Laboratorian Interpretation of Drug Testing Results in Pain Management: Lessons From College of American Pathologists Proficiency Testing.

CONTEXT.­: Accurate interpretation of drug test results is key to appropriate patient care in numerous settings including pain management. Despite recommendations that providers should consult laboratory professionals for guidance when necessary, literature demonstrating laboratorian expertise in drug test interpretation is lacking. OBJECTIVE.­: To evaluate participating laboratories' performance on the case-based, interpretive ("dry") challenge included with each Drug Monitoring for Pain Management proficiency testing program from 2012-2023. DESIGN.­: All challenges (n = 23) required participants to identify if drug test results were consistent or inconsistent with prescribed medications in the case history. Relevant medications, presumptive and confirmatory drug test results, and participant responses were extracted from program summary reports and examined for performance and common themes. RESULTS.­: Overall, 91.8% (6821 of 7431) of participant responses correctly identified whether drug testing was consistent with medications. There were 8 challenges with participant scores below 91.8% (range, 59.8% [49 of 82 responses] to 88.9% [193 of 217 responses]). Common knowledge gaps identified in these challenges included false-positive presumptive (screening) results, minor metabolism of opiates, and recognizing that presence of a nonprescribed drug is inconsistent with prescribed medications. Although some participants repeatedly responded incorrectly, there were no associations between laboratory type, personnel responding, or analytical performance with incorrect responses to interpretative challenges. CONCLUSIONS.­: Program participants performed well overall, but several concerning educational gaps were identified. Laboratorians have a role in providing interpretative guidance for drug testing and should emphasize ongoing education to ensure competence in the setting of constantly changing prescribed and nonprescribed drug use.

Artificial elevation of phosphatidylethanol due to red blood cell transfusion.

INTRODUCTION: Phosphatidylethanol (PEth) is a marker of alcohol consumption used in clinical and forensic settings. PEth positivity in individuals expected to abstain from alcohol can have serious consequences. PEth is located on erythrocytes, thus packed red blood cell (pRBC) transfusion is a potential cause of false-positive results. This report is the first to demonstrate this phenomenon in an authentic patient who was negative for PEth immediately prior to transfusion. METHODS: Residual blood samples collected for clinical testing before and after pRBC transfusion and citrated pRBC segments were tested for PEth homologues 16:0/18:1 (POPEth) and 16:0/18:2 (PLPEth) by liquid chromatography - tandem mass spectrometry with limit of quantitation 10 ng/mL (0.01 µmol/L). CASE: A 56-year-old male with new-onset leukemia required transfusion of 4 pRBC units on hospital days 1-2. Blood collected at admission (day 0) showed POPEth and PLPEth < 10 ng/mL (<0.01 µmol/L). Blood collected after completion of the fourth pRBC transfusion demonstrated POPEth = 57 ng/mL (0.08 µmol/L), PLPEth = 38 ng/mL (0.05 µmol/L). One citrated segment demonstrated extremely elevated PEth, supporting pRBC transfusion as the source. DISCUSSION: This case demonstrates pRBC transfusion elevating PEth to concentrations associated with moderate alcohol consumption. Studies suggest that healthy individuals (potential donors) could have PEth concentrations sufficient to cause significant elevation of PEth from a single pRBC unit. This is concerning for populations such as liver transplant candidates who are required to abstain from alcohol, but whose disease sequelae may require pRBC transfusion. CONCLUSIONS: pRBC transfusion can artificially elevate PEth into clinically and forensically relevant ranges. Individuals interpreting toxicology testing should consider recent pRBC transfusion when evaluating PEth concentrations.

Nicotine and Tobacco Alkaloid Testing and Challenges: Lessons Learned From a College of American Pathologists Proficiency Testing Survey.

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.

The North American opioid epidemic: opportunities and challenges for clinical laboratories.

Since 1999, the opioid epidemic in North America has resulted in over 1 million deaths, and it continues to escalate despite numerous efforts in various arenas to combat the upward trend. Clinical laboratories provide drug testing to support practices such as emergency medicine, substance use disorder treatment, and pain management; increasingly, these laboratories are collaborating in novel partnerships including drug-checking services (DCS) and multidisciplinary treatment teams. This review examines drug testing related to management of licit and illicit opioid use, new technologies and test strategies employed by clinical laboratories, barriers hindering laboratory response to the opioid epidemic, and areas for improvement and standardization within drug testing. Literature search terms included combinations of "opioid," "opiate," "fentanyl," "laboratory," "epidemic," "crisis," "mass spectrometry," "immunoassay," "drug screen," "drug test," "guidelines," plus review of PubMed "similar articles" and references within publications. While immunoassay (IA) and point-of-care (POC) test options for synthetic opioids are increasingly available, mass spectrometry (MS) platforms offer the greatest flexibility and sensitivity for detecting novel, potent opioids. Previously reserved as a second-tier application in most drug test algorithms, MS assays are gaining a larger role in initial screening for specific patients and DCS. However, there are substantial differences among laboratories in terms of updating test menus, algorithms, and technologies to meet changing clinical needs. While some clinical laboratories lack the resources and expertise to implement MS, many are also slow to adopt available IA and POC tests for newer opioids such as fentanyl. MS-based testing also presents challenges, including gaps in available guidance for assay validation and ongoing performance assessment that contribute to a dramatic lack of standardization among laboratories. We identify opportunities for improvement in laboratory operations, reporting, and interpretation of drug test results, including laboratorian and provider education and laboratory-focused guidelines. We also highlight the need for collaboration with providers, assay and instrument manufacturers, and national organizations to increase the effectiveness of clinical laboratory and provider efforts in preventing morbidity and mortality associated with opioid use and misuse.

Therapeutic Drug Monitoring of Second- and Third-Generation Antiepileptic Drugs.

CONTEXT.­: Therapeutic drug monitoring has traditionally been widely used for first-generation antiepileptic drugs (AEDs) such as carbamazepine and phenytoin. The last 2 decades have seen the introduction of second- and third-generation AEDs (eg, lamotrigine, levetiracetam, and topiramate) into clinical practice. OBJECTIVE.­: To use data from the College of American Pathologists Therapeutic Drug Monitoring, Extended Proficiency Testing Survey to determine the performance of assays used for therapeutic drug monitoring of newer AEDs, including comparison of enzyme immunoassay and chromatographic techniques. DESIGN.­: Six years of proficiency testing surveys were reviewed (2013-2018). RESULTS.­: Steady growth was seen in participant volumes for newer AEDs. The analytical performance of automated enzyme immunoassays for lamotrigine, levetiracetam, and topiramate was similar to that of chromatographic methods, consistent with published literature using patient samples for comparisons. The majority of participating laboratories now use enzyme immunoassays to measure levetiracetam. CONCLUSIONS.­: Survey results reflect steadily growing interest in therapeutic drug monitoring of newer AEDs. The increasing availability of robust immunoassays for new AEDs should facilitate their clinical utility, especially for clinical laboratories that do not perform chromatographic assays for therapeutic drug monitoring.

Ethanol Biomarker Testing and Challenges.

CONTEXT.­: Clinical and forensic testing for ethanol biomarkers, including ethyl glucuronide (EtG) and ethyl sulfate (EtS), is used to discern alcohol use from abstinence. These markers can be key in major decisions, including transplant eligibility or retaining licensure after alcohol misuse. Accuracy, precision, and recognition of the implications of reporting cutoffs are all essential for correct interpretation. OBJECTIVE.­: To evaluate trends in testing for EtG and EtS, including how laboratories perform testing and how comparable participant results are. DESIGN.­: The study examined the College of American Pathologists ethanol biomarker proficiency testing survey from 2013 to 2019. Trends in methodology, reporting cutoffs, and participant performance were evaluated for qualitative and quantitative EtG testing and for quantitative EtS testing. RESULTS.­: There was little consensus in reporting cutoffs, which ranged from 10 to 1000 ng/mL for EtG and 10 to 1500 ng/mL for EtS. Although median EtG and EtS compared well with target concentrations, individual participants' results varied widely. For quantitative enzyme immunoassay, accuracy and precision were best in EtG challenges less than 1500 ng/mL. For EtG or EtS by mass spectrometry, overall accuracy was good over a wide concentration range, but variability between participants was high. Approximately 10% (409 of 4059) of results were unacceptable, which for mass spectrometry corresponded to more than 35% above or below the group mean. CONCLUSIONS.­: Although many participants performed well, there was insufficient consensus in reporting cutoffs, and a consistent fraction of laboratories failed to achieve survey standards. Guidelines for assay performance and reporting could greatly benefit laboratories and end users.

Gradient-Based Rapid Digital Immunoassay for High-Sensitivity Cardiac Troponin T (hs-cTnT) Detection in 1 µL Plasma.

Rapid and sensitive detection of biomarkers is the key to the diagnosis of acute diseases. One example is the detection of troponin in myocardial infarction. Here, we report a gradient-based digital immunoassay method, which can achieve high-sensitivity cardiac troponin T (hs-cTnT) detection with only 1 µL of plasma sample. We designed a multizone microfluidic channel functionalized with capture antibody specific to troponin. Taking advantage of limited sample volume, a troponin concentration gradient is created along the channel because of binding induced depletion. We quantified the concentration gradient by counting the detection antibody conjugated gold nanoparticles bound to different test zones with optical imaging. Differential counting between the zones removes most common noises and nonspecific bindings. The total analytical time is about 30 min, and the limit of quantification is 6.2 ng/L. We examined 41 clinical plasma samples from 15 patients and the change in hs-cTnT concentration in serial samples showed good linear correlation with clinical results (R2 = 0.98). Therefore, this simple and sensitive gradient-based digital immunoassay method is a promising technology for clinical hs-cTnT detection and could be adapted for detection of other biomarkers.

Updated ferritin reference intervals for the Roche Elecsys® immunoassay.

Reference intervals (RI) for ferritin are the subject of some controversy, with indications that changes in lifestyle and demographics (e.g., obesity) have limited the validity of RIs established decades ago. Package insert RIs for the Roche Elecsys® immunoassay do not include expected values for pediatric (<17-20 years) or geriatric (>60 years) individuals; furthermore the female ranges were established in mostly premenopausal volunteers. To establish more robust RIs, we utilized 5 years of retrospective patient data from physician-ordered ferritin measurements and excluded results from patients with diagnoses known to affect ferritin concentrations. Ferritin results from 1438 unique patients aged 7 months to 91 years were included in the study. Continuous RIs were fitted for females (n = 951) and males (n = 487) as a function of age; these were then divided into clinically relevant sex-specific age breaks. RIs were established for pre-adolescent (<10 years), adolescent (10-17 years) and adult males, and for pediatric (<18 years), adult (18-50 years) and older (>50 years) females. Established RIs were verified using specimens obtained from healthy donors.

Altered metabolism of mothers of young children with Autism Spectrum Disorder: a case control study.

BACKGROUND: Previous research studies have demonstrated abnormalities in the metabolism of mothers of young children with autism. METHODS: Metabolic analysis was performed on blood samples from 30 mothers of young children with Autism Spectrum Disorder (ASD-M) and from 29 mothers of young typically-developing children (TD-M). Targeted metabolic analysis focusing on the folate one-carbon metabolism (FOCM) and the transsulfuration pathway (TS) as well as broad metabolic analysis were performed. Statistical analysis of the data involved both univariate and multivariate statistical methods. RESULTS: Univariate analysis revealed significant differences in 5 metabolites from the folate one-carbon metabolism and the transsulfuration pathway and differences in an additional 48 metabolites identified by broad metabolic analysis, including lower levels of many carnitine-conjugated molecules. Multivariate analysis with leave-one-out cross-validation allowed classification of samples as belonging to one of the two groups of mothers with 93% sensitivity and 97% specificity with five metabolites. Furthermore, each of these five metabolites correlated with 8-15 other metabolites indicating that there are five clusters of correlated metabolites. In fact, all but 5 of the 50 metabolites with the highest area under the receiver operating characteristic curve were associated with the five identified groups. Many of the abnormalities appear linked to low levels of folate, vitamin B12, and carnitine-conjugated molecules. CONCLUSIONS: Mothers of children with ASD have many significantly different metabolite levels compared to mothers of typically developing children at 2-5 years after birth.

Interpretation and Utility of Drug of Abuse Screening Immunoassays: Insights From Laboratory Drug Testing Proficiency Surveys.

CONTEXT.­: Urine drug testing is frequently ordered by health care providers. Immunoassays are widely used for drug testing, yet have potential limitations, including variable cross-reactivity. The last decade has seen worsening of a prescription drug abuse epidemic. OBJECTIVE.­: To use data from a College of American Pathologists proficiency testing survey, Urine Drug Testing, Screening, to determine and summarize the characteristics, performance, and limitations of immunoassays. DESIGN.­: Seven years of proficiency surveys were reviewed (2011-2017). RESULTS.­: Rapid growth was seen in participant volumes for specific immunoassays for synthetic opioids (eg, buprenorphine, fentanyl, oxycodone) and 3,4-methylenedioxymethamphetamine ("ecstasy"). Participant volumes remained high for immunoassays targeting less commonly abused drugs such as barbiturates and phencyclidine. For opiate immunoassays, the number of laboratories using a 2000 ng/mL positive cutoff remained stable, and an increasing number adopted a 100 ng/mL cutoff. Opiate and amphetamine immunoassays showed high variability in cross-reactivity for drugs other than the assay calibrator. Assays targeting a single drug or metabolite generally performed well on drug challenges. CONCLUSIONS.­: Survey results indicate strong clinical interest in urine drug testing and some adoption of new assays. However, urine drug testing availability does not parallel prevailing patterns of drug prescribing and abuse patterns. In particular, specific immunoassays for synthetic opioids and a lower positive cutoff for opiate immunoassays may be underused, whereas immunoassays for barbiturates, methadone, propoxyphene, and phencyclidine may be overused. Laboratories are encouraged to review their test menu, cutoffs, and assay performance and adjust their test offerings based on clinical needs and technical capabilities.

Evaluating Tiered Hemolysis Thresholds to Facilitate Rapid Reporting of High-Sensitivity Cardiac Troponin.

BACKGROUND: Cardiac troponin (cTn) assays are used for the diagnosis of acute myocardial infarction and frequently require serial measurements. Recollection of unacceptable specimens for hemolysis can delay results and disrupt timing of serial measurements. This study was designed to assess the influence of hemolysis on a high-sensitivity cTnT assay in granular detail at clinically important concentrations. These were used in consultation with the clinical practice to evaluate risk-based thresholds for acceptable amounts of hemolysis. METHODS: Plasma pools ranging from <10 to >100 ng/L cTnT were spiked with hemolysate to various degrees of hemolysis and measured using the Elecsys Troponin T Gen 5 STAT assay. The impact of accepting expanded hemolysis thresholds was completed using retrospective data of 12225 serial cTnT results and an additional 4651 baseline cTnT results. RESULTS: The mean percent change in cTnT was consistent for a given degree of hemolysis regardless of the initial nonhemolyzed cTnT concentration. Tiered hemolysis thresholds were evaluated for low-risk patients (apparent cTnT ≤8 ng/L), intermediate-risk patients (thresholds for 9-37 ng/L, 38-66 ng/L, and 67-99 ng/L cTnT), and high-risk patients (≥100 ng/L cTnT). The influence of tiered hemolysis thresholds was calculated for patients with serial (0 and 2 h) cTnT results, which demonstrated that the majority of 2-h delta interpretations were unchanged. Implementation of tiered thresholds dramatically decreased recollections for hemolyzed cTnT samples. CONCLUSION: Tiered hemolysis cutoffs minimized disruption to patient care for low- and high-risk patients, while maintaining the integrity of serial measurements for those with intermediate cTnT concentrations.

"Rainbow Draws" in the Emergency Department: Clinical Utility and Staff Perceptions.

BACKGROUND: Collecting a predefined set of blood tubes (the "rainbow draw") is a common but controversial practice in many emergency departments (EDs), with limited data to support it. We determined the actual utilization of rainbow draw tubes at a single facility and evaluated the perceptions of ED staff regarding the utility of rainbow draws. METHODS: We analyzed 2 weeks of ED visits (1326 visits by 1240 unique patients) to determine blood tube utilization for initial and add-on testing, as well as the incidence of additional venipunctures. We also surveyed ED staff regarding aspects of ED phlebotomy and test ordering. Utilization data analysis was structured to satisfy specific concerns addressed in the ED staff survey. RESULTS: Observed tube utilization data showed that fluoride/oxalate, citrate, and serum separator tubes were frequently discarded unused, and that the actual utility of the rainbow draw for add-on testing and avoiding additional venipunctures was low. ED staff perceived that the rainbow draw was highly valuable, both to expedite add-on testing and to avoid additional venipunctures. Contrasting the objective (utilization data) and subjective (survey results) to drive changes in the standard ED blood collection reduced the estimated waste blood by 175 L/year. CONCLUSIONS: Comparison of perceptions and objective utilization data drove process changes that were mutually agreeable to ED and laboratory staff. Although specifics of ED and laboratory work flows vary between institutions, the principles and strategy of this study are widely applicable.

False-Positive Serum Cocaine Screening Results in Patients Undergoing Evaluation for Renal Transplant.

Drug screening during pre-transplant evaluations can have major implications for patient care, particularly because drug abuse has been associated with poor transplant outcomes. Although urine drug screening is usually preferred, serum testing is available for situations such as anuria due to end stage renal disease. However, there are few studies evaluating serum drug screening in specific populations such as patients undergoing kidney transplant evaluation. All serum drug screens ordered between January 2015 and November 2017 on patients being evaluated for renal transplant were compared against a large population of serum drug screens ordered from other institutions. Cocaine screening and confirmation results were evaluated to determine false positives. Cocaine screens were positive in 23 of 537 (4.3%) pre-transplant samples, and 211 of 5,115 (4.1%) comparison samples. Confirmation testing demonstrated that 14 (60.9%) pre-transplant samples were false positives, which was significantly (P < 0.01) higher than the rate of false positives in the comparison group (47/211, 22.3%). No common medication or other cross-reacting substance could be identified in the pre-transplant cohort to explain the false-positive results. Although serum cocaine screening had a low overall false-positive rate, the proportion of false positives was significantly higher in pre-transplant patients. Given the poor transplant outcomes associated with drug abuse, failure to properly interpret screening results as being false positives could negatively affect patient care. All members of the transplant team should recognize the importance of confirmation testing in this setting, to avoid unintended consequences due to false-positive screening results.

An Introduction to Drug Testing: The Expanding Role of Mass Spectrometry.

Measurement of drugs and their metabolites in biological fluids is the foundation of both therapeutic drug monitoring (TDM) and toxicology. The introduction of methods based on mass spectrometry (MS), coupled with gas or liquid chromatography, has revolutionized these areas. This chapter will introduce the reader to the application of MS to TDM and toxicology, the steps that should be considered during implementation and the processes that should be implemented to assure continued quality. Points of emphasis include advances and recent trends since the publication of the first edition of this book, such as high-resolution mass spectrometry and increased interest in alternate matrices.

False-positive amphetamine results on several drug screening platforms due to mexiletine.

OBJECTIVE: False-positive urine drug of abuse screening (UDS) results can have serious implications in clinical practice, particularly when confirmation assay results are not immediately available to providers making medical decisions. Often it is not possible to identify the specific medication or other interfering compound that is responsible for the false-positive UDS result. Even when a potential interference is reported in the literature or package insert for one assay, the applicability to other UDS platforms/assays is often unknown. Mexiletine has been suggested as a cause of false-positive amphetamine results, but never confirmed as the causative agent in previous reports. The goal of this study was to confirm this drug as a cross-reacting compound in amphetamine screening tests. METHODS: We evaluated several amphetamine screening assays: the Syva EMIT II Plus and the Roche KIMS automated immunoassays, along with the Noble Split-Specimen and Synchron QuikScreen point-of-care assays. RESULTS: Urine samples from two patients treated with mexiletine were positive on all amphetamine screens but confirmed negative by mass spectrometry. Drug-free urine spiked with mexiletine caused positive results on all assays, although the EMIT II Plus and KIMS assays cross-reacted at lower mexiletine concentrations than the point-of-care assays. CONCLUSION: This report confirms that mexiletine can cross-react on several amphetamine screening assays. Assay manufacturers are encouraged to evaluate mexiletine cross-reactivity, and providers and laboratories should be aware of the potential for false-positive amphetamine screening results in patients taking mexiletine.

Rapid reduction of extremely high kappa free light chains in a patient with myeloma cast nephropathy.

This report describes a patient with light chain myeloma and acute renal injury. Serum kappa free light chain (FLC) was extremely elevated, >33,000 mg/dL. Treatment with therapeutic plasma exchange (TPE) started day 2 for biopsy-confirmed cast nephropathy. Bortezomib-containing chemotherapy was initiated on day 5, and hemodialysis for tumor lysis syndrome on day 7. TPE alone decreased kappa FLC >70% by day 5, indicating direct FLC removal was successful in this patient. A total of 25 TPE procedures were performed in a 31-day hospitalization. Hemodialysis was discontinued after 3 months, and the patient's renal function and kappa FLC remain stable. Although the use of TPE for FLC removal is controversial, recent evidence supports its use as adjuvant therapy for acute renal injury secondary to myeloma cast nephropathy. TPE can be effective for rapidly reducing FLC; however, several TPE procedures might be required to reduce the risk of hemodialysis dependency.

A Flow Cytometry-Based Assay for Procoagulant Platelet Polyphosphate.

BACKGROUND: Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets. METHODS AND RESULTS: Recombinant Escherichia coli-expressed exopolyphosphatase deletion mutant PPX_Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX_Δ12-Alexa488-signal dose-dependently increased with long-chain polyphosphate binding to platelets. In contrast, short-chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre-incubation abolished PPX_Δ12-Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12-Alexa488 signal in a dose-dependent manner. CONCLUSION: This study indicates that long-chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet-rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society.