Validation of Population Pharmacokinetic Models for Clozapine Dosage Prediction.

BACKGROUND: Clozapine is unique in its capacity to ameliorate severe schizophrenia but at high risk of toxicity. A relationship between blood concentration and clinical response and evidence for concentration-response relationships to some adverse effects justify therapeutic drug monitoring of clozapine. However, the relationship between drug dose and blood concentration is quite variable. This variability is, in part, due to inductive and inhibitory interactions varying the activity of cytochrome P450 1A2 (CYP1A2), the principal pathway for clozapine elimination. Several population pharmacokinetic models have been presented to facilitate dose selection and to identify poor adherence in individual patients. These models have faced little testing for validity in independent populations or even for persisting validity in the source population. METHODS: Therefore, we collected a large population of clozapine-treated patients (127 patients, 1048 timed plasma concentrations) in whom dosing and covariate information could be obtained with high certainty. A population pharmacokinetic model was constructed with data collected in the first 6 weeks from study enrolment (448 plasma concentrations), to estimate covariate influences and to allow alignment with previously published models. The model was tested for its performance in predicting the concentrations observed at later time intervals up to 5 years. The predictive performances of 6 published clozapine population models were then assessed in the entire population. RESULTS: The population pharmacokinetic model based on the first 6 weeks identified significant influences of sex, smoking, and cotreatment with fluvoxamine on clozapine clearance. The model built from the first 6 weeks had acceptable predictive performance in the same patient population up to the first 26 weeks using individual parameters, with a median predictive error (PE) of -0.1% to -15.9% and median absolute PE of 22.9%-27.1%. Predictive performance fell progressively with time after 26 weeks. Bayesian addition of plasma concentration observations within each prediction period improved individual predictions. Three additional observations extended acceptable predictive performance into the second 6 months of therapy. When the published models were tested with the entire data set, median PE ranged from -8% to +35% with a median absolute PE of >39% in all models. Thus, none of the tested models was successful in external validation. Bayesian addition of single patient observations improved individual predictions from all models but still without achieving acceptable performances. CONCLUSIONS: We conclude that the relationship between covariates and blood clozapine concentrations differs between populations and that relationships are not stable over time within a population. Current population models for clozapine are not capturing influential covariates.

The Addition of Hand-specific Skeletal Maturity Parameters Does Not Improve Skeletal Maturity Estimation Accuracy of the Modified Fels Wrist System.

BACKGROUND: The Modified Fels Wrist system is potentially the most accurate clinically accessible skeletal maturity system utilizing hand or wrist radiographs. During development, parameters distal to the metacarpals were excluded. We attempted to further optimize the Modified Fels wrist system through the inclusion of hand parameters distal to the metacarpals. METHODS: Forty-three new anteroposterior (AP) hand radiographic parameters were identified from the Fels and Greulich and Pyle (GP) skeletal maturity systems. Twelve parameters were eliminated from further evaluation for poor correlation with skeletal maturity, poor reliability, and lack of relevance in the peripubertal years. In addition to the 8 previously described Modified Fels Wrist parameters, 31 hand radiographic parameters were evaluated on serial peripubertal AP hand radiographs to identify the ones most important for accurately estimating skeletal age. This process produced a "Modified Fels hand-wrist" model; its performance was compared with (1) GP only; (2) Sanders Hand (SH) only; (3) age, sex, and GP; (4) age, sex, and SH; and (5) Modified Fels Wrist system. RESULTS: Three hundred seventy-two radiographs from 42 girls and 38 boys were included. Of the 39 radiographic parameters that underwent full evaluation, 9 remained in the combined Modified Fels Hand-Wrist system in addition to chronological age and sex. Four parameters are wrist specific, and the remaining 5 are hand specific. The Hand-Wrist system outperformed both GP and SH in estimating skeletal maturity ( P <0.001). When compared with the Modified Fels Wrist system, the Modified Fels Hand-Wrist system performed similarly regarding skeletal maturity estimation (0.36±0.32 vs. 0.34±0.26, P =0.59) but had an increased (worse) rate of outlier predictions >1 year discrepant from true skeletal maturity (4.9% vs. 1.9%, P =0.01). CONCLUSIONS: The addition of hand parameters to the existing Modified Fels Wrist system did not improve skeletal maturity estimation accuracy and worsened the rate of outlier estimations. When an AP hand-wrist radiograph is available, the existing Modified Fels wrist system is best for skeletal maturity estimation. LEVEL OF EVIDENCE: Level III.

Risk Factors for Chronic Atrial Fibrillation Following Lung Lobectomy.

INTRODUCTION: Postoperative atrial fibrillation (POAF) is a common complication following lung lobectomy and is associated with increased risk of stroke, mortality, and prolonged hospital length of stay. The purpose of this study was to define the risk factors for POAF after lobectomy, hypothesizing that operative approach would be associated with risk of chronic POAF. METHODS: The TriNetX database was used to identify adult patients with no history of arrythmia receiving elective lung lobectomy for cancer from 7/6/2003-7/6/2023. Patients were categorized by approach: video-assisted thoracoscopic surgery (VATS) or open. The outcome of interest was the presence of POAF occurring at 1-3 months ("early") and 12-24 months postop ("chronic"). Propensity matching was performed to reduce bias between cohorts. RESULTS: We identified 22,998 patients: 8472 (36.8%) who received open and 14,526 (63.2%) VATS lobectomy. The rate of early POAF was 3.7% of VATS and 5.3% of open patients. The rate of chronic POAF was 5.5 % of VATS patients and 6.2% of open lobectomy patients. Propensity matching decreased bias between the approach groups, creating 7942 pairs for analysis. After matching, the risk of early POAF was greater in the open approach (5.5% open vs 3.4% VATS, risk ratio 1.607 (95% confidence interval 1.385-1.865), P < 0.001). Chronic POAF was (also) higher in the open approach (6.3% open vs 5.2% VATS, Risk Ratio 1.211 (95%CI 1.067-1.374), P = 0.003). CONCLUSIONS: Postoperative atrial fibrillation (POAF) occurs more commonly after open lobectomy, both acutely and chronically. Providers should counsel patients about the risk of chronic arrythmia after lung resection.

Methionol as an aggregation-sex pheromone of the desert-dwelling beetle Eustromula valida (Coleoptera: Cerambycidae).

As part of a long-term project on unraveling the use of pheromones in the large beetle family Cerambycidae, field trials were conducted with generic blends of known cerambycid pheromones at a desert site in southern California. In the first year of testing (2022), the species Eustromula valida (LeConte) (subfamily Cerambycinae, tribe Elaphidiini) and Aethecerinus latecinctus (Horn) (Cerambycinae, Trachyderini) were weakly attracted to one of the lure blends. In follow-up trials in 2023, only E. valida were caught, and collection of volatiles from both sexes of E. valida determined that males sex-specifically produced 3-methylthiopropan-1-ol (methionol), a compound that was not in the tested lure blends. Beetles of both sexes were strongly and specifically attracted to this compound in field bioassays, verifying that it is an aggregation-sex pheromone. No sympatric species were attracted to methionol while it was deployed in the field. Several recent studies have identified methionol as a pheromone component for other cerambycid species in both North and South America, suggesting it may represent another common pheromone component within the Cerambycidae.

Assessing and Identifying Improvements for Lung Cancer Screening in a Rural Population: A Human-Centered Design and Systems Approach.

Although lung cancer claims more lives than any other cancer in the United States, screening is severely underutilized, with <6% of eligible patients screened nationally in 2021 versus 76% for breast cancer and 67% for colorectal cancer. This article describes an effort to identify key reasons for the underutilization of lung cancer screening in a rural population and to develop interventions to address these barriers suitable for both a large health system and local community clinics. Data were generated from 26 stakeholder interviews (clinicians, clinical staff, and eligible patients), a review of key systems (Electronic Health Record and billing records), and feedback on the feasibility of several potential interventions by health care system staff. These data informed a human-centered design approach to identify possible interventions within a complex health care system by exposing gaps in care processes and electronic health record platforms that can lead patients to be overlooked for potentially life-saving screening. Deployed interventions included communication efforts focused on (1) increasing patient awareness, (2) improving physician patient identification, and (3) supporting patient management. Preliminary outcomes are discussed.

(1R,4R)-Quercivorol, a Male-Produced Aggregation-Sex Pheromone of the Beetle Elytrimitatrix undata (F.) (Coleoptera: Disteniidae).

The beetle family Disteniidae is currently considered to be closely related to the much larger family Cerambycidae, the longhorned beetles. The 300 + species of disteniids are mostly native to tropical and subtropical regions, with the only described North American species north of Mexico being Elytrimitatrix undata (F.). Here we describe the identification and field testing of (1R,4R)-quercivorol as a male-produced aggregation-sex pheromone component for E. undata. This is the first pheromone identified for any species within the family Disteniidae.

Contemporary outcomes of surgical resection for chest wall chondrosarcoma.

Objective: Chondrosarcoma is the most common primary malignant chest wall tumor and is historically associated with poor prognosis. Recommendations regarding surgical excision are on the basis of small, single-institution studies. We used a large national database to assess outcomes of surgery for chest wall chondrosarcoma (CWC) hypothesizing that surgical excision remains standard of care. Methods: The National Cancer Databases for bone and soft tissue were merged to identify patients with chondrosarcoma from 2004 to 2018. Clinical and demographic characteristics of CWC were compared with chondrosarcoma from other sites. The primary outcome was overall survival described using Kaplan-Meier estimate. Univariable and multivariable Cox analysis was used to determine risk factors for poor survival among CWC patients who underwent surgery. Multivariable analysis of predictors of margin status was performed because of worse prognosis associated with positive margins. Results: Among 11,925 patients with chondrosarcoma, 1934 (16.2%) had a CWC. Relative to other sites, CWC was associated with older age, male sex, White race, surgical resection, and care at a nonacademic institution. CWC was associated with 1-, 3-, 5-, and 10-year survival of 91.5%, 82.0%, 75.5%, and 62.7%, respectively. In univariable analysis, survival was associated with surgery (hazard ratio, 0.02; P < .001) and adversely affected by positive margins (hazard ratio, 2.66; P < .001). Multivariable analysis showed larger tumor size was independently associated with increased risk for positive margins (odds ratio, 1.04; 95% CI, 1.011-1.075). Conclusions: CWC represents a different cohort of patients relative to chondrosarcoma from other sites. Surgical excision remains the optimal treatment, and positive margins are associated with poor prognosis.

Identification of a Female-Produced Sex Attractant Pheromone of the Winter Firefly, Photinus corruscus Linnaeus (Coleoptera: Lampyridae).

Firefly flashes are well-known visual signals used by these insects to find, identify, and choose mates. However, many firefly species have lost the ability to produce light as adults. These "unlighted" species generally lack developed adult light organs, are diurnal rather than nocturnal, and are believed to use volatile pheromones acting over a distance to locate mates. While cuticular hydrocarbons, which may function in mate recognition at close range, have been examined for a handful of the over 2000 extant firefly species, no volatile pheromone has ever been identified. In this study, using coupled gas chromatography - electroantennographic detection, we detected a single female-emitted compound that elicited antennal responses from wild-caught male winter fireflies, Photinus corruscus. The compound was identified as (1S)-exo-3-hydroxycamphor (hydroxycamphor). In field trials at two sites across the species' eastern North American range, large numbers of male P. corruscus were attracted to synthesized hydroxycamphor, verifying its function as a volatile sex attractant pheromone. Males spent more time in contact with lures treated with synthesized hydroxycamphor than those treated with solvent only in laboratory two-choice assays. Further, using single sensillum recordings, we characterized a pheromone-sensitive odorant receptor neuron in a specific olfactory sensillum on male P. corruscus antennae and demonstrated its sensitivity to hydroxycamphor. Thus, this study has identified the first volatile pheromone and its corresponding sensory neuron for any firefly species, and provides a tool for monitoring P. corruscus populations for conservation and further inquiry into the chemical and cellular bases for sexual communication among fireflies.

Disparities in Access to Thoracic Surgeons among Patients Receiving Lung Lobectomy in the United States.

OBJECTIVE: Lung lobectomy is the standard of care for early-stage lung cancer. Studies have suggested improved outcomes associated with lobectomy performed by specialized thoracic surgery providers. We hypothesized that disparities would exist regarding access to thoracic surgeons among patients receiving lung lobectomy for cancer. METHODS: The Premier Hospital Database was used to identify adult inpatients receiving lung lobectomy from 2009 to 2019. Patients were categorized as receiving their lobectomy from a thoracic surgeon, cardiovascular surgeon, or general surgeon. Sample-weighted multivariable analysis was performed to identify factors associated with provider type. RESULTS: When adjusted for sampling, 121,711 patients were analyzed, including 71,709 (58.9%) who received lobectomy by a thoracic surgeon, 36,630 (30.1%) by a cardiovascular surgeon, and 13,373 (11.0%) by a general surgeon. Multivariable analysis showed that thoracic surgeon provider type was less likely with Black patients, Medicaid insurance, smaller hospital size, in the western region, and in rural areas. In addition, non-thoracic surgery specialty was less likely to perform minimally-invasive (MIS) lobectomy (cardiovascular OR 0.80, p < 0.001, general surgery OR 0.85, p = 0.003). CONCLUSIONS: In this nationally representative analysis, smaller, rural, non-teaching hospitals, and certain regions of the United States are less likely to receive lobectomy from a thoracic surgeon. Thoracic surgeon specialization is also independently associated with utilization of minimally invasive lobectomy. Combined, there are significant disparities in access to guideline-directed surgical care of patients receiving lung lobectomy.

A pleiotropic chemoreceptor facilitates the production and perception of mating pheromones.

Optimal mating decisions depend on the robust coupling of signal production and perception because independent changes in either could carry a fitness cost. However, since the perception and production of mating signals are often mediated by different tissues and cell types, the mechanisms that drive and maintain their coupling remain unknown for most animal species. Here, we show that in Drosophila, behavioral responses to, and the production of, a putative inhibitory mating pheromone are co-regulated by Gr8a, a member of the Gustatory receptor gene family. Specifically, through behavioral and pheromonal data, we found that Gr8a independently regulates the behavioral responses of males and females to a putative inhibitory pheromone, as well as its production in the fat body and oenocytes of males. Overall, these findings provide a relatively simple molecular explanation for how pleiotropic receptors maintain robust mating signaling systems at the population and species levels.

Hematocrit Correction of Whole Blood Phosphatidylethanol Concentrations to Estimate Erythrocyte PEth Concentrations: Sensitivity, Specificity and Influence on Test Utility.

Phosphatidylethanol (PEth) forms in erythrocyte membranes after alcohol consumption, offering a persisting biomarker, that is measurable in whole blood, washed erythrocytes and dried blood spots. For a predominantly erythrocyte-restricted analyte, erythrocyte concentrations seem to have most validity in patients who are anemic through alcoholism or other pathologies, despite preparation increasing assay complexity. Differences in specimen preparation alter PEth concentrations for the same patient, meaning that criteria for interpreting PEth results should relate to specimen type, presenting a barrier to achieving harmonization. We therefore tested whether erythrocyte PEth might be validly calculated by hematocrit correction of a whole blood PEth measurement. PEth testing primarily serves to distinguish drinkers from non-drinkers. In choosing between specimen types, it is important to compare their utility in separating those two groups. We therefore processed 281 blood samples from 17 non-drinkers and 61 drinkers, to prepare matched whole blood and washed erythrocyte specimens. These were assayed by liquid chromatography-tandem mass spectrometry and compared in identifying alcohol consumption. The erythrocyte PEth concentration in the whole blood specimens was also calculated by correcting whole blood concentration by the specimen's hematocrit, as an alternative to prepare washed erythrocytes. The hematocrit-corrected erythrocyte concentrations were included in these comparisons. Predictably, this work found that sensitivity was consistently better at the lower cut-off of 8 µg/L than at 20 µg/L. Sensitivities were also higher for washed erythrocytes than whole blood, explained by the lower erythrocyte mass in the same volume of whole blood. Hematocrit-corrected whole blood PEth concentrations correlated with erythrocyte concentrations, except for the four highest values, which did not influence comparative sensitivity. Specificity was 100% for washed erythrocytes, whole blood and hematocrit-corrected whole blood at either cut-off because non-drinkers had undetectable PEth. We conclude that hematocrit correction of whole blood PEth concentrations theoretically provides an alternative to the preparation of washed erythrocytes.

Two-Photon Polymerization: Fundamentals, Materials, and Chemical Modification Strategies.

Two-photon polymerization (TPP) has become a premier state-of-the-art method for microscale fabrication of bespoke polymeric devices and surfaces. With applications ranging from the production of optical, drug delivery, tissue engineering, and microfluidic devices, TPP has grown immensely in the past two decades. Significantly, the field has expanded from standard acrylate- and epoxy-based photoresists to custom formulated monomers designed to change the hydrophilicity, surface chemistry, mechanical properties, and more of the resulting structures. This review explains the essentials of TPP, from its initial conception through to standard operating principles and advanced chemical modification strategies for TPP materials. At the outset, the fundamental chemistries of radical and cationic polymerization are described, along with strategies used to tailor mechanical and functional properties. This review then describes TPP systems and introduces an array of commonly used photoresists including hard polyacrylic resins, soft hydrogel acrylic esters, epoxides, and organic/inorganic hybrid materials. Specific examples of each class-including chemically modified photoresists-are described to inform the understanding of their applications to the fields of tissue-engineering scaffolds, micromedical, optical, and drug delivery devices.

Comparing Thoracoscopic and Robotic Lobectomy Using a Nationally Representative Database.

BACKGROUND: Studies of robotic lobectomy (Robot-L) have been performed using data from high-volume, specialty centers which may not be generalizable. The purpose of this study was to compare mortality, length of stay (LOS), and cost between Robot-L and thoracoscopic lobectomy (VATS-L) using a nationally representative database hypothesizing they would be similar. METHODS: The Premier Healthcare Database was used to identify patients receiving elective lobectomy for lung cancer from 2009 to 2019. Patients were categorized as receiving Robot-L or VATS-L using ICD-9/10 codes. Survey methodology and patient level weighting were used to correct for sampling error and estimation of a nationally representative sample. A propensity match analysis was performed to reduce bias between the groups. Primary outcome of interest was in-hospital mortality. Secondary outcomes were LOS and patient charges. RESULTS: Among 62 698 patients, 19 506 (31.1%) underwent Robot-L and 43 192 (68.9%) underwent VATS-L. Differences between the groups included age, race, comorbidities, and insurance type. A propensity matched cohort demonstrated similar in-hospital mortality for Robot-L and VATS-L (.9% vs .9%, respectively, P = .91). Patients who underwent Robot-L had a shorter LOS (4 vs 5d, respectively, P < .001) but higher patient charges (90 593.0 vs 72 733.3 USD, respectively, P < .001). CONCLUSIONS: In a nationally representative database, Robot-L and VATS-L had similar mortality. Although Robot-L was associated with shorter hospitalization, it was also associated with excess charges of almost $20,000. As Robot-L is now the most common approach for lobectomy in the U.S., further study into the cost and benefit of robotic surgery is warranted.

Identification and Bioassays of Sex-Specific Compounds From a Nuisance Net-Spinning Caddisfly Smicridea fasciatella (Trichoptera: Hydropsychidae).

Municipalities in Arizona and Nevada along the Colorado River are subject to seasonal mass emergences of a nuisance net-spinning caddisfly, Smicridea fasciatella McLachlan (Trichoptera: Hydropsychidae). Here, we describe the characterization and field testing of S. fasciatella extracts to evaluate their potential as lures in baited traps. Solvent extracts of external (i.e., full body-cuticular hydrocarbon, abdominal hexane washes) and internal (i.e., crushed abdomen) parts of adult S. fasciatella were prepared from both sexes, and analyzed by coupled gas chromatography-mass spectrometry (GCMS). Several sex-specific compounds were identified, including (6Z,9Z)-6,9-nonadecadiene and (3Z,6Z,9Z)-3,6,9-nonadecatriene from males, and 2-undecanone, 2-tridecanone, and a heptadecene isomer from females. Extracts from both sexes were also analyzed by coupled gas chromatography-electroantennographic detection (EAD) using antennae of males for detection. Antennae of males weakly responded to 2-undecanone and 2-tridecanone, and to their corresponding alcohols, 2-undecanol and 2-tridecanol, which were included in field tests. Extracts of adult males did not elicit a response from male antennae, suggesting that males do not produce aggregation pheromones attractive to other males. The synchronized, dense populations of lekking males and other possible mating signals (e.g., visual recognition) may have contributed to the minimal attraction seen to test lures deployed in PHEROCON 1C traps. Overall, our results suggest that for this species, attractant pheromones have minimal or no role in bringing the sexes together for mating.

Identification and Synthesis of Leptotriene, a Unique Sesquiterpene Hydrocarbon from Males of the Leaffooted Bugs Leptoglossus zonatus and L. occidentalis.

The leaffooted bugs Leptoglossus zonatus and L. occidentalis (Hemiptera: Coreidae) cause substantial damage in tree nut crops in North America and pine seed orchards in North America and Europe, respectively. Sexually mature males of both species produce a number of aldehydes, esters, and sesquiterpenes, which are hypothesized to constitute an aggregation pheromone attractive to both sexes. Among the volatiles produced by males of both species, we identified a unique sesquiterpene hydrocarbon, given the common name "leptotriene" (5), which elicited strong responses from antennae of both sexes in electroantennogram assays. Here, we report its structure and its synthesis from (-)-(E)-ß-caryophyllene (1).

Healthcare quality during pediatric mental health boarding: A qualitative analysis.

BACKGROUND: Following initial evaluation and management, youth requiring inpatient psychiatric care often experience boarding, defined as being held in the emergency department or another location while awaiting inpatient care. Although mental health boarding is common, little research has examined the quality of healthcare delivery during the boarding period. OBJECTIVE: This study aimed to explore the perspectives and experiences of multidisciplinary clinicians and parents regarding mental health boarding and to develop a conceptual model to inform quality improvement efforts. DESIGN, SETTING, & PARTICIPANTS: We conducted semistructured interviews with clinicians and parents of youth experiencing boarding. Interviews focused on experiences of care and perceived opportunities for improvement were continued until thematic saturation was reached. Interviews were recorded, transcribed, and analyzed to identify emergent themes using a general inductive approach. Axial coding was used to inform conceptual framework development. RESULTS: Interviews were conducted with 19 clinicians and 11 parents. Building on the Donabedian structure-process-outcome model of quality evaluation, emergent domains, and associated themes included: (1) infrastructure for healthcare delivery, including clinician training, healthcare team composition, and the physical environment; (2) processes of healthcare delivery, including clinician roles and responsibilities, goals of care, communication with families, policies/procedures, and logistics of inter-facility transfer; and (3) measurable outcomes, including patient safety, family experience, mental health status, timeliness of care, and clinician moral distress. CONCLUSION: This qualitative study summarizes clinician and family perspectives about care for youth experiencing boarding. The conceptual model resulting from this analysis can be applied to implement and evaluate quality improvement endeavors to support this vulnerable population.

A Symmetrical Diester as the Sex Attractant Pheromone of the North American Click Beetle Parallelostethus attenuatus (Say) (Coleoptera: Elateridae).

Hexanoic acid, 1-octanol, 1,8-octanediol, octyl hexanoate, 1,8-octanediol monohexanoate, and 1,8-octanediol dihexanoate were identified in headspace volatiles collected from the crushed abdomen of a female click beetle of the species Parallelostethus attenuatus (Say) (Elaterinae, tribe Elaterini). In field trials carried out in Illinois, South Carolina, North Carolina, and Virginia, adult male beetles were strongly attracted to 1,8-octanediol dihexanoate alone. Blends of the dihexanoate with one or more of the other compounds proved to be less attractive than the dihexanoate alone, suggesting that the pheromone of this species may consist of a single compound. The symmetrical diester structure of the pheromone is a novel natural product and appears to be structurally unique among insect pheromones.

Methionol, a Sulfur-Containing Pheromone Component from the North American Cerambycid Beetle Knulliana cincta cincta.

We describe the identification and field testing of 3-methylthiopropan-1-ol (methionol) as a male-produced aggregation-sex pheromone for the cerambycid beetle Knulliana cincta cincta (Drury) (subfamily Cerambycinae, tribe Bothriospilini). The corresponding sulfoxide, 3-methylsulfinylpropan-1-ol, was also produced sex-specifically by males, but its function remains unclear because the measured release rates of this compound from five different types of release devices were very low to undetectable. Unexpectedly, adults of the cerambycine Elaphidion mucronatum (Say) (Elaphidiini), primarily females, also were attracted by methionol, despite males of this species producing an aggregation-sex pheromone of entirely different structure, (2E,6Z,9Z)-2,6,9-pentadecatrienal.

Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for erythrocyte phosphatidylethanol revealing critical considerations for its use as a clinical biomarker.

Erythrocyte membrane-incorporated phosphatidylethanol (PEth) forms only in the presence of ethanol and, once formed, provides a persisting marker for historical alcohol consumption. Relationships between PEth concentration, extent of consumption and time from consumption are under investigation. Threshold values of PEth have been proposed as indicators for any, or for harmful alcohol consumption. Here, we describe an assay for erythrocyte PEth 16:0/18:1 that offers the efficiency needed for routine clinical deployment, in the context of a fully validated methodology. However, we observe that conventional procedures for validating assay methodology are insufficient where the analyte of interest, membrane-incorporated PEth 16:0/18:1, has different physicochemical properties to the soluble PEth 16:0/18:1 and PEth 16:0/18:1-d5 that are used for making calibrator, controls and internal standards. Whereas the internal standard did fully correct for differences in matrix effects and recovery when different extraction solvents were applied to calibrators and controls (in soluble form), it failed to correct for a 1.5-fold difference in the relative efficiency of two solvents, in this case, acetonitrile and isopropanol in extracting PEth from erythrocyte membrane in clinical samples. Differences in the efficiency of the extraction of membrane-bound PEth translate to different results from the same specimen. That can mean that threshold values derived by one methodology cannot be safely generalised to another. That hampers the generalisability of individual laboratory's experience with PEth assay results. Harmonising extraction methodology between laboratories becomes very important where membrane-incorporated PEth itself remains unavailable as an assay standard.