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Accurate serum cortisol quantification is required for the correct diagnosis and management of adrenal pathologies. Presently, most laboratories use immunoassay to measure serum cortisol with proficiency schemes demonstrating a wide dispersion of results. Here, we investigate the effects of sex, matrix, and antibody specificity on serum cortisol quantification in 6 routine assays. Surplus serum was obtained before disposal and the following cohorts were created: males, nonpregnant females, pregnant patients, and patients prescribed either metyrapone or prednisolone. Samples were anonymized and distributed to collaborating laboratories for cortisol analysis by 6 routine assays. Cortisol was also measured in all samples using an LC-MS/MS candidate reference measurement procedure (cRMP); cortisol-binding globulin (CBG) was measured in the nonpregnant and pregnant female cohorts. Considerable inter- and intraassay variation was observed across the male and nonpregnant female cohorts relative to the cRMP. Four immunoassays underrecovered cortisol in the pregnancy cohort, and CBG was found to be significantly higher in this cohort than in the nonpregnant females. In the metyrapone and prednisolone cohorts, all immunoassays overestimated cortisol. The first generation Roche E170 and Siemens Centaur XP were particularly prone to overestimation. In all cohorts the routine LC-MS/MS assay aligned extremely well with the cRMP. Despite the clinical importance of serum cortisol, the performance of routine immunoassays remains highly variable. Accurate quantification is compromised by both matrix effects and antibody specificity. Underpinning this study with a cRMP has highlighted the deficiencies in standardization across routine cortisol immunoassays.

Phenytoin is an anti-epileptic drug that has a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Only the free fraction is pharmacologically active, and in some cases, accurate determination of the free phenytoin concentration may be essential to prevent phenytoin toxicity. Although it is possible to measure free phenytoin concentration, often only the total concentration is measured, with equations used to estimate the free fraction. Several equations are quoted in the literature with no overall consensus with regard to accuracy. This study aimed to assess the correlation between total and free phenytoin in a mixed patient population, and to compare the accuracy of several different equations used to estimate the free phenytoin concentration. Fifty-one serum samples were analysed for total phenytoin, free phenytoin and albumin. The measured free phenytoin concentrations were compared against those estimated using five selected equations, identified through a literature search. This study showed poor correlation between the total and measured free phenytoin concentrations, and between the estimated and measured free concentrations. The overall correlation was concentration-dependent, but a correction factor could not be applied to improve the accuracy consistently. The equations assessed showed wide variability between the estimated and measured free phenytoin concentrations, with several showing a clinically significant negative bias when compared with the measured free fraction. This study highlights the disparity of the free phenytoin concentrations generated by the equations. Underestimation of free phenytoin concentrations using these equations may result in phenytoin toxicity, bringing into question the safety of using calculated values for patient management in place of physical measurement of free phenytoin concentration by ultra-performance liquid chromatography tandem mass spectrometry.

Metastatic neuroendocrine tumors secrete serotonin and other vasoactive substances that are responsible for carcinoid syndrome and carcinoid heart disease. We sought to evaluate the discriminatory utility of diagnostic biomarkers in determining the presence and severity of carcinoid heart disease in patients with metastatic neuroendocrine tumors. A cross-sectional study of patients with neuroendocrine tumors with documented liver metastases and/or carcinoid syndrome between April 2009-October 2012 in 5 tertiary referral centers. Serum was analyzed for Chromogranin A, Chromogranin B and N-terminal pro Brain Natriuretic Peptide (NT-proBNP). Plasma was analyzed for Neurokinin A and 5-Hydroxyindoleacetic acid (5HIAA). Echocardiography was used to determine the presence and severity of carcinoid heart disease. Non-parametric receiver operating characteristic curves were constructed for biomarkers, and the area under the curve determined. The severity of cardiac involvement was correlated with the concentration of each biomarker. A total of 187 patients were identified of whom 37 (20%) had carcinoid heart disease. Significantly higher median values of all biomarkers were found in the patients with cardiac involvement. NT-proBNP and plasma 5HIAA had the highest areas under the curve for the prediction of carcinoid heart disease [NT-proBNP 0.82 (95% confidence interval 0.74-0.90, p<0.0001) and 5HIAA 0.85 (95% confidence interval 0.78-0.92, p<0.0001]. NT-proBNP was moderately correlated (r = 0.48, p<0.001) whereas plasma 5HIAA was only weakly correlated (r = 0.34, p<0.001) with the echocardiographic severity score. NT-proBNP and plasma 5HIAA are both sensitive and specific biomarkers for the presence of carcinoid heart disease whereas only NT-proBNP is moderately correlated with disease severity.

BackgroundA standard cut-off of ≥2 CSF-specific OCBs is used in multiple sclerosis but some studies suggest ≥3 CSF OCBs maybe more specific.AimTo determine if a cut-off of ≥2 or ≥3 CSF OCBs should be used in MSMaterials and MethodsOligoclonal bands were tested using standardised methods incorporating iso- electric focussing. Samples sent between July 2018-June 2019 were included. CSF OCB number (1, ≥2,≥3, or unclear) was assessed by two blinded raters and case records were reviewed.ResultsCSF OCBs were detected in 196/746 (26%) samples (CSF OCB=1; 14/196 (7%), ≥2=182/196 (93%), ≥3=169/196 (86%). Clinical data were available in 157 patients (MS=101, other inflammatory=37, unclear significance=19). Excluding CSF OCB results, 68/101 (67%) cases fulfilled MS diagnostic criteria. Of patients with 1, ≥2 and ≥3 CSF OCBs, 1/8 (12%), 100/182 (55%) and 98/169 (58%) had MS respectively. The positive predictive value of 1, ≥2, and ≥3 OCBs was 0.35, 0.89 and 0.92 respectively in CNS inflammatory disease.ConclusionDetection of CSF OCBs was necessary to fulfil MS diagnostic criteria in approximately 30% of cases. Detection of ≥3 OCBs marginally improved the likelihood of an MS diagnosis. Overall measures of sensitivity, specificity and receiver operator curves will be presented.giovanna.campagna@thewaltoncentre.nhs.uk

Background: Carcinoid heart disease (CHD) is an important complication of metastatic neuroendocrine disease, requiring regular monitoring to enable intervention prior to right heart failure. We aimed to identify the most appropriate echocardiographic scoring systems for the quantitative assessment of CHD. Methods: In this prospective study conducted between April and October 2012 in two European Neuroendocrine Tumor Society (ENETS) Centres of Excellence, patients with neuroendocrine tumours with liver metastases and/or carcinoid syndrome underwent transthoracic echocardiography and blood sampling for serum N-terminal pro-brain natriuretic peptide (NT-proBNP) and plasma 5-hydroxyindoleacetic acid (5-HIAA). Each patient was assessed according to six echocardiographic scoring systems. The individual scoring systems' feasibility, observer variability, sensitivity, specificity and correlation with the concentration biomarkers were determined. Results: 100 patients were included; 21% had echocardiographic evidence of CHD. All scores discriminated highly between those with/without CHD, with no single score performing significantly better than another. The severity, determined using all of the scoring systems, correlated with the concentration of both biomarkers, but the strongest correlations were seen between the Bhattacharyya score and serum NT-proBNP. Conclusion: All scoring systems are comparable in terms of sensitivity and specificity for the detection of CHD. There is a variation in the feasibility of the scoring systems due to varying complexity of the score components. All scores correlate with NT-proBNP and plasma 5-HIAA. The Westberg score appears to be the most optimal scoring system for use in screening of CHD whereas the more complex scoring systems are more suited to the patient with established disease who may require surgical intervention.

Background: Dehydroepiandrosterone sulphate (DHEAS) is a steroid that is increasingly being recognized as a potential drug of abuse in many countries. This is due to its reputation as a hormone that may be able to retard the ageing process. The measurement of DHEAS is useful in the diagnosis of medical conditions such as congenital adrenal hyperplasia and polycystic ovary syndrome. Thus, a liquid chromatography-tandem mass spectrometry method has been developed to determine DHEAS concentrations in human serum. Method: The chromatography was performed using a WatersTM 2795 Alliance HT LC system coupled to a Mercury Fusion-RP column fitted with a SecurityGuardTM column. Results: DHEAS and the internal standard, deuterated DHEAS, both had a retention time of 1.5 min. The transition determined by the Micromass QuattroTM tandem mass spectrometer for DHEAS was m/z 367.3>96.7 and for the internal standard m/z 369.3>96.6. The method was linear up to 20 µmol/L; the lower limit of detection and the lower limit of quantitation were both 1 µmol/L. The intra- and interassay imprecision were <11% over a concentration range of 1-18 µmol/L for the in-house quality control and <12% for the intra- and interassay imprecision for the Bio-Rad Lyphocheck QC. Conclusion: The measurement of DHEAS by liquid chromatography-tandem mass spectrometry is robust and has a simple sample preparation procedure with a rapid cycle time of only 4 min.

Background. The decay of human immunodeficiency virus type 1 (HIV-1)-infected cells during early combination antiretroviral therapy (cART) in infected infants is not defined. Methods. HIV-1 DNA, including 2-long terminal repeat (2-LTR) circles, and multiply spliced (ms-) and unspliced (us-) HIV-1 RNA concentrations were measured at 0, 24, 48, and 96 weeks of cART in infants from the IMPAACT P1030 trial receiving lopinavir-ritonavir-based cART. The ratio of HIV-1 DNA concentrations to replication-competent genomes was also estimated. Linear mixed effects models with random intercept and linear splines were used to estimate patient-specific decay kinetics of HIV-1 DNA. Results. The median HIV-1 DNA concentration before cART at a median age of 2 months was 3.2 log10 copies per million PBMC. With cART, the average estimated patient-specific change in HIV-1 DNA concentrations was −0.040 log10/week (95% confidence interval [CI], −.05, −.03) between 0 and 24 weeks and −0.017 log10/week between 24 and 48 weeks (95% CI, −.024, −.01). 2-LTR circles decreased with cART but remained detectable through 96 weeks. Pre-cART HIV-1 DNA concentration was correlated with time to undetectable plasma viral load and post-cART HIV-1 DNA at 96 weeks; although HIV-1 DNA concentrations exceeded replication-competent HIV-1 genomes by 148-fold. Almost all infants had ms- and usRNA detected pre-cART, with 75% having usRNA through 96 weeks of cART. Conclusions. By 2 months of age, a large pool of HIV-1-infected cells is established in perinatal infection, which influences time to undetectable viral load and reservoir size. This has implications for informing novel approaches aimed at early restriction of HIV-1 reservoirs to enable virologic remission and cure.

Background: Black sexual minority men (BSMM) are disproportionately affected by HIV incidence and have lower rates of HIV care engagement (e.g., retention in care, viral suppression), particularly in the US South. The COVID-19 pandemic exacerbated co-occurring psychosocial syndemic factors (e.g., depression, violence, substance use) that disproportionately impact BSMM living with HIV (BSMM+), but the impact of the pandemic on HIV care engagement among BSMM+ in the US South has not been explored in depth. Methods: BSMM+ (n = 27) were recruited from a longitudinal cohort in Dallas and Houston, Texas. Participants with less-than-optimal HIV care engagement, previous experiences of intimate partner violence (IPV), and prevalence of self-reported substance use were purposively selected for in-depth interviews from June 2022 to August 2023. Interviews lasted 54 min on average and were audio-recorded, transcribed, and de-identified before applying a thematic analysis approach. Results: Over half of participants experienced a relationship with IPV, used illicit substances in the past 60 days, and were depressed. Interviews highlighted that, during the COVID-19 pandemic, heightened levels of depression, substance use, and IPV individually and synergistically inhibited ART adherence and HIV care engagement. Participants described how IPV served to worsen their mental health and substance use, which, in turn, were the core drivers of poor HIV care engagement. Conclusions: The findings suggest that the COVID-19 pandemic exacerbated the syndemics of IPV, mental health, and substance use, and these acted as barriers to HIV care engagement. Future adherence interventions should synergistically address syndemic factors to maximize effectiveness.