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Objectives: This study determined the performance characteristics of a quantitative glucose-6-phosphate dehydrogenase (G6PD) assay with automated lysis and evaluated the robustness of the operational workflow following implementation in a hospital laboratory., Methods: The G6PD activity was measured in whole blood using an enzymatic quantitative test on a Roche cobas c501 analyzer with onboard lysis configuration and normalized to hemoglobin (Hb). The performance characteristics of the method and stability of G6PD in whole blood collected in EDTA-containing tubes were evaluated, and the reference interval was established on a population of healthy individuals (n = 279). The robustness of this automated workflow for sample lysis was evaluated during validation and after implementation for routine clinical use for 18 months and in 2,181 patients., Results: The G6PD assay was linear from 0.7 to 16.5 U/g Hb. Inter- and intra-assay precision using control and patient samples was below 12%. The G6PD results correlated well with a reference laboratory method (r = 0.96, y = 0.9615x - 1.222). The reference interval in our population was 9.8 to 15.5 U/g Hb. There were no interferences by lipemia and icteria, although grossly hemolyzed specimens may be affected. The testing workflow requires analyzing samples within minutes from mixing and loading into the instrument to avoid sample sedimentation. Measures to repeat samples with Hb 8.0 g/dL or less identified sedimented samples. In our patient population, 10.6% and 5.8% of the total males and females tested were G6PD deficient, respectively., Conclusions: The G6PD assay with automated lysis is acceptable for patient testing. Several measures ensured the robustness of this workflow in a hospital laboratory., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

In unconscious appearing patients with acute brain injury, wilful brain activation to motor commands without behavioural signs of command following, known as cognitive motor dissociation (CMD), is associated with functional recovery. CMD can be detected by applying machine learning to EEG recorded during motor command presentation in behaviourally unresponsive patients. Identifying patients with CMD carries clinical implications for patient interactions, communication with families, and guidance of therapeutic decisions but underlying mechanisms of CMD remain unknown. By analysing structural lesion patterns and network level dysfunction we tested the hypothesis that, in cases with preserved arousal and command comprehension, a failure to integrate comprehended motor commands with motor outputs underlies CMD. Manual segmentation of T2-fluid attenuated inversion recovery and diffusion weighted imaging sequences quantifying structural injury was performed in consecutive unresponsive patients with acute brain injury (n = 107) who underwent EEG-based CMD assessments and MRI. Lesion pattern analysis was applied to identify lesion patterns common among patients with (n = 21) and without CMD (n = 86). Thalamocortical and cortico-cortical network connectivity were assessed applying ABCD classification of power spectral density plots and weighted pairwise phase consistency (WPPC) to resting EEG, respectively. Two distinct structural lesion patterns were identified on MRI for CMD and three for non-CMD patients. In non-CMD patients, injury to brainstem arousal pathways including the midbrain were seen, while no CMD patients had midbrain lesions. A group of non-CMD patients was identified with injury to the left thalamus, implicating possible language comprehension difficulties. Shared lesion patterns of globus pallidus and putamen were seen for a group of CMD patients, which have been implicated as part of the anterior forebrain mesocircuit in patients with reversible disorders of consciousness. Thalamocortical network dysfunction was less common in CMD patients [ABCD-index 2.3 (interquartile range, IQR 2.1-3.0) versus 1.4 (IQR 1.0-2.0), P < 0.0001; presence of D 36% versus 3%, P = 0.0006], but WPPC was not different. Bilateral cortical lesions were seen in patients with and without CMD. Thalamocortical disruption did not differ for those with CMD, but long-range WPPC was decreased in 1-4 Hz [odds ratio (OR) 0.8; 95% confidence interval (CI) 0.7-0.9] and increased in 14-30 Hz frequency ranges (OR 1.2; 95% CI 1.0-1.5). These structural and functional data implicate a failure of motor command integration at the anterior forebrain mesocircuit level with preserved thalamocortical network function for CMD patients with subcortical lesions. Amongst patients with bilateral cortical lesions preserved cortico-cortical network function is associated with CMD detection. These data may allow screening for CMD based on widely available structural MRI and resting EEG., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Studies evaluating stroke following varicella zoster virus (VZV) infection are limited, and the utility of zoster vaccination against this phenomenon is unclear. This study aimed to determine the risk of stroke 30 days following zoster infection and to evaluate the impact of zoster vaccinations on the risk of stroke in VZV-infected patients., Methods: This retrospective case-control study was conducted from January 2010 to January 2020 utilizing nationwide patient data retrieved from the Veterans Affairs' Corporate Data Warehouse., Results: A total of 2 165 505 patients ≥18 years of age who received care at a Veterans Affairs facility were included in the study, of whom 71 911 had a history of zoster infection. Zoster patients were found to have 1.9 times increased likelihood of developing a stroke within 30 days following infection (odds ratio [OR], 1.93 [95% confidence interval {CI}, 1.57-2.4]; P < .0001). A decreased risk of stroke was seen in patients who received the recombinant zoster vaccine (OR, 0.57 [95% CI, .46-.72]; P < .0001) or the live zoster vaccine (OR, 0.77 [95% CI, .65-.91]; P = .002)., Conclusions: Patients had a significantly higher risk of stroke within the first month following recent herpes zoster infection. Receipt of at least 1 zoster vaccination was found to mitigate this increased risk. Vaccination may therefore be viewed as a protective tool against the risk of neurologic postinfection sequelae., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2022.)

Background: Accurate reference intervals are essential for the interpretation of laboratory test results. Typically, they are determined by the central 95% range of test results from a predefined reference population. As these direct studies can face practical and ethical challenges, indirect methods using routine measurements offer an alternative approach., Methods: We provide step-by-step guidance on how to apply an indirect method in practice using refineR, the most recently published indirect method, and showcase the application by evaluating real-world data of 12 prespecified analytes. Measurements were retrieved from ARUP Laboratories' data warehouse, and were obtained from routine patient testing on cobas c502 or e602 analyzers. Test results were prefiltered and cleaned and, if necessary, physiologically partitioned prior to estimating reference intervals using refineR. Estimated reference intervals were then compared to established intervals provided by the manufacturer., Results: For most analytes, the reference intervals estimated by refineR were comparable to those provided by the manufacturer, shown by overlapping confidence intervals at both reference limits, or only the upper or lower limit. For thyroid-stimulating hormone, refineR estimated higher reference limits, while estimates for prealbumin were lower compared to the established reference interval., Conclusions: We applied the refineR algorithm to a variety of real-world data sets resulting in reference intervals similar to intervals previously established by direct methods. We further provide practical guidance and a code example on how to apply an indirect method in a real-world scenario facilitating their access and thus their use in laboratory settings., (© American Association for Clinical Chemistry 2023.)

Background: The free hormone (FH) hypothesis states that hormone action and the corresponding biological effects are mediated by the unbound (free) fraction of hormone in circulation. The in vivo relationship between protein-bound and FH is complex and dynamic. In most individuals, measurement of total hormone (TH) is usually adequate to reflect the hormone status; however, certain physiological conditions and/or medications can affect protein binding and alter FH concentration. In these cases, measurement of FH will provide a better measure of the bioactive hormone status than measurement of the TH. Measurement of FH presents many challenges, as the concentrations are very low and there are number of pitfalls, which may affect the measured concentrations., Content: In this review, we discuss techniques used in the separation and direct quantitation of FH concentrations in biological samples using mass spectrometry for analysis. We also highlight clinical situations in which FH analysis is warranted and when mass spectrometry should be the preferred methodology over immunoassays., Summary: Equilibrium dialysis, ultrafiltration, or size-exclusion separation coupled with liquid chromatography-tandem mass spectrometry provides a sensitive and specific method to measure FH concentrations. These direct methods are useful in iatrogenic or physiological states that alter hormone binding or metabolism., (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Streptavidin-to-biotin binding is one of the strongest noncovalent interactions in nature and incorporated into many immunoassays. Biotin-streptavidin coupling assays are susceptible to interference from free biotin in patient specimens, which may falsely decrease or increase results. To prevent biotin interference, we evaluated a method to preconjugate biotinylated antibodies to the assay's streptavidin solid surface before adding patient specimen and compared this technique to a biotin depletion protocol., Methods: Biotin interference in 3 manual ELISAs and 2 automated immunoassays was established. Mitigation of biotin interference by preincubation was evaluated in each assay by adding biotinylated antibody to the streptavidin-coated surface before adding biotin- or PBS-spiked serum. Lastly, the preincubation method was compared to a biotin-depletion protocol to compare the effectiveness of mitigating biotin interference., Results: In the presence of 400 µg/L biotin, analyte detection was reduced to 10% to 15% of total in the ELISA assays and to 15.2% in the automated sandwich (thyroglobulin) immunoassay. In the automated competitive (free thyroxine) immunoassay, biotin caused an increased detection of 551.6%. Preconjugation of the biotinylated capture antibody to the streptavidin surface in the ELISA assays resulted in 84% to 99% activity recovery, compared to 84% to 97% by a biotin depletion protocol. Similarly, automated sandwich and competitive immunoassays obtained 97.1% and 116.5% recovery by preconjugation, compared to 95.6% and 100.3% by the depletion method, respectively., Conclusion: This study demonstrates how assay redesign to include preconjugation of biotinylated capture antibody to streptavidin is an effective alternative to biotin-depletion methods to mitigate biotin interference., (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Biochemical testing of urinary metanephrines is useful in the diagnosis and monitoring of pheochromocytoma and paragangliomas. We investigated the feasibility of mixture decomposition (ie, indirect) methods in verifying clinically derived reference intervals for urinary deconjugated metanephrine metabolites., Methods: Urinary 24-hour metanephrine and normetanephrine excretion results were extracted from our data warehouse and intervals were estimated by the modern variant of the Hoffmann method, maximum likelihood estimation (MLE), and gamma mixture model using R software., Results: Hoffmann, MLE, and gamma mixture models provided metanephrine and normetanephrine intervals that closely matched those derived from clinical studies. However, three-component MLE and gamma models were required for normetanephrine in adult women because the Hoffmann method was not suitable. Some data transformations caused blending of the mixed distributions and subsequent widening of the reference interval estimation, emphasizing the importance of careful data transformation for Hoffmann and MLE analyses. Gamma mixture models gave overall good agreement without the need for data transformation., Conclusions: Indirect methods have utility in verifying reference intervals in 24-hour urine specimens collected by patients. We emphasize the benefits of applying multiple decomposition methods to corroborate findings and careful application of data transformation when using Gaussian-based models., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The psychoactive component of cannabis, tetrahydrocannabinol (THC), is one of many cannabinoids present in the plant. Since cannabinoids have extensive structural similarity, it is important to be aware of potential cross-reactivity with immunoassays designed to detect THC metabolite. This is especially important as cannabinoid products are increasingly marketed as legal supplements. The objective of this study was to assess the cross-reactivity of 2 commercial immunoassays designed to detect THC metabolite with 4 cannabinoids: cannabidiol, cannabinol, cannabichromene, and cannabigerol., Methods: Deidentified residual patient urine samples that tested negative for THC metabolite on initial testing were pooled and fortified with the above compounds to detect cross-reactivity. We next tested a range of CBN concentrations to determine what concentration of CBN was required to trigger a positive immunoassay result. Finally, we tested whether CBN has an additive effect with THC in the immunoassay by adding CBN to 21 samples weakly positive for THC by a mass spectrometry method but negative by the EMIT II Plus immunoassay., Results: Both the EMIT II Plus assay and the Microgenics MultiGent assay demonstrated cross-reactivity with CBN. For the EMIT II Plus assay, about 5-fold more CBN than THC metabolite was required to produce an assay signal equivalent to the cutoff concentration, and CBN displayed an additive effect with THC metabolite. For the Microgenics assay, 20-fold more CBN than THC metabolite was required to cross the cutoff concentration., Conclusions: These data may help guide the need for confirmatory testing when results of THC metabolite testing by immunoassay are inconsistent with expectations., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Little is known about the prevalence or incidence of Zika virus (ZIKV) infection in settings affected by the 2015-2016 Zika pandemic and associated risk factors. We assessed these factors among household contacts of patients with ZIKV disease enrolled in a cohort study in Puerto Rico during 2016-2017., Methods: Household contacts of index case patients completed a questionnaire and gave specimens for real-time polymerase chain reaction (RT-PCR) and immunoglobulin M enzyme-linked immunosorbent assay testing to detect ZIKV infection. We measured the prevalence of ZIKV infection among contacts and associated individual and household factors, examined sexual transmission using a sexual-networks approach, and assessed incident infection among initially uninfected household contacts 2-4 months later., Results: Of 366 contacts, 34.4% had evidence of ZIKV infection at enrollment, including 11.2% by RT-PCR. Having open doors and windows that were either screened (prevalence ratio [PR], 2.1 [95% confidence interval {CI}, 1.2-3.6]) or unscreened (PR, 2.5 [95% CI, 1.5-4.1]) was associated with increased prevalence. Sexual partners were more likely to both be RT-PCR positive relative to other relationships (odds ratio, 2.2 [95% CI, 1.1-4.5]). At follow-up, 6.1% of contacts had evidence of incident infection., Conclusions: This study identified sexual contact as a risk factor for ZIKV infection. Persons living with ZIKV-infected individuals should be a focus of public health efforts., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Zika virus (ZIKV) has recently caused a large epidemic in the Americas that is associated with birth defects. Although ZIKV is primarily transmitted by Aedes mosquitoes, ZIKV RNA is detectable in blood and semen of infected individuals for weeks or months, during which sexual and other modes of transmission are possible. However, viral RNA is usually detectable longer than infectious virus is present. We determined the frequency of isolation of infectious virus from semen and serum samples prospectively obtained from a cohort of patients in Puerto Rico. We confirmed isolation of infectious virus on the basis of a tissue culture cytopathic effect, an increase in virus genome copy equivalents (GCE), and positive results of immunofluorescence analysis; virus in infected cells was quantitated by flow cytometry. These criteria confirmed the presence of infectious virus in semen specimens from 8 of 97 patients for up to 38 days after initial detection when virus loads are >1.4 × 106 genome copy equivalents/mL. Two serum isolates were obtained from 296 patients. These findings can help guide important prevention guidelines for persons that may potentially be infectious and transmit ZIKV sexually.

A prospective cohort of women with reverse transcription polymerase chain reaction (RT-PCR) confirmed Zika virus infection aged 18-39 years in Puerto Rico found that pregnant women have about a 3-fold longer estimated median detection of Zika virus RNA in serum, which can increase definitive diagnosis of infection and facilitate timely and appropriate clinical management.

Some amphetamine (AMP) and ecstacy (MDMA) urine immunoassay (IA) kits are prone to false-positive results due to poor specificity of the antibody. We employed two techniques, high-resolution mass spectrometry (HRMS) and an in silico structure search, to identify compounds likely to cause false-positive results. Hundred false-positive IA specimens for AMP and/or MDMA were analyzed by an Agilent 6230 time-of-flight (TOF) mass spectrometer. Separately, SciFinder (Chemical Abstracts) was used as an in silico structure search to generate a library of compounds that are known to cross-react with AMP/MDMA IAs. Chemical formulas and exact masses of 145 structures were then compared against masses identified by TOF. Compounds known to have cross-reactivity with the IAs were identified in the structure-based search. The chemical formulas and exact masses of 145 structures (of 20 chemical formulas) were compared against masses identified by TOF. Urine analysis by HRMS correlates accurate mass with chemical formulae, but provides little information regarding compound structure. Structural data of targeted antigens can be utilized to correlate HRMS-derived chemical formulas with structural analogs., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: To analyze the demand for services from the nation's medical laboratories, which is predicted to dramatically increase as our citizens age and millions receive insurance coverage through the Affordable Care Act., Methods: A systematic review of relevant publications and databases was conducted to assess the current state of the nation's medical laboratory workforce and to examine the impact of population demographics and health reform on workforce development to address the future demand for laboratory services., Results: Building a Laboratory Workforce to Meet the Future, a new report from the American Society for Clinical Pathology (ASCP), provides a comprehensive strategy to address the future workforce needs of the nation's medical laboratories to meet this demand to provide timely, accurate, and safe patient care and to fully realize the benefits of personalized medicine., Conclusions: The report, from the ASCP Task Force on the Laboratory Professionals Workforce, is a comprehensive review of the myriad of factors affecting recruitment and retention of qualified laboratory professionals and provides a set of thoughtful recommendations outlining a multifaceted approach to bolster the pipeline of potential candidates for the profession as well as leadership in health care.

The Drosophila wing consists of a transparent wing membrane supported by a network of wing veins. Previously, we have shown that the wing membrane cuticle is not flat but is organized into ridges that are the equivalent of one wing epithelial cell in width and multiple cells in length. These cuticle ridges have an anteroposterior orientation in the anterior wing and a proximodistal orientation in the posterior wing. The precise topography of the wing membrane is remarkable because it is a fusion of two independent cuticle contributions from the dorsal and ventral wing epithelia. Here, through morphological and genetic studies, we show that it is the dorsal wing epithelium that determines wing membrane topography. Specifically, we find that wing hair location and membrane topography are coordinated on the dorsal, but not ventral, surface of the wing. In addition, we find that altering Frizzled Planar Cell Polarity (i.e., Fz PCP) signaling in the dorsal wing epithelium alone changes the membrane topography of both dorsal and ventral wing surfaces. We also examined the wing morphology of two model Hymenopterans, the honeybee Apis mellifera and the parasitic wasp Nasonia vitripennis. In both cases, wing hair location and wing membrane topography are coordinated on the dorsal, but not ventral, wing surface, suggesting that the dorsal wing epithelium also controls wing topography in these species. Because phylogenomic studies have identified the Hymenotera as basal within the Endopterygota family tree, these findings suggest that this is a primitive insect character.

According to the multi-process theory of prospective memory (ProM), time-based tasks rely more heavily on strategic processes dependent on prefrontal systems than do event-based tasks. Given the prominent frontostriatal pathophysiology of HIV infection, one would expect HIV-infected individuals to demonstrate greater deficits in time-based versus event-based ProM. However, the two prior studies examining this question have produced variable results. We evaluated this hypothesis in 143 individuals with HIV infection and 43 demographically similar seronegative adults (HIV-) who completed the research version of the Memory for Intentions Screening Test, which yields parallel subscales of time- and event-based ProM. Results showed main effects of HIV serostatus and cue type, but no interaction between serostatus and cue. Planned pair-wise comparisons showed a significant effect of HIV on time-based ProM and a trend-level effect on event-based ProM that was driven primarily by the subset of participants with HIV-associated neurocognitive disorders. Nevertheless, time-based ProM was more strongly correlated with measures of executive functions, attention/working memory, and verbal fluency in HIV-infected persons. Although HIV-associated deficits in time- and event-based ProM appear to be of comparable severity, the cognitive architecture of time-based ProM may be more strongly influenced by strategic monitoring and retrieval processes.

Background: Polyamines (e.g., putrescine, spermidine, and spermine) are required for optimal cell growth. Inhibition of polyamine synthesis suppresses carcinogen-induced epithelial cancers, including colon cancer, in animal models. In a short-term phase IIa trial, we determined that low doses of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (an enzyme involved in polyamine synthesis), reduced the polyamine content of normal-appearing rectal mucosa of subjects with a prior history of resected colon polyps. In a follow-up study, we have attempted to determine the lowest dose of DFMO that can suppress the polyamine content of rectal mucosa over a course of 1 year with no or minimal side effects., Methods: Participants were randomly assigned to daily oral treatment with a placebo or one of three doses (0.075, 0.20, or 0.40 g/m2) of DFMO. Baseline and serial determinations of polyamine levels in rectal mucosa and extensive symptom monitoring (including audiometric measurements, since DFMO causes some reversible hearing loss at higher doses) were performed over a 15-month period., Results: DFMO treatment reduced putrescine levels in a dose-dependent manner. Following 6 months of treatment, doses of 0.20 and 0.40 g/m2 per day reduced putrescine levels to approximately 34% and 10%, respectively, of those observed in the placebo group. Smaller decreases were seen in spermidine levels and spermidine:spermine ratios. Polyamine levels increased toward baseline values after discontinuation of DFMO. Although there were no statistically significant differences among the dose groups with respect to clinically important shifts in audiometric thresholds and nonaudiologic side effects, statistically significant higher dropout and discontinuation rates were observed in the highest dose group., Conclusions: Polyamine levels in rectal mucosa can be continuously suppressed by daily oral doses of DFMO that produce few or no side effects. A dose of 0.20 g/m2 can be used safely in combination phase IIb or single-agent phase III chemoprevention trials.