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.

Development of an LC-MS/MS assay with automated sample preparation for phosphatidylethanol (PEth)- Not your typical clinical marker.

Phosphatidylethanol (PEth) is a group of phospholipids formed exclusively in the presence of ethanol on the erythrocyte membrane, making it a direct biomarker for long-term ethanol consumption for which a clinical reference interval has been established. Here, we describe an assay for quantitation for two most abundant PEth homologues, PEth 16:0/18:1 and PEth 16:0/18:2, from human whole blood, and present challenges overcome throughout the development process. Since PEth is localized within erythrocyte membranes, a reliable sample preparation technique is an important aspect of PEth analysis. Therefore, various erythrocyte lysing agents for recovery of exogenously spiked standards and controls were evaluated to identify one that performed comparably to the recovery of endogenous analytes found in authentic samples. A supported liquid extraction (SLE) technique was employed for sample cleanup and enrichment which together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis enabled automated sample preparation, appropriate chromatographic resolution, and minimal system carryover. This resulted in a laboratory developed test with an analytical measurement range (AMR) of 10-1000 ng/mL (slope = 0.9902-1.0138, R2 = 0.9958-0.9972), that was precise (intra-day precision: 3.4-4.1%; inter-day precision: 4.4-8.2% over the AMR), accurate when compared with an available external laboratory test (slope = 0.9943-1.0206, R2 = 0.9635-0.9678, no lower decision point interpretation changes), with effective analyte recovery (77.2-83.5%), and established stability characteristics, while chromatographically separating the analytes to ensure no additive effects due to the isotopic distribution of the opposing analyte.

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.

Determination of the Duplicated CYP2D6 Allele Using Real-Time PCR Signal: An Alternative Approach.

CYP2D6 duplication has important pharmacogenomic implications. Reflex testing with long-range PCR (LR-PCR) can resolve the genotype when a duplication and alleles with differing activity scores are detected. We evaluated whether visual inspection of plots from real-time-PCR-based targeted genotyping with copy number variation (CNV) detection could reliably determine the duplicated CYP2D6 allele. Six reviewers evaluated QuantStudio OpenArray CYP2D6 genotyping results and the TaqMan Genotyper plots for seventy-three well-characterized cases with three copies of CYP2D6 and two different alleles. Reviewers blinded to the final genotype visually assessed the plots to determine the duplicated allele or opt for reflex sequencing. Reviewers achieved 100% accuracy for cases with three CYP2D6 copies that they opted to report. Reviewers did not request reflex sequencing in 49-67 (67-92%) cases (and correctly identified the duplicated allele in each case); all remaining cases (6-24) were marked by at least one reviewer for reflex sequencing. In most cases with three copies of CYP2D6, the duplicated allele can be determined using a combination of targeted genotyping using real-time PCR with CNV detection without need for reflex sequencing. In ambiguous cases and those with >3 copies, LR-PCR and Sanger sequencing may still be necessary for determination of the duplicated allele.

Drug testing in support of the diagnosis of neonatal abstinence syndrome: The current situation.

Illicit drug use during pregnancy is a concern worldwide, with many international studies describing attempted strategies to mitigate this problem. Drug misuse during pregnancy is associated with significant maternal as well as perinatal complications, which include a high incidence of stillbirths, fetal distress, neonatal abstinence syndrome (NAS) and increased neonatal mortality. Unfortunately, the identification of a drug-exposed mother or neonate is challenging. Maternal disclosure of drug use is often inaccurate, principally due to psychosocial factors including behavioral denial or the fear of the consequences resulting from such admissions. Likewise, many infants who have been exposed to drugs in utero may appear normal at birth and initially show no overt manifestations of drug effects. Thus, the identification of the drug-exposed infant requires a high index of clinical suspicion. Conversely, analytical testing is an objective means of determining drug exposure when it may be necessary to document proof of the infant's exposure to illicit drugs. The review will discuss the different matrices that are most commonly used for testing (e.g., maternal urine, neonatal urine, meconium, and umbilical cord), the strengths and limitations for each matrix, which drugs and metabolites are appropriate for testing, the various testing methods, and the advantages and disadvantages of each method.

Therapeutic Drug Monitoring and Dosage Adjustments of Immunosuppressive Drugs When Combined With Nirmatrelvir/Ritonavir in Patients With COVID-19.

ABSTRACT: Nirmatrelvir/ritonavir (Paxlovid) consists of a peptidomimetic inhibitor (nirmatrelvir) of the SARS-CoV-2 main protease and a pharmacokinetic enhancer (ritonavir). It is approved for the treatment of mild-to-moderate COVID-19. This combination of nirmatrelvir and ritonavir can mediate significant and complex drug-drug interactions (DDIs), primarily due to the ritonavir component. Indeed, ritonavir inhibits the metabolism of nirmatrelvir through cytochrome P450 3A (CYP3A) leading to higher plasma concentrations and a longer half-life of nirmatrelvir. Coadministration of nirmatrelvir/ritonavir with immunosuppressive drugs (ISDs) is particularly challenging given the major involvement of CYP3A in the metabolism of most of these drugs and their narrow therapeutic ranges. Exposure of ISDs will be drastically increased through the potent ritonavir-mediated inhibition of CYP3A, resulting in an increased risk of adverse drug reactions. Although a decrease in the dosage of ISDs can prevent toxicity, an inappropriate dosage regimen may also result in insufficient exposure and a risk of rejection. Here, we provide some general recommendations for therapeutic drug monitoring of ISDs and dosing recommendations when coadministered with nirmatrelvir/ritonavir. Particularly, tacrolimus should be discontinued, or patients should be given a microdose on day 1, whereas cyclosporine dosage should be reduced to 20% of the initial dosage during the antiviral treatment. Dosages of mammalian target of rapamycin inhibitors (m-TORis) should also be adjusted while dosages of mycophenolic acid and corticosteroids are expected to be less impacted.

Endogenous and iatrogenic sources of variability in response to opioid therapy in Post-Surgical and injured orthopedic patients.

BACKGROUND: Hydrocodone is the most prescribed opioid in the US. The objective was to evaluate associations between genetic, intrinsic, and extrinsic patient factors, plasma hydrocodone and metabolites, common side effects, and pain scores in a cohort of orthopedic surgery patients. METHODS: Data for each patient was collected by review of the electronic hospital record (EHR), and patient interview. Patients were recruited from those with trauma or undergoing scheduled elective surgery for total knee replacement or total hip at the University of Louisville Hospital, Baptist East Hospital, and Jewish Hospital, Louisville, KY. Plasma opiate concentrations and a targeted genotyping panel was performed. RESULTS: There were statistically significant correlations with daily (p < 0.001) and total dose (p = 0.002) of hydrocodone in hospital and duration of opioid therapy. The length of opioid administration was significantly shorter in CYP2D6 EM/UM versus CYP2D6 PM/IM patients (p = 0.018). Subjects with the OPRM1 c.118G variant were also on opioids longer (p = 0.022). The effect of co-administration of a CYP2D6 inhibitor had a significant effect on the length of opioid therapy (P < 0.001). And not surprisingly the effect of the inhibitor adjusted CYP2D6 phenotype was greater in both the hospital stay period and days of opioid use post hospital discharge (p < 0.001). CONCLUSIONS: Based on this study, patients should be evaluated for the use of inhibitors of CYP2D6, during hydrocodone therapy can alter the phenotype of the patient (phenocopy) and increase the probability that the patient will be on opioids for longer periods of time.

Personalized Therapy for Mycophenolate: Consensus Report by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.

ABSTRACT: When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.

Assessment of mass spectrometry-based pain management testing in clinical laboratories across North America.

BACKGROUND: Urine drug testing (UDT) is a useful tool in monitoring compliance to prescribed medication and can also help identify behaviors of drug misuse, abuse, and diversion. Mass spectrometry (MS)-based screening is recommended as the first-line of UDT for pain management patients; however, this testing comes with an inherent lack of standardization in methodologies and various analytical challenges. The objective of this study was to assess the current state of UDT for pain management in a cross-section of clinical laboratories in North America. MATERIALS AND METHODS: A total of 10 blinded urine samples were sent to 6 laboratories across the United States and Canada. Urine samples containing drugs and/or metabolites of interest were included to represent different clinical scenarios commonly seen in pain management settings. Assessment was based on the ability of the laboratories to correctly identify drugs and provide a meaningful interpretation of the findings (when offered by the performing laboratory). RESULTS: Across the laboratories involved in the study, 85% of tests correctly identified and appropriately reported the drugs present in the urine samples. Similarly, 84% of samples were considered to have an accurate interpretation included in the UDT report. Out of the total number of drugs included in the samples, 11% were not offered on every test menu. CONCLUSIONS: This study revealed the lack of standardization in pain management UDT performed in a limited cross-section of clinical laboratories across North America.

Volumetric Microsampling of Capillary Blood Spot vs Whole Blood Sampling for Therapeutic Drug Monitoring of Tacrolimus and Cyclosporin A: Accuracy and Patient Satisfaction.

BACKGROUND: Immunosuppressant therapeutic drug monitoring (TDM) usually requires outpatient travel to hospitals or phlebotomy sites for venous blood collection; however Mitra® Microsampling Device (MSD) sampling could allow self-collection and shipping of samples to a laboratory for analysis. This study examined the feasibility of using volumetric microsampling by MSD for TDM of tacrolimus (TaC) and cyclosporin A (CsA) in transplant patients, along with their feedback on the process. METHODS: MSD was used to collect TaC and CsA from venous (VB) or capillary (CB) blood. The MSDs were rehydrated, extracted, and analyzed using on-line solid phase extraction coupled to tandem mass spectrometry (SPE-MS/MS). We report an abbreviated method validation of the MSD including: accuracy, precision, linearity, carry-over, and stability using residual venous whole blood (VB) samples. Subsequent clinical validation compared serially collected MSD + CB against VB (200 µL) from transplant patients. RESULTS: Accuracy comparing VB vs. MSD+VB showed high clinical concordance (TaC = 89% and CsA = 98%). Inter- and intra-precision was ≤11.5 %CV for TaC and CsA. Samples were stable for up to 7 days at room temperature with an average difference of <10%. Clinical validation with MSD+CB correlated well with VB for CsA (slope = 0.95, r2 = 0.88, n = 47) and TaC (slope = 0.98, r2 = 0.82, n = 49). CB vs. VB gave concordance of 94% for CsA and 79% for TaC. A satisfaction survey showed 82% of patients preferred having the capillary collection option. CONCLUSION: Transplant patients favored having the ability to collect capillary samples at home for TaC/CsA monitoring. Our results demonstrate good concordance between MSD+CB and VB for TaC and CsA TDM, but additional studies are warranted.

Pharmacologic Treatment of Transplant Recipients Infected With SARS-CoV-2: Considerations Regarding Therapeutic Drug Monitoring and Drug-Drug Interactions.

BACKGROUND: COVID-19 is a novel infectious disease caused by the severe acute respiratory distress (SARS)-coronavirus-2 (SARS-CoV-2). Several therapeutic options are currently emerging but none with universal consensus or proven efficacy. Solid organ transplant recipients are perceived to be at increased risk of severe COVID-19 because of their immunosuppressed conditions due to chronic use of immunosuppressive drugs (ISDs). It is therefore likely that solid organ transplant recipients will be treated with these experimental antivirals. METHODS: This article is not intended to provide a systematic literature review on investigational treatments tested against COVID-19; rather, the authors aim to provide recommendations for therapeutic drug monitoring of ISDs in transplant recipients infected with SARS-CoV-2 based on a review of existing data in the literature. RESULTS: Management of drug-drug interactions between investigational anti-SARS-CoV-2 drugs and immunosuppressants is a complex task for the clinician. Adequate immunosuppression is necessary to prevent graft rejection while, if critically ill, the patient may benefit from pharmacotherapeutic interventions directed at limiting SARS-CoV-2 viral replication. Maintaining ISD concentrations within the desired therapeutic range requires a highly individualized approach that is complicated by the pandemic context and lack of hindsight. CONCLUSIONS: With this article, the authors inform the clinician about the potential interactions of experimental COVID-19 treatments with ISDs used in transplantation. Recommendations regarding therapeutic drug monitoring and dose adjustments in the context of COVID-19 are provided.

The challenge that chlorine presented during the development of a benzodiazepine assay.

During the routine validation of a benzodiazepine method (performed on a Liquid Chromatography - Tandem Mass Spectrometer), it was noted that lorazepam, triazolam, and α-hydroxytriazolam showed a quadratic shift/bias in the calibration curve, particularly at high concentrations. The ultimate cause of this bias was determined to be due to the natural presence of chlorine (Cl) isotopes (35Cl and 37Cl) in these benzodiazepines. The presence of the heavy (37Cl) isoforms of Cl resulted in the analyte's mass being the same as the internal standard which, in turn, caused the internal standard to appear "falsely increased", thus skewing the calibration curve. One solution to this potential issue was to take advantage of this natural phenomenon and use the Cl heavy isoforms of the respectively labeled internal standards.

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.

Targeted Opioid Screening Assay for Pain Management Using High-Resolution Mass Spectrometry.

The use and adherence monitoring of opioids in pain management is recommended by numerous clinical practice guidelines. Many physicians use urine immunoassay screening tests, which suffer from a lack of sensitivity and specificity, to verify compliance to pain medications. However, several immunoassay tests are required to comprehensively detect the synthetic, semisynthetic, and natural opioids due to the limited cross-reactivity of each assay. Superior testing strategies are required to specifically identify low concentrations of opioids found in adherent pain management patients. Therefore we present a method for the qualitative identification of 33 opioids and metabolites (codeine, codeine-6-ß-glucuronide, morphine, morphine-6-ß-glucuronide, 6-acetylmorphine, hydrocodone, norhydrocodone, dihydrocodeine, hydromorphone, hydromorphone-3-ß-glucuronide, oxycodone, noroxycodone, oxymorphone, oxymorphone-3-ß-glucuronide, noroxymorphone, meperidine, normeperidine, methadone, EDDP, propoxyphene, norpropoxyphene, tramadol, O-desmethyltramadol, tapentadol, tapentadol-ß-glucuronide, N-desmethyltapentadol, buprenorphine, norbuprenorphine, norbuprenorphine glucuronide, naloxone, naloxone glucuronide, fentanyl, and norfentanyl) in unhydrolyzed urine using a liquid chromatography tandem mass spectrometry (LC-MS/MS) with high-resolution, accurate-mass Orbitrap detection.

Simultaneous Determination of Tacrolimus and Cyclosporine A in Whole Blood by Ultrafast LC-MS/MS.

Numerous methods for the measurement of tacrolimus and cyclosporine A involving traditional liquid chromatography tandem mass spectrometry (LC-MS/MS) have previously been described. The majority of these methods use solid-phase extraction, liquid-liquid extraction, or protein precipitation extraction with instrument run times greater than 15 s per sample. Continued demands in clinical labs for greater efficiency and throughput have put increased stress on traditional technologies such as high-performance liquid chromatography-ultraviolet detection (HPLC-UV) and traditional LC-MS/MS. As an improvement to the existing methods, we describe a sensitive ultrafast LC-MS/MS with run times of less than 15 s per sample.

Unidentified anion gap metabolic acidosis.

A 35-month-old female with nonketotic hyperglycinemia (NKH) presented to the Emergency department with severe hypoglycemia, fever, and several episodes of seizures. Due to worsening respiratory status, additional seizures and anion gap worsening metabolic acidosis the patient was transferred to the pediatric intensive care unit. The useful mnemonics for causes of high anion gap metabolic acidosis are the classic MUDPILES (representing Methanol, Uraemia, Diabetes, Paraldehyde, Iron (and Isoniazid), Lactate, Ethylene glycol, and Salicylate) and the more recently proposed GOLD MARK (Glycols [ethylene and propylene], Oxoproline, l-lactate, d-lactate, Methanol, Aspirin, Renal failure, and Ketoacidosis) as causes of the anion gap metabolic acidosis were all ruled out. Relatively stable concentrations of salicylate (approximately 10 mg/dL, 0.7 mmol/L) were noted, despite no evidence the patient received aspirin Therefore further laboratory testing was performed. A Basic-Acid-Neutral (BAN) gas chromatography mass-spectroscopy (GC-MS) Drug screen of urine was undertaken. A large benzoic acid peak was identified by spectral match, which supported the clinical history that the patient was taking sodium benzoate powder 1175 mg as a dietary supplement three times a day. However, salicylate was not identified. This patient had benzoic acid concentration in excess of 2000 µg/mL. Given that benzoic acid is a weak acid, with a pK of approximately 4 it is almost completely ionized at pH 7. Therefore, the large amount of benzoic acid was not only thought to be contributing to the patient's anion gap metabolic acidosis, but the source of the interference in the salicylate assay.

Intravenous Ribavirin for Parainfluenza and Respiratory Syncytial Virus in an Infant Receiving Extracorporeal Membrane Oxygenation and Continuous Renal Replacement Therapy.

BACKGROUND: Viral bronchiolitis remains a significant cause of hospitalization as well as morbidity and mortality during the first year of life, with treatment options beyond supportive care being limited. In cases of severe illness, ribavirin may offer therapeutic benefit. OBJECTIVE: We report the use of intravenous (IV) ribavirin in an infant requiring concomitant venovenous extracorporeal membrane oxygenation (VV-ECMO) and continuous venovenous hemofiltration (CVVH) for respiratory syncytial virus (RSV) and parainfluenza virus (PIV) coinfection. PATIENTS AND METHODS: A 5-week-old male former 33-week preterm infant was admitted with respiratory failure and subsequently tested positive for RSV and PIV-type 1 infection. Progressive clinical deterioration subsequently required the initiation of both VV-ECMO and CVVH. Although the patient received combined VV-ECMO and CVVH, IV ribavirin was administered, and serial plasma and ultrafiltrate samples were obtained for pharmacokinetic analyses after the first dose (collection period 1) and again after an estimated 5 half-lives (collection period 2). RESULTS: Pharmacokinetics for collection period 1 demonstrated a calculated Cmax of 11.99 mg/L, an AUC0-24 of 43.32 mg·hr/L, ke 0.26 hr-1, t½ 2.69 hr, Vd 10.04 L (2.92 L/kg, using patient's dosing weight 3.43 kg), CLT 43.47 mL/min, and CLCVVH 6.75 mL/min. Pharmacokinetics for collection period 2 demonstrated a calculated Cmax of 10.31 mg/L, AUC0-6 of 52.55 mg· hr/L, ke 0.06 hr-1, t½ 10.69 hr, Vd 17.5 L (5.1 L/kg), and CLT 17.44 mL/min. The sieving coefficient during collection period 1 was 1.17 (range, 1.07-1.37). The percent decline between prefilter and postfilter oxygenator was 19.1%. CONCLUSION: Our patient demonstrated therapeutic concentrations of ribavirin, despite drug removal via CVVH and the ECMO oxygenator. Standard ribavirin dosing used and resultant concentrations achieved were associated with viral clearance and clinical improvement.