Context.—

Consequences related to nicotine (NIC) use remain a major health concern, leading to demand for testing to detect NIC, metabolites such as cotinine (COT), and related tobacco alkaloids, including anabasine (ANAB). NIC-related testing is not standardized among laboratories, nor are there clinical or regulatory guidelines to inform decisions such as appropriate screening cutoffs or limits of quantitation.

Objective.—

To evaluate analytical performance and reporting practices of laboratories that perform NIC-related testing by reviewing participant responses to the Nicotine and Tobacco Alkaloid (NTA) Proficiency Testing Survey.

Design.—

NTA results were retrieved from 2017 (the first year of the survey) through 2020. Survey participants, methodologies, and results were evaluated for all analytes, and simulated grading was performed for COT. Additional data, including limits of quantitation, qualitative cutoffs, and reasons for testing, were reviewed.

Results.—

Participant growth was steady for qualitative COT testing. Participation was stable for NIC, ANAB, and quantitative COT testing. Overall, participants performed well on survey challenges. However, reporting thresholds were widely divergent, ranging from 10 to 3000 ng/mL and 0.5 to 300 ng/mL, respectively, for qualitative and quantitative COT testing. Screening cutoffs were as high as 100 ng/mL for ANAB and 1000 ng/mL for NIC.

Conclusions.—

Although participating laboratories performed well on the NTA Survey, the wide diversity of qualitative and quantitative reporting thresholds creates substantial risk for misinterpretation of results, and could lead to analytical concerns such as excessively high false-negative or false-positive rates. NIC-related testing would benefit from evidence-based guidelines to drive standardization of reporting.

Cigarettes and other products containing nicotine (NIC) remain a significant public health issue, with well-characterized harmful effects on the respiratory, cardiovascular, and other organ systems.1  In addition to traditional combustible tobacco products (eg, cigarettes, cigars, and pipes) and chewing tobacco, electronic cigarettes (e-cigarettes) and other noncombustible modes of NIC delivery have emerged in the last 2 decades.2  There are also NIC replacement products such as chewing gum and dermal patches, used primarily for management of NIC dependence and smoking cessation programs.3 

In addition to long-term health risks, cigarette smoking impairs surgical wound healing and recovery from burns.48  The most detailed surgical literature on adverse effects of cigarette smoking relates to cosmetic and plastic surgery,4,7  but effects in other surgeries have been documented as well. NIC delivered by e-cigarettes or replacement products does not appear to reduce the risk of perioperative complications as compared with active smoking in plastic surgery patients.9  Cigarette smoking is also a negative prognostic factor in the success of solid organ transplantation.1013  Because of the greater health risks associated with NIC, life insurance companies charge higher premiums for users applying for permanent life insurance.14,15 

NIC distributes rapidly to target organs and has an elimination half-life of approximately 2 hours.16  The major metabolite of NIC is cotinine (COT), which has an average elimination half-life of 20 hours. COT accounts for 70% of circulating compounds related to NIC. Additional minor metabolites of NIC include nornicotine and downstream metabolites of COT (eg, 3-hydroxycotinine). Nornicotine may be found in tobacco independently from NIC, as well as being a minor NIC metabolite.16,17  Ratios of NIC metabolites, particularly 3-hydroxycotinine/COT, serve as phenotypic markers of cytochrome P450 2A6 (CYP2A6) activity, which varies based on genetics, sex, race, and environmental factors. Metabolic ratios are relatively stable over time, and may predict success in NIC cessation treatments.18,19  Some NIC cessation treatments require participants to abstain from active smoking and use replacement products; the above-mentioned metabolites cannot distinguish these sources of NIC exposure. In contrast, anabasine (ANAB) and anatabine are alkaloids structurally related to NIC, also commonly found in tobacco products.17,20  ANAB and anatabine are not part of the NIC metabolic pathways and are thereby proposed to indicate exposure to tobacco products as opposed to purified NIC products.17,2023  However, the utility of ANAB for this purpose is limited by poor sensitivity and specificity.24 

Measurement of NIC and its metabolites in body fluids (most commonly urine, saliva, or serum/plasma) may be used for a variety of purposes.2123,25  A detailed quantitative profile of NIC and its metabolites can help ascertain the likelihood of whether the patient is an active tobacco user, one who recently abstained, a nonuser, or someone exposed to passive (secondhand) smoking.21  Evaluation of NIC, NIC metabolites, and other tobacco alkaloids may be useful in testing related to qualification for elective surgery, organ transplant recipient risk assessment, NIC cessation programs, and life insurance health risk determination.26  In contrast, simpler testing focused on detection of COT alone may be useful for rapid evaluations such as just prior to elective surgery.27  Concentrations of NIC and metabolites are similar regardless of whether administration occurs via transdermal patch, e-cigarettes, or smoking.9 

Immunoassays (IAs) approved by the Food and Drug Administration are available for either qualitative or quantitative measurement of COT.28  Some of these are classified by the Clinical Laboratory Improvement Amendments as waived complexity (eg, test strips), whereas others are moderate-complexity assays intended for clinical chemistry analyzers common in hospital or reference clinical laboratories. Chromatographic methods coupled to mass spectrometric detectors, especially liquid chromatography–tandem mass spectrometry (LC-MS/MS) and less commonly gas chromatography–mass spectrometry (GC-MS), are used for the more detailed NIC and tobacco alkaloid profiles. These assays tend to be high complexity under Clinical Laboratory Improvement Amendments. Published limits of quantification (LOQs) for LC-MS/MS assays vary tremendously and may be as low as 0.1 ng/mL.29 

A variety of analytical and clinical cutoffs have been proposed for interpretation of NIC and tobacco alkaloid testing.21,26  An example of this variability is illustrated by comparing options for panels of NIC metabolite and tobacco alkaloid testing in urine available at 4 large commercial reference laboratories in the United States (ARUP Laboratories, Labcorp, Mayo Clinic Laboratories, and Quest Diagnostics).3033  Each offers a panel including NIC and COT plus 0, 2, or 4 additional NIC metabolites or tobacco alkaloids in urine. ANAB is included in panels for 3 of the 4 reference laboratories. Nornicotine and 3-hydroxycotinine are included in the panel by only 2 of the 4 laboratories, and norcotinine is offered by only 1 of the laboratories. Positive cutoffs for NIC in these urine panels are 5, 15, 17, and 100 ng/mL. Positive cutoffs for COT are 5, 15, 20, and 200 ng/mL. Two of the laboratories (ARUP and Labcorp) offer stand-alone screens targeted at only COT, with cutoffs of 100 and 300 ng/mL, respectively.34,35 

Laboratory cutoffs and LOQs are often chosen based largely on analytical performance, although there are some clinical studies to guide these decision limits. For example, a urine COT cutoff of 100 ng/mL is close to the concentrations of COT in urine associated with smokers who show adverse effects on airway epithelium from smoking.36  A urine COT cutoff of 300 ng/mL was recommended to distinguish active smoking from passive exposure for a study of 1545 adults, wherein approximately 20% reported tobacco use. In this study, 81% of tobacco users and 67% of nonusers were correctly classified.37  There are fewer clinical studies addressing interpretive cutoffs for other NIC metabolites or tobacco alkaloids, leaving analytical performance as the primary guidance for establishing LOQs.

Proficiency testing is one method used by laboratories to demonstrate adequate assay performance and personnel competency. The College of American Pathologists (CAP) proficiency testing surveys are used by laboratories worldwide for this purpose. In response to the growing number of laboratories performing NIC and tobacco alkaloid testing, the Nicotine and Tobacco Alkaloid (NTA) Survey was introduced in 2017 to evaluate a laboratory's ability to qualitatively or quantitatively measure NIC, COT, and ANAB in urine. This article summarizes 4 years of data from the NTA Survey, focusing in particular on the choices of cutoffs and LOQs by laboratories and analytical performance of participants.

The NTA Survey is sent out twice a year (A and B mailings). Each mailing consists of 3 samples (challenges) containing varying amounts of NIC, COT, and ANAB. Challenges are prepared by spiking drug-free urine at concentrations defined by the CAP Toxicology Committee; because of the nature of NIC-related testing, zero challenges (ie, lacking one or more analytes) are used more frequently in NTA compared with many other CAP surveys. Participants can report any of the 3 analytes qualitatively and/or quantitatively.

NTA Survey data, including comments, were extracted for 2017–2020 (8 mailings, total of 24 challenges per analyte). Data analyzed for all mailings included participant numbers, methodologies, reporting thresholds, and test results for each analyte and reporting mechanism (qualitative and quantitative). Additional survey data were also reviewed, including optional supplemental questions asked with the 2019-A mailing to gain testing-related information from participants (112 participants in the mailing; 95 responses received). The NTA Survey has been classified as educational (ungraded), with grading scheduled to begin in 2022 for NIC and COT. Grading for quantitative reporting will be all-method mean ± 2 SD; qualitative grading will be based on 80% consensus by cutoff.

Participant responses are grouped by methodology and cutoff for qualitative results or methodology and LOQ for quantitative results. Descriptive statistics for quantitative results were calculated by mailing and analyte for specimens with a peer group size of at least 10 laboratories. Prior to calculating summary statistics, quantitative results reported by participating laboratories were screened for outliers. An interquartile range (IQR)–based procedure was applied to screen for outlying values, defined as being more extreme than 1.5 times the IQR of the distribution of quantitative results. Results of zero (0.0), if reported by participants, were included in SD and coefficient of variation (CV) calculations unless removed by the IQR screen. All outlier screening, visual graphics, and comparative analyses were performed and generated using SAS 9.4 (SAS Institute, Cary, North Carolina).

Mock grading was performed retrospectively for this study. For quantitative testing, all-method means and CVs were calculated for each challenge; individual participant responses were then assessed using criteria of ±2 SD, ±3 SD, or ±25% from the all-method mean. Unacceptable responses were reported as the percentage of total responses for that challenge. For qualitative testing, participant responses were evaluated according to the reporting cutoff provided. Unacceptable results were reported as the percentage of total participants using each cutoff, for all challenges where that cutoff-specific peer group reached consensus (≥80% agreement).

Survey Participants

The number of participating laboratories reporting qualitative results for COT has steadily increased since the NTA Survey began, from 57 participants in the 2017-A mailing to 104 participants in the 2020-B mailing (83% increase; Figure 1, A). In contrast, the number of participants for quantitative COT (Figure 1, B) testing rose initially, then subsequently plateaued, with 18 participants in 2017-A and 31 in 2020-B (72% increase). Participation for ANAB and NIC has remained steady but low (Figure 1, A and B). In 2020-B there were 3 qualitative and 10 quantitative responses for ANAB and 8 qualitative and 17 quantitative responses for NIC; these represented increases of only 1 to 3 participants since 2017-A.

Figure 1

Survey participation for (A) qualitative (qual) and (B) quantitative (quant) reporting of anabasine (ANAB), cotinine (COT), and nicotine (NIC) in the Nicotine and Tobacco Alkaloid (NTA) Survey, 2017–2020.

Figure 1

Survey participation for (A) qualitative (qual) and (B) quantitative (quant) reporting of anabasine (ANAB), cotinine (COT), and nicotine (NIC) in the Nicotine and Tobacco Alkaloid (NTA) Survey, 2017–2020.

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Throughout the study period, the overwhelming majority of participating laboratories were from the United States, with most associated with medical clinics or hospitals rather than industry or reference laboratories. Responses to a supplemental question on the 2019-A survey outlined the 2 most common indications for testing as (1) monitoring tobacco use, exposure, or cessation (57 of 95 participants; 60%) and (2) prequalification for surgery, including organ transplantation (42 of 95; 44%; participants could choose more than one response).

For ANAB, LC-MS/MS was the most common methodology used for both qualitative and quantitative reporting; throughout the study period, at most 1 participant per mailing reported use of an IA or GC-MS to detect ANAB. Participants reporting quantitative results for NIC and COT primarily used LC-MS/MS, with occasional (≤2 per mailing) use of LC–time-of-flight or GC-MS. Only 2 to 5 laboratories in each survey mailing reported quantitative COT results from IAs; no laboratories reported NIC quantitatively from IAs. In contrast, NIC and COT qualitative assays showed somewhat more diversity of methodologies. IAs still predominated for qualitative COT testing, with 4 to 7 participants per mailing reporting qualitative results from GC-MS and LC-MS/MS, whereas qualitative NIC testing was nearly evenly divided between IA and MS-based methods.

Cutoffs and LOQs

Cutoffs for qualitative screening assays and LOQs for quantitative testing ranged widely for all analytes in the NTA Survey. For instance, at least 8 different COT screening cutoffs were reported during the study period; 2020 survey results are shown as a representative example (Figure 2, A). Qualitative COT cutoffs ranged from 10 to 3000 ng/mL during the study period. COT cutoffs of 200 and 500 ng/mL predominated; for example, in 2020-B these were used by 24 (37.5%) and 27 participants (42.2%), respectively, of the 64 who provided their cutoff concentrations. The most common methodologies used at these cutoffs were lateral flow IA (200 ng/mL cutoff) and enzyme IA or homogeneous enzyme IA (500 ng/mL cutoff). Laboratories using MS-based methods reported qualitative COT cutoffs ranging from 10 to 500 ng/mL; most IA cutoffs were 100 ng/mL or higher, but at least 1 participant reported an IA cutoff of 10 ng/mL. Few participants provided their qualitative cutoffs for ANAB or NIC, but the cutoffs reported ranged as high as 100 ng/mL for ANAB and 1000 ng/mL for NIC.

Figure 2

Proportion of participants reporting (A) qualitative cutoff and (B) lower limit of quantitation (LOQ) for cotinine (COT) in the 2020-A and 2020-B Nicotine and Tobacco Alkaloid (NTA) Survey mailings. Note: data are not shown for participants who did not specify their qualitative cutoff (n = 2 for 2020-A, n = 1 for 2020-B) or LOQ (n = 3 for 2020-A, n = 2 for 2020-B).

Figure 2

Proportion of participants reporting (A) qualitative cutoff and (B) lower limit of quantitation (LOQ) for cotinine (COT) in the 2020-A and 2020-B Nicotine and Tobacco Alkaloid (NTA) Survey mailings. Note: data are not shown for participants who did not specify their qualitative cutoff (n = 2 for 2020-A, n = 1 for 2020-B) or LOQ (n = 3 for 2020-A, n = 2 for 2020-B).

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Quantitative limits also varied substantially among participants; for example, COT LOQs ranged from 0.5 to 300 ng/mL during the study period (2020 results shown as representative, Figure 2, B; no participants reported LOQs of 0.5 or 300 ng/mL in 2020). As noted above, few participants reported COT quantitative results from IAs; LOQs provided by these laboratories ranged from 100 to 300 ng/mL. Unlike qualitative reporting, no particular LOQs predominated for quantitative COT testing; at least 8 to 10 different LOQs were used by participating laboratories for the 2020 mailings. Interestingly, the same number of participants (5 in 2020-A, 4 in 2020-B) reported the lowest and highest LOQs in the 2020 mailings (2 and 100 ng/mL, respectively). Comparatively few participants reported quantitative results for the other survey analytes; NIC LOQs ranged from 2 to 200 ng/mL, whereas ANAB LOQs were lower (≤10 ng/mL).

In addition to the variety of cutoffs and LOQs, reporting algorithms were inconsistent among participants. For example, one laboratory reporting results for COT in 2020 had a greater than 80-fold difference between its qualitative test cutoff and its LOQ, whereas another laboratory used the same concentration for both qualitative and quantitative reporting. At least 1 participant used a borderline range for reporting screening results, rather than a single cutoff between positive and negative. The NTA Survey structure did not address whether laboratories set their quantitative reporting or interpretive cutoffs at the LOQ. It also did not address test algorithms such as reflexing, that is, testing samples with MS-based methods only if initially positive using a qualitative test, similar to the approach commonly used in urine drug testing.

Performance on Survey Challenges

Quantitative and qualitative performance on survey challenges were evaluated retrospectively, as the NTA Survey was educational (ie, ungraded) during the study period. For participants reporting quantitative results, all-method means compared well with the target values of challenges for both NIC and COT (Figure 3, A and B). Imprecision data, as measured by CVs for NIC and COT, were generally less than 15% (Table 1). There were not enough participants reporting ANAB values to plot all-method means for comparison against target concentrations for most challenges.

Figure 3

Comparison of all-method mean to target concentration for participants reporting quantitative results for nicotine (A) and cotinine (B). Lines of identity are shown.

Figure 3

Comparison of all-method mean to target concentration for participants reporting quantitative results for nicotine (A) and cotinine (B). Lines of identity are shown.

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Table 1

Quantitative Results for Anabasine (ANAB), Cotinine (COT), and Nicotine (NIC), Nicotine and Tobacco Alkaloids Survey 2017–2020a

Quantitative Results for Anabasine (ANAB), Cotinine (COT), and Nicotine (NIC), Nicotine and Tobacco Alkaloids Survey 2017–2020a
Quantitative Results for Anabasine (ANAB), Cotinine (COT), and Nicotine (NIC), Nicotine and Tobacco Alkaloids Survey 2017–2020a

Mock grading was performed to evaluate the analytical performance of participants. This analysis focused on COT because of the small numbers of laboratories reporting NIC or ANAB. For quantitative testing, CAP uses either statistical (typically ±2–3 SD) or absolute (eg, ±25%) grading criteria. The NTA Survey is not accuracy based; thus, simulated grading was compared against the mean of participant results rather than target concentration (Table 2). Grading against all-method mean ± 3 SD showed no unacceptable responses. Participants reporting quantitative COT results showed similar performance on ±2 SD and ±25% criteria, with averages of 6.41% and 5.15% unacceptable responses, respectively. Performance was similar across COT target concentrations from 20 to 5000 ng/mL.

Table 2

Simulated Grading of Cotinine Quantitative Challenge Data, Nicotine and Tobacco Alkaloids Survey 2017–2020

Simulated Grading of Cotinine Quantitative Challenge Data, Nicotine and Tobacco Alkaloids Survey 2017–2020
Simulated Grading of Cotinine Quantitative Challenge Data, Nicotine and Tobacco Alkaloids Survey 2017–2020

Mock grading of qualitative performance was again focused on COT because of low participation in qualitative reporting for NIC and ANAB. The 2 largest COT peer groups (cutoffs of 200 and 500 ng/mL) were evaluated for agreement on all survey challenges (Table 3). Using 80% or greater consensus by cutoff as the grading criterion, participant performance is graded as acceptable or unacceptable only if at least 80% of peer group responses agree for a given challenge. Nonconsensus (ie, <80% agreement among participants in the peer group) results in no grading. During the study period, the 500 ng/mL peer group met 80% consensus for all challenges, including 2 targeted near the group cutoff at 400 ng/mL and 600 ng/mL. In contrast, the 200 ng/mL peer group did not reach consensus for 3 of 4 challenges targeted between 120 and 250 ng/mL. For challenges where consensus was achieved, 90% of participants or more would have been scored as acceptable in all but 1 or 2 challenges for each peer group.

Table 3

Results for Cotinine Qualitative Challenge Data, Nicotine and Tobacco Alkaloids Survey 2017–2020a

Results for Cotinine Qualitative Challenge Data, Nicotine and Tobacco Alkaloids Survey 2017–2020a
Results for Cotinine Qualitative Challenge Data, Nicotine and Tobacco Alkaloids Survey 2017–2020a

This publication and others demonstrate that cutoffs and LOQs for NIC and metabolite testing are widely variable.21,26  Screening cutoffs ranged as high as 3000 ng/mL for COT and 1000 ng/mL for NIC, significantly increasing the likelihood of false-negative results. On the other hand, LOQs for quantitative assays were as low as 0.5 to 2 ng/mL, well within the range of secondhand exposure and potentially prone to analytical challenges such as contamination, carryover, or (particularly for IA) cross-reactivity with other NIC metabolites. For example, urine COT exceeded the 10 ng/mL cutoff in 40% of samples collected in a study of adolescents ages 12 to 21 years who reported abstinence from NIC-containing products.29  Misclassification was also common in studies of adults using a 100 ng/mL cutoff.37,38  Although most NTA Survey participants used 200 or 500 ng/mL cutoffs for qualitative COT reporting (likely driven by manufacturer recommendations), quantitative responses showed no predominant LOQs.

Although there are no clinical or forensic guidelines to inform choice of reporting thresholds, one study38  suggested that COT interpretive cutoffs between 50 and 200 ng/mL have approximately equal sensitivity and that normalization to creatinine is not helpful. Another37  demonstrated that a cutoff of 500 ng/mL may serve younger populations better than the generalized cutoff proposed of 300 ng/mL, although neither sensitivity nor specificity was terribly different between cutoffs of 100 and 500 ng/mL. The utility of lower reporting thresholds is questionable, and might be limited to specific indications, such as assessing secondhand exposure.

The NTA Survey does not directly address how testing is performed (eg, reflex from screening); however, individual differences such as the choice of LOQ and cutoff within a single laboratory suggest that reporting practices are widely disparate among participants. As with drug of abuse or alcohol testing, appropriate reporting thresholds and test algorithms for NIC-related testing can vary depending on the patient population being tested, reasons for testing, and the specific clients served by each laboratory. Although this study demonstrated that participants compare well overall against target analyte concentrations, it could not address whether the cutoffs, LOQs, or algorithms used were appropriate for the intended purposes of testing. Unfortunately, the lack of standardization in reporting and test methodology is a source of confusion for individuals interpreting results and a concern for patients, life insurance enrollees, and others who could be affected by an inappropriate cutoff or poorly performing assay. Evidence-based guidelines addressing NIC-related analytes and reporting thresholds appropriate to specific purposes for testing are needed.

Despite the wide range of cutoffs and LOQs, this report demonstrates good quantitative performance from participants in the NTA Survey. During the 4 years of the study period, mock grading using either all-method mean ± 2 SD or all-method mean ± 25% would have resulted in an average of less than 6.5% of laboratories being scored as unacceptable. Mean concentrations for both COT and NIC were uniformly close to targets, with CVs generally less than 15%, indicating consistent performance across survey participants. Previous smaller studies have shown similar consistency among laboratories; for example, a coordinated exchange of NIC, COT, and 3-hydroxycotinine in urine among 11 laboratories in 6 countries performing testing by MS-based methods demonstrated mean bias of less than 3.5%, consistent with results from this larger set of comparisons.39 

In contrast, although qualitative performance was good at high COT concentrations, 3 of 4 COT challenges near the 200 ng/mL cutoffs showed nonconsensus. This could reflect variable detection among manufacturers or assay lots, or challenges associated with visually interpreting lateral flow IA. Given regulatory requirements and the implications of inaccurate results for patients or clients, laboratories should regularly evaluate assay performance near the cutoff. There were fewer challenges targeted near the 500 ng/mL cutoff and more participants using automated IA, but the same requirements to assess performance near the cutoff apply.

Issues with IA targeting NIC metabolites have been previously demonstrated. For example, a study evaluating 10 263 authentic samples demonstrated detection of COT at approximately half of the stated cutoff.40  IAs are known for cross-reactivity to 3-hydroxycotinine, which often appears as the most concentrated of urine analytes in active smokers.18,22  Because the NTA Survey has not specifically included 3-hydroxycotinine in any challenge sample, the data presented here may be biased to suggest better IA performance than would be observed with authentic urine. This might also explain one reason why, despite availability of several commercial IAs, some participants use MS-based assays for qualitative reporting of NIC and COT.

Although COT testing is quite common, there are several reasons that likely contribute to the observation that few laboratories routinely perform NIC and ANAB testing. Unlike COT, NIC has a short half-life and can be prone to external contamination. Analytically, NIC and ANAB are isobaric and thereby require chromatographic separation. In addition, the anticipated concentrations in urine collected from active smokers are typically much lower for ANAB than for NIC and COT, which could complicate analysis of all analytes in a single assay.17,22  Specificity of ANAB is also a concern because it is present in other nightshade family plants such as eggplant and potatoes, as well as some insecticides.37  Anatabine is another minor alkaloid of tobacco. Both ANAB and anatabine are thought to be present in tobacco products, but the content is known to vary based on the plant used to manufacture tobacco products. Further, little is known about the metabolism of these alkaloids, which may introduce bias in the utility of these markers.20,22 

The NTA Survey is currently limited to urine, and may need to be extended to represent alternative matrices such as oral fluid. Several studies demonstrate good discrimination between active smokers and passive exposure or nonsmokers based on COT, with a cutoff of 10 ng/mL in oral fluid, although a cutoff of 30 ng/mL has also been proposed.40,41  Blood (serum or plasma) is also a good alternative specimen to urine, and is often preferred for estimation of metabolic ratios or impairment. However, neither ANAB nor anatabine appears in detectable concentrations in oral fluid or blood, making these specimens unhelpful if distinction between active tobacco use and replacement products is desired.

This study demonstrates wide divergence in qualitative and quantitative reporting thresholds, which is a potentially significant concern for appropriate test selection and interpretation. Laboratories should evaluate the needs of practices and clients ordering NIC-related testing to ensure that both the methodology and the reporting algorithm are appropriate. Given the growth in participation for qualitative COT testing, laboratories should also familiarize themselves and those interpreting results with the limitations of screening tests, including variable performance near reporting cutoffs and potential IA cross-reactivity with other NIC metabolites. Selection of specimen, analytical approach, and reporting thresholds are all critical to any testing in which NIC use or exposure is used to make decisions about patient care or eligibility for benefits. The NTA Survey is one mechanism by which analytical performance of testing may be evaluated and monitored, but cannot replace the need for clinical assessments.

We would like to thank Barbarajean Magnani, PhD, MD, who, as chair of the Toxicology Committee, encouraged the scientific investigation of trends in proficiency testing.

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Author notes

The authors have no relevant financial interest in the products or companies described in this article.