Cutoff Ranges and Result Interpretation

Even though, a lot of test panels depend on the program they are being applied to, most commonly used are the 5 paneled tests which are according to the federal mandate. (See Table 2.3) those panels that use additional 2-5 drugs which are available are indicated in Table 2.4. The type of assay system incorporated by a laboratory depends on many factors such as the specificity and sensitivity of the assay, the ease of use, format, volume of specimens used, cost and the equipment available. Many dealers and types of assay system are available to choose from.

The administrative concentration cutoff range in the workplace drugs-of-abuse testing program have been changing over a period of time. These depend on the technology available, as well as what is clinically important for the application. The performance traits of the reagents are very important wile considering in formulating thresholds to make decisions and run the program efficiently. For instance, the cross reactivity of target compounds such as codeine, morphine, amphetamine, methamphetamine, etc may cause the assay’s cutoff to range higher than the confirmatory cutoff so that there is more correlation between the two methods. More so, in many cases, the thresholds are set such that they minimize the issues that impact result interpretation. For instance, the initial cutoff which was established for opiates in these programs was set up to 300 ng/mL which was revised to 2000 ng/ml in the year 1994 to lower the number of positive results that had lower morphine concentration due to ingestion of poppy seeds in certain foodstuff. The DOT and NRC formulates rules and regulations for the workplace drug testing programs that standardize the cutoff ranges so that the testing is conducted according to the given guidelines. Some states tend to establish their own threshold and criteria to conduct testing. Many corporate policies have emerged leading way to drug-testing policies which establish and negotiate with work groups to dictate and set cutoff ranges. Manufacturers have test kits which match conventional cutoffs. These assays kits are used in a semi quantitative mode to give more flexibility while selecting cutoffs. Having the laboratory perspective, the accuracy of the test should be determined around the cutoff and monitored routinely along with using standard cutoff so that testing is done on more practical terms.

Since it is a standard that confirmatory tests are deemed necessary after the initial screening results arrive, the initial results are basically used to find whether the extra confirmatory tests are needed or not. The final results are interpreted in the laboratory by certified scientists who include consideration of initial and confirmed test to ensure consistency and accuracy. To make appropriate decisions, it is essential that a certified scientist fully understands the assay’s performance. Some important considerations for common assay tests are summarized below:

Amphetamines

Immunoassays are now made in such a way that they are able to detect more than one drugs of this class of compounds. More focus is on amphetamines, followed by methamphetamine while limiting their cross reactivity to other compounds such as amines and isomers of amphetamines. It is also shown in figure 2.10 that the structure of amphetamines is similar to many other compounds that lie in the class of amine drugs which are commonly available as OTC drugs. Even though immunoassay reagents are modified to increase their focus and specification to amphetamine or methamphetamine, it can be seen from the diagrams why their structural similarity has been such a problem.

Comparative information has been provided in Table 2.5 related to the selected amphetamine reagents. The cross reactivities which are documented can be different from each other depending on their test optimization and according to the dealers. Even though the reagents such as Microgenics and Siemens can be made such that they have equal cross reactivity to amphetamine and methamphetamines, the reagents made by Abbott and Roche are more specific to their drugs having minimal cross reactivity with methamphetamines. Since amphetamine is there in urine, due to amphetamine usage and as a by-product of methamphetamine usage, its lacking or cross reactivity to methamphetamine doesn’t impact the capability of amphetamine specific reagents to be able to detect the usage of methamphetamine. Even though some cross reactivity to OTC drugs is still prominent in Siemens, Roche and Microgenics reagents, the ones made by Abbott are however comparatively more efficient to eliminate any cross reactivity that occurs due to the addition of buffer having sodium per iodate before the assay reagents are added. This removes β-hydroxyamine compounds which can cause cross reactivity. The AxSYM FPIA reagents are used as secondary screen in order to further decrease the OTC interference and to improve specificity so that the number of specimen which are uselessly submitted for confirmation tests can be reduced.

Many drugs including methamphetamines exist in the form of chiral isomers having ‘l’ and ‘d’ forms to show different specifications and actions. The d-isomer of this drug has central nervous system activity which is controlled and is likely to be sought out for while doing the drug testing programs. The l-isomers have peripheral effects and are not controlled substances. Many assay kits are still cross reactive to l-isomers even though the degree of their activity can vary according to different kits. (See Table 2.5). The l-isomers are usually more associated with the use of Nasal inhalers found OTC or due to the drug Selegiline® which is normally prescription. This drug can also be present due to the presence of illicitly used methamphetamine. Therefore, it is important that confirmatory tests are performed to identify the isomers of amphetamine in order to interpret the results properly. Even though, currently, many positive results have been obtained, the l-isomer of methamphetamines has been quite low comparatively. Recently, Selegiline®, used for adult depression is causing increasing incidence of l-isomers in the drug-related testing programs at workplaces.

Cross reactions to amphetamine with the designer drug MDA – MethyleneDioxyAmphetamine and MDMA – MethyleneDioxyMethAmphetamine in the assay kits also differs according to different formulations. If the laboratory is conducting tests for people where the designer drugs are prevalent, then both Siemens (Emit) and Microgenics (CEDIA,DRI) have made kits which are specific for MDMA, also commonly called as ‘Ecstasy’.

Lastly, another polyclonal amphetamine test kit is available to be used to detect a broader spectrum of compounds in the class of sympathomimetic drugs. Even though the detection of these drugs is not really important in the workplace drug testing, it is used commonly in some important clinical uses where it is needed to identify broader aspects of cross reactivity. Therefore, even vast aspects of cross reactivity help to better understand the results of amphetamine immunoassays.

Barbiturates

Even though, short term barbiturates are broadly metabolized to convert into inactive compounds, the therapeutic doses of the drugs cause sufficient quantity of the parent drugs in urine to be detected in standard assays. Not being embraced in the panel of federal drug testing program, Barbiturates are however commonly tested in the non-regulated panels. Many commercial assays optimize with secobarbital, and have different reactivities with other barbiturates (Table 2.6). No important cross reactions that are unexpected have been reported and the results are easily interpreted.

Benzodiazepines

Commonly prescribed in US, the drugs Benzodiazepines come in the urine as hydroxylated and N-dealkylated by-products, mainly as glucorunide conjugates. The ring substituent materials and metabolites are diverse and it is highly difficult to detect all the present compounds which are there in the class. This is also quite challenging for confirmatory tests. The immunoassays, therefore, are mostly oriented towards common drug identification within that class. It is also well acknowledged that benzodiazepines also share the same end products which give enough specificity to be detected in workplaces. (See Table 2.7). Some of the low dose, esoteric benzodiazepines include ‘date rape’ drug flunitrazepam which need to be checked for cross reactivity under a different profile.

The false negative and false positive tests can be problematic for benzodiazepines. Cross reaction with the antidepressant Sertraline and anti-inflammatory Oxaprozin can be misleading. If the drugs show limited cross reactivity, they can result in false negative results. Sometimes, glucuronidase is added to enhance the detection of these drugs.

Cannabinoids

The detection of cannabinoids in the immunoassays of urine testing is focused to detect 11-carboxy-THC compound as only minute quantities of un reacted THC compound are there in the matrix. Therefore, it is important to know that the parent and the metabolite ratio along with the individual ratio varies according to the matrix tested and those immunoassays which are particular to different types of matrices are needed to detect maximum amounts of the compounds efficiently.

Moreover, other than 11-OH-carboxy-THC and other amounts of parent drugs, the THC along with some other acidic metabolites are there in the urine after using marijuana. Even though the assays are focused to trace primary compound i.e. 11-carboxy-THC, the assay kits have enough cross reactivity to the metabolite so that it is easily detected in the testing. Table 2.8 shows cross reactivity studies on the cannabinoid reagents.

Even though it is not listed in the inserts of the assay kits, all the assays show cross reactivity to cannabinoid compounds which can cause a resultant screening of 50 ng/mL with the carboxy concentration being less than 50 ng/mL. This shows the basis for having cutoff lower than 50 ng/mL for the GC-MS confirmatory tests which show different specifications in the two tests. It is a complicated procedure to interpret the test results of THC metabolite as the drug is also acquired via passive inhaling and consuming THC contained foods or supplements. It can also be consumed via using antiemetic drugs which are legally prescribed such as Nabilone (Cesamet) or Dronabinol (Marinol). The initial assay tests in this condition would be resulted as positive for cannabinoid presence. However, cutoff ranges are set to decrease the presence of ‘passive inhaling positive’ cannabinoids. Therefore, the initial assay cutoff is 50 ng/mL where as the confirmatory test cutoff is 15 ng/mL which tends to decrease the incidence of passive inhalation appearance of cannabinoids.Interferences which are not specific are also identified in some reagents such as Gen I Online® which were formulated from anti-HIV drug Efavirenz (Sustiva®) and some proton pump inhibitors such as pantoprazole and omeprazole. Gen II Online® reagents are the newer reagents that have solved the problems which appeared with Gen I.

Cocaine

Cocaine is the drug which is quickly metabolized to form compounds such as Benzoylecgonine (BE) and ecgonine methyl ester which are detected in the urine assays after the usage of this drug. Therefore, many assays are targeted to detect BE, not Cocaine other than CEDIA reagents which show limited cross reactions with cocaine. While testing for other matrices in which the Cocaine concentration might be significant or where the detection of cocaine is needed, then immunoassays having alternate specifications should be used.

On the other hand, commercial assays to detect metabolite of cocaine as highly specific, there have not been any undesirable cross reactions. The concentration of Benzoylecgonine in urine varies dramatically. However, the relation between the initial immunoassay test and the GC-MS confirmatory test is often quite excellent given the assay range. Many commercial assays have therefore; been quite effective to be used as indicators to meet the dilution needs for GC-MS tests.

The data of cocaine cross reactivity is given in Table 2.9 below:

Methadone

Methadone has been newly introduced in the US drug market, with its use increasing as a treatment to opiate addiction and it was uncommon to detect methadone in routine tests. However, the use of methadone to treat and manage chronic pain has increased and so, nowadays, the general population is showing increasing prevalence of this drug.

Methadone and its metabolites EMDP (2-ethyl-5-methyl-3,3-diphenylpyrrolin) and EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) are present in the urine sample after ingesting methadone, with the relative amounts being affected by the pH of urine. Many commercial assays are maximized to detect the parent compound, showing no cross reactivity with the metabolites. This is depicted clearly in Table 2.10 below. However, in normal cases, this mode is acceptable, where as the individuals who are monitored for compliance with methadone treatment programs might need an assay test with metabolite specificity, which is available via Microgenics for both CEDIA® and DRI® techniques. These techniques are used in situations where drug diversion is of concern. However, it is also useful in circumstances where lower doses are required to ensure conformity.

Many different compounds are identified as interferences to methadone assays over quite some time, such as doxylamine and diphenhydramine. Moreover, some package inserts also denote the cross reactivity of these assays to amitriptyline and chlorpromazine. Therefore, in this regard, many laboratories show individual experiences which depend on the occurrence of significantly interfering compounds found in donors.

Methaqualone

A sedative hypnotic drug, notoriously known for its abuse, Methaqualone was used frequently in the US in the 1970s but removed from the market from 1984 onwards. Even though, it is not frequently found while testing for workplace drug abuse, but many panels still include to test this drug. In some conditions, these programs are included in the contract which might be difficult to change whereas in some cases, employers do not tend to change long standing procedures. Methaqualone assay tests equally cross react with the parent drugs and their primary metabolites. So far, no significant cross reactions to unrelated or structurally similar drugs have been noted.

Opiates

Present day assays are targeted to detect morphine along with cross reactivity to other opiate compounds such as hydrocodone, codeine, hydromorphone and heroin compound known as 6-acetylmorphine. Table 2.11 shows the cross reactions of this class of drugs. Many of these drugs are excreted in the form of glucuronide conjugates, therefore, cross reaction to these conjugates are also noted. There are some cross reactions which are identified outside the opiate groups. However, confirmatory rates of opiate assays often suffer, due to limitations in the breadth of compounds which is monitored in the confirmatory tests and the application of administrative cutoffs at concentrations which are identical to the initial assay tests. However, a problem that was faced earlier in the drug testing regime was the possibility of positive results of morphine due to the ingestion of poppy seeds found in different foods. Poppy seeds normally contain codeine and morphine, depending on the amounts of commercial seeds. Studies have been conducted to monitor the quantity of morphine and codeine in the urine samples after the consumption of foodstuffs having poppy seeds. These assays result in positive initial and confirmatory GC-MS tests, making them more challenging to interpret the positive results for workplace programs. Normally, the concentration found is moderate, it is usually seen that the impact of positive results due to poppy seeds is minimized by increasing the cutoff from 300 ng/mL to 2000 ng/mL. This standard cutoff is needed in many federal guidelines as some employers adopt a higher cutoff too. However, raising the cutoffs might not be suitable in all the situations and so, laboratories need to maintain multiple threshold of testing to suit different clients.

The cross reactions of opiate tests are limited as compared to synthetic opoids such as oxymorphone, oxycodone and meperidine. Therefore, if the detection of these and other esoteric opioids is needed, then many specific assays are available (Eg: Microgenics DRI® Oxycodone Assay).

The reaction of opiates is given in Table 2.11 below.

Phencyclidine

Although the prevalence of PCP in the US in comparatively low, the testing of PCP is normally utilized in drug test panels, as it is available as veterinary tranquilizer and its abuse potential along with its history is very well known in a majority of workplace drug testing programs. The immunoassays target to identify the parent drug long with fewer cross reactions which are identified and shown in Table 2.12. Therefore, the confirmatory rate for PCP assays is quite low, which indicates that nonspecific cross reactions can be problematic. Literature shows that cross reaction can occur from thioridazine, venlafaxine and diphenhydramine which are not re-confirmed using other techniques by many laboratories. Some geographic ‘pockets’ show that PCP is being used in the US. For instance, the Department of Justice shows that PCP is being produced and distributed limitedly but primarily in South California. New England and Mid Atlantic States such as Pennsylvania, Washington DC, New York and Virginia show that PCP is being distributed there and many laboratories which receive samples can more likely identify positive PCP results.

Propoxyphene

A lot of non-regulated tests drug tests include Propoxyphene. Many commercial assays are focused to detect this parent drug as well as having cross reactions to its metabolites such as norpropoxyphene which are present in the urine after usage. This is depicted clearly in Table 2.13. Many commercial reagent kits exhibit cross reaction with Propoxyphene metabolites. Even though, many dealers include these reagents while defining their portfolios. However, Abbott’s AxSYM® doesn’t have Propoxyphene detecting kit.

Table 2.13:

Other Miscellaneous Tests

Many drug testing panels include most of the drugs which are mentioned above. However, some recent assay tests need to be mentioned. These assays test to detect esoteric compounds which are not normally found in majority of the population. However, they occur with a greater frequency in some special populations that undergo pain management, substance abuse management or are medical professionals. These special populations might be subjected to employment related tests or compliance monitoring. Therefore, the new reagents include tests for narcotic analgesics such as fentanyl, meperidine and tramadol and some segative hypnotic drugs such as meprobamate, carisoprodol and zolpidem. These tests can be used as homogenous and ELISA formats and are currently undergoing clearance process by FDA. The laboratory first evaluates if these reagents satisfy the client’s and the regulations’ requirements before putting these tests in their menu.

Whether alone or combined with naloxone, Buprenorphine is now accredited by the US as a treatment for opiate addiction and unlike methadone; this drug can be prescribed by a physician. Buprenorphine might also be used to treat chronic pain. Even though this drug has not achieved significant frequency, it is bound to have an increase in its prevalence over a period of time. Currently, two manufacturers have homogenous assay reagents to test for buprenorphine.

The immunoassay for EtG – Ethyl Glucuronide is also developed to be added to the microgenics menu. EtG is a metabolite of alcohol consumption which is detected in urine for some days after consuming alcohol. Initially, it was analyzed by chromatographic techniques. However, due to the rising incidence of the use of this biomarker to evaluate alcohol abstinence, the immunoassay for this biomarker would be shortly available to give quicker and less expensive screening as compared to chromatography.

Lastly, immunoassays for LSD are available in ELISA and homogenous kit formats. The prevalence of LSD, is however not applicable in general population and therefore, it is not used in routine workplace drug testing programs.

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