use of GC-MS to screen and then LC-MS/MS to confirm).
126.96.36.199 A Note on Terminology
55. I note that forensic toxicologists often refer to positive results from an immuno- assay-based test such as ELISA as a “presumptive positive.” Other terms that are used include “unconfirmed positive” or “tentative positive.”
56. However, it is important to be clear about the terminology used to describe results from an ELISA screening test as opposed to those from a confirmation test. Forensic toxicology requires documentation and terminology that serve the needs of the partici- pants in the justice system. Because any misunderstanding of the meaning of the results could have severe consequences, information provided by forensic toxicologists must be
communicated to the justice system in a way that is accurate, fully comprehensible, and
57. Although, to forensic toxicologists, it is implied and understood that a “presumptive positive” test is preliminary only and must be followed by a confirmation test, I do not think the phrase is sufficiently clear and unambiguous for use in the justice system or by this Independent Review. The phrase “presumptive positive” suggests that the result is positive or at least probably positive unless it has been disproved by a confirmation test. However, such a suggestion would be wrong; the ELISA test produces a preliminary result, not a presumptive one. As a result, for clarity, I will use “preliminary,” “uncon- firmed,” or “tentative” to refer to ELISA test results that have not been confirmed.
58. Similarly, the term “confirmation test” implies that the test is designed to confirm the presence of a drug or metabolite that may be present. A confirmation test does not “con- firm” anything; it determines at first instance if a drug or metabolite is present and, if so, the concentration. Put another way, without a confirmation test there is no identification of the drug in the hair. However, because the science refers to these tests as confirmation tests, I have continued to use that term in this Report.
3 .2 .4 Testing for Alcohol Markers
59. Unlike for drugs of abuse, no immunoassay-based screening tests for alcohol marker testing in hair are commercially available. Hair analysis for FAEEs routinely involves HS-SPME (described above) to extract the FAEEs from the sample before testing using GC-MS or LC-MS/MS. EtG is tested using GC-MS or LC-MS/MS following a process similar to the one used for drugs.
3 .3 Analytical Concepts
60. Several other analytical concepts need to be understood in assessing the adequacy and reliability of a laboratory’s analytical methods.
3 .3 .1 Calibration
61. All analytical instruments need to be calibrated. Calibration involves the testing of several samples that contain known quantities of the target compound (referred to as
“calibrators”). The results from testing the calibrators are plotted on a graph, called a “cali-
bration curve,” which is then used to calculate the amount of identified drug in the sample.
62. The number of calibrators required depends on whether the test is being run for qualitative or quantitative purposes. A qualitative test simply seeks to determine if the test result is positive or negative, while a quantitative test determines the concentration of the target compound present. If testing for qualitative purposes, only two calibrators are required – one blank calibrator to establish the standard for a negative result, and one calibrator with a known quantity of the target compound to establish the standard for a positive result. If testing for quantitative purposes, at least five calibrators are usually needed – from low to high concentrations – in order to plot a sufficiently reliable calibration curve.
3 .3 .2 Quality Controls
63. Laboratories are required to use quality controls to assess if their tests have been run properly. This process typically involves running tests using a negative control (a blank sample that does not contain the target compound) and a positive control (a sample containing a known quantity of the target compound) with every batch. If the test has been run properly, the negative control should produce a negative result, and the positive control should produce a positive result close to the known concentration.
64. It is also common practice for laboratories to test more than one sample at a time, a practice referred to as “batch testing.” Monitoring batch-to-batch performance is import- ant to ensure that the data generated are consistent with one another. Quality control performance across batches is assessed using a Levey-Jennings chart, which plots the dates of the test on the X-axis, and the results for the positive and negative controls on the Y-axis. The chart allows laboratories to identify and investigate statistically significant deviations that may be caused, for example, by errors in or other problems with the calibration.
3 .3 .3 Internal Standards
65. When carrying out GC-MS or LC-MS/MS analysis, laboratories use compounds called “internal standards” to correct for any potential differences in recovery arising from the sample preparation and the extraction process. Internal standards are drugs or metabolites that are almost identical to the target drug or metabolite. The most common
(and recommended) ones are known as “deuterated internal standards” – their molecu- lar makeup is identical to that of the target drug or metabolite, except the hydrogen ion has been replaced by deuterium (also called heavy hydrogen). For example, for cocaine, a deuterated internal standard referred to as “cocaine-d3” is used, in which three deute-rium ions replace the hydrogen ions on the molecule.
66. Internal standards are added to every sample before it is tested using GC-MS or LC-MS/MS. The same concentration of the internal standard is used in every sam-ple. After the test has been run, the laboratory calculates the concentration of the target drug in the sample by comparing the ratio of the drug to the internal standard with the same ratio in the calibrators. In this way, the internal standard corrects for any differences that may arise in the sample preparation and extraction process.
67. Because the GC-MS or LC-MS/MS instrument separates out the compounds in a sample, it can detect the internal standards separate and apart from the target drug or metabolite in the sample in order to carry out these calculations. By contrast, because ELISA responds not only to the target drug or metabolite but also to structurally related compounds (and ELISA tests may therefore cross-react with the internal standards for the target drug), internal standards are not used in tests carried out using ELISA.
3 .3 .4 Cut-offs
68. The way to determine if a test result is positive or negative is by assessing if the result falls above or below a certain cut-off. Two limits can affect the appropriate cut-off for a drug or metabolite:
1. The limit of detection (LOD) is the lowest concentration of the substance that the instrument can reliably detect.
2. The limit of quantification (LOQ) is the lowest concentration of the substance that the instrument can reliably quantify.
69. It is up to individual laboratories to determine, through instrument validation, the LOD and the LOQ for each of the tests they carry out.
70. The SoHT has recommended cut-offs for reporting positive results for drugs of abuse; however, those cut-offs are only guidelines, and it is ultimately up to the laboratory
to develop its own cut-offs for assessing the results that it generates. For example, to elim- inate small positive results that could have occurred through external contamination, some laboratories may choose to adopt a cut-off that is higher than the LOQ of their instrument. Other laboratories may adopt the LOQ as their cut-off and report the con- centration of any sample that is higher than the LOQ.
71. For alcohol markers only, the SoHT has developed cut-offs on alcohol use. These cut- offs, which are unrelated to an instrument’s cut-off, are described below.
3 .4 Quality Management System
72. In addition to running quality controls with each batch to ensure that the specific test has been properly carried out, laboratories should have a quality management system, which refers to the overarching processes that exist to ensure quality in the results that the laboratory reports. Core components of a quality management system include, at a minimum, documentation of the laboratory’s policies and procedures – among them, its analytical methods – in standard operating procedures. As part of the quality man- agement system, laboratories should also have a system in place to identify and correct errors in the testing process.
Scientific Studies on Drug Screening Equipment:
Douglas J. Beirness & D'Arcy R. Smith (2017) An assessment of oral fluid drug screening devices, Canadian Society of Forensic Science Journal, 50:2, 55-63, DOI: 10.1080/00085030.2017.1258212