To improve on the quality of preclinical studies and their clinical translatability, patient-derived xenograft (PDX) models are increasingly used because they reflect inter- and intra-patient heterogeneity as well as human-specific tumor cell characteristics. However, the endocrine milieu of human patients, which affects grafted tumor cells may differ from mice. This is a growing concern as evidence of sex-specific biology in cancer has accumulated and an increase in the incidence of endocrine-related cancers has been observed highlighting the need to correctly reflect the hormonal milieu in PDX models. Here, we address the need to better model different female endocrine milieus in xenograft studies. Using an improved Liquid Chromatography-Mass Spectrometry (LC-MS) protocol for concomitant analysis of four different ovarian steroids in low volume plasma samples, we show that female mice of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) strain frequently used for xenografts have 17-β-estradiol (E2) and testosterone (T) levels comparable to widely used C57Bl6 strain but higher progesterone (P4) levels. While NSG E2 levels are comparable, T levels are lower and P4 levels higher compared to those observed in menopausal women. Following ovariectomy, T levels increase to those found in postmenopausal women. Subcutaneous implantation of E2 and combined E2 and P4 silicon pellets mimic ovarian hormone levels of premenopausal women in follicular and luteal phase of the menstrual cycle. Thus, straightforward procedures can effectively humanize the endocrine environment of experimental animals and improve physiologic relevance in women’s health-related research.

Objectives: Current LC-MS/MS applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for DHEAS. We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification.Methods: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued.Results: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within +/- 20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively.Conclusions: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Background: Salivary cortisol is a safe and non-invasive measure of hypothalamic-pituitary-adrenal axis function and is used as a biomarker of the human stress response. Natural environments are recognized to contribute to help reduce the effect of stress. Objective: To determine the feasibility of a salivary cortisol collection protocol for acute severely brain injured patients, and to explore the influence of exposure to natural settings on salivary cortisol concentration as an index of stress level. Methods: An exploratory study on 17 acute patients with severe brain injury was performed. We collected salivary samples in a closed hospital ward and a therapeutic garden at the start of the session and after 30 minutes of rest time. Physiological parameters, level of communication, and subjective well-being were also assessed. Results: The primary objectives regarding the feasibility of the protocol were met overall. We found no significant differences in cortisol values when including the whole population. However, cortisol values were significantly higher in the indoor environment in patients with communication attempts. Conclusions: A salivary collection protocol with brain-injured patients in the acute phase is feasible and safe, and this type of measurement could pave the way for future research supporting the benefits of nature as an additional resource in their neurorehabilitation.

Abstract Objectives Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. Methods Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. Results Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from −5.6 to 12.3% for corticosterone, −14.6 to 12.4% for 11-deoxycortisol and −4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. Conclusions Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization.

The dysregulation of the hormone cortisol is related to several pathological states, and its monitoring could help prevent severe stress, fatigue, and mental diseases. While wearable antibody-based biosensors could allow real-time and simple monitoring of antigens, an accurate and low-cost antibody-based cortisol detection through electrochemical methods is considerably challenging due to its low concentration and the high ionic strength of real biofluids. Here, a label-free and fast sensor for cortisol detection is proposed based on antibody-coated organic electrochemical transistors. The developed devices show unprecedented high sensitivities of 50 μA/dec for cortisol sensing in high-ionic-strength solutions with effective cortisol detection demonstrated with real human sweat. The sensing mechanism is analyzed through impedance spectroscopy and confirmed with electrical models. Compared to existing methods requiring bulky and expensive laboratory equipment, these wearable devices enable point-of-care cortisol detection in 5 min with direct sweat collection for personalized well-being monitoring.

Abstract Objectives Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. Methods Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. Results Using CAL1, intra-laboratory CVs ranged between 2.8–7.4%, 4.4–18.0% and 5.2–22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from −6.6 to 6.9%, −17.2 to 7.8% and −12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. Conclusions Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)