Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

The impact of mitotane therapy on serum-free proteins in patients with adrenocortical carcinoma.

Introduction: Adrenocortical carcinoma (ACC) is a rare malignancy of the adrenal cortex. Whilst surgery is the preferred treatment, adjunctive therapy with mitotane may be offered post-surgically to minimise the risk of recurrence or, in the absence of surgery, to attenuate progression. Aim: The objective was to evaluate the effects of mitotane treatment on serum protein concentrations in patients treated for ACC with mitotane therapy and compare this to patients with other adrenal neoplasms and a normal pregnant cohort. Methods: Serum cortisol, thyroid function tests, adrenocorticotrophic hormone (ACTH), cortisol-binding globulin (CBG), thyroxine-binding globulin (TBG), gonadotrophins and androgens were measured on plasma and serum samples. Thirty-five patients with ACC were included, and mitotane levels were noted to be sub-/supra-therapeutic. Data were tested for normality, reported as mean ± s.d., and compared to other two cohorts using paired-sample t-test with a 5% P-value for significance and a 95% CI. Results: Patients on mitotane therapy had a higher mean serum CBG concentration compared to the adrenal neoplasm group (sub-therapeutic: 79.5 (95% CI: 33.6, 125.4 nmol/L), therapeutic: 85.3 (95% CI: 37.1-133.6 nmol/L), supra-therapeutic: 75.7 (95% CI: -19.3, 170.6 nmol/L) and adrenal neoplasm: 25.5 (95% CI: 17.5, 33.5 nmol/L). Negative correlations between serum cortisol and CBG concentration were demonstrated within the supra-therapeutic plasma mitotane and adrenal neoplasm groups. Conclusion: Patients with ACC and therapeutic plasma mitotane concentrations had higher serum CBG concentrations compared to those with adrenal neoplasms or pregnant women, and higher serum cortisol. Whilst there was no direct correlation with cortisol and mitotane level, the negative correlation of cortisol with CBG may suggest that the direct effect of mitotane in increasing cortisol may also reflect that mitotane has a direct adrenolytic effect.

Twelve tips to develop entrustable professional activities.

Entrustable professional activities (EPAs), units of professional practice that require proficient integration of multiple competencies and can be entrusted to a sufficiently competent learner, are increasingly being used to define and inform curricula of health care professionals. The process of developing EPAs can be challenging and requires a deep yet pragmatic understanding of the concepts underlying EPA construction. Based on recent literature and the authors' lessons learned, this article provides the following practical and more or less sequential recommendations for developing EPAs: [1] Assemble a core team; [2] Build up expertise; [3] Establish a shared understanding of the purpose of EPAs; [4] Draft preliminary EPAs; [5] Elaborate EPAs; [6] Adopt a framework of supervision; [7] Perform a structured quality check; [8] Use a Delphi approach for refinement and/or consensus; [9] Pilot test EPAs; [10] Attune EPAs to their feasibility in assessment; [11] Map EPAs to existing curriculum; [12] Build a revision plan.

The logic behind entrustable professional activity frameworks: A scoping review of the literature.

INTRODUCTION: Entrustable professional activities (EPAs), discrete profession-specific tasks requiring integration of multiple competencies, are increasingly used to help define and inform curricula of specialty training programmes. Although guidelines exist to help guide the developmental process, deciding what logic to use to draft a preliminary EPA framework poses a crucial but often difficult first step. The logic of an EPA framework can be defined as the perspective used by its developers to break down the practice of a profession into units of professional work. This study aimed to map dominant logics and their rationales across postgraduate medical education and fellowship programmes. METHODS: A scoping review using systematic searches within five electronic databases (Medline, Embase, Google Scholar, Scopus and Web of Science) was performed. Dominant logics of included papers were identified using inductive coding and iterative analysis. RESULTS: In total, 42 studies were included. Most studies were conducted in the United States (n = 22; 52%), Canada (n = 6; 14%) and the Netherlands (n = 4; 10%). Across the reported range of specialties, family medicine (n = 4; 10%), internal medicine (n = 4; 10%), paediatrics (n = 3; 7%) and psychiatry (n = 3; 7%) were the most common. Three dominant logics could be identified, namely, 'service provision', 'procedures' and/or 'disease or patient categories'. The majority of papers (n = 37; 88%) used two or more logics when developing EPA frameworks (median = 3, range = 1-4). Disease or patient groups and service provision were the most common logics used (39% and 37%, respectively). CONCLUSIONS: Most programmes used a combination of logics when trying to capture the essential tasks of a profession in EPAs. For each of the three dominant logics, the authors arrived at a definition and identified benefits, limitations and examples. These findings may potentially inform best practice guidelines for EPA development.

International consensus on initial screening and follow-up of asymptomatic SDHx mutation carriers.

Approximately 20% of patients diagnosed with a phaeochromocytoma or paraganglioma carry a germline mutation in one of the succinate dehydrogenase (SDHx) genes (SDHA, SDHB, SDHC and SDHD), which encode the four subunits of the SDH enzyme. When a pathogenic SDHx mutation is identified in an affected patient, genetic counselling is proposed for first-degree relatives. Optimal initial evaluation and follow-up of people who are asymptomatic but might carry SDHx mutations have not yet been agreed. Thus, we established an international consensus algorithm of clinical, biochemical and imaging screening at diagnosis and during surveillance for both adults and children. An international panel of 29 experts from 12 countries was assembled, and the Delphi method was used to reach a consensus on 41 statements. This Consensus Statement covers a range of topics, including age of first genetic testing, appropriate biochemical and imaging tests for initial tumour screening and follow-up, screening for rare SDHx-related tumours and management of elderly people who have an SDHx mutation. This Consensus Statement focuses on the management of asymptomatic SDHx mutation carriers and provides clinicians with much-needed guidance. The standardization of practice will enable prospective studies in the near future.

Biochemical abnormalities in COVID-19: a comparison of white versus ethnic minority populations in the UK.

AIMS: Public Health England has identified that in COVID-19, death rates among ethnic minorities far exceeds that of the white population. While the increase in ethnic minorities is likely to be multifactorial, to date, no studies have looked to see whether values for routine clinical biochemistry parameters differ between ethnic minority and white individuals. METHODS: Baseline biochemical data for 22 common tests from 311 SARS-CoV-2 positive patients presenting to hospital in April 2020 in whom ethnicity data were available was retrospectively collected and evaluated. Data comparisons between ethnic minority and white groups were made for all patient data and for the subset of patients subsequently admitted to intensive care. RESULTS: When all patient data were considered, the ethnic minority population had statistically significant higher concentrations of C reactive protein (CRP), aspartate aminotransferase and gamma-glutamyl transferase, while troponin T was higher in the white group. A greater proportion of ethnic minority patients were subsequently admitted to intensive care, but when the presenting biochemistry of this subset of patients was compared, no significant differences were observed between ethnic minority and white groups. CONCLUSION: Our data show for the first time that routine biochemistry at hospital presentation in COVID-19 differs between ethnic minority and white groups. Among the markers identified, CRP was significantly higher in the ethnic minority group pointing towards an increased tendency for severe inflammation in this group.

The recommended description of an entrustable professional activity: AMEE Guide No. 140.

Entrustable professional activities (EPAs) have received much attention in the literature since they were first proposed in 2005. Useful guidelines, workshops, courses, and conferences have supported faculty in developing programs and designing assessment procedures using EPAs and entrustment decision-making. Yet, the need for clarification remains, particularly as more programs make the step from design to implementation.Well-written EPAs provide a natural construct to establish the outcome of training. To be useful, EPAs require more than a suitable title. This AMEE Guide elaborates eight sections of a full EPA description, and provides explanations and justifications for each. These sections are: title; specification and limitations; risks in case of failure; most relevant competency domains; knowledge, skills, attitudes and experiences; information sources to assess progress and support summative entrustment; entrustment/supervision level expected at which stage of training; and time period to expiration if not practiced.Constructing fully elaborated EPAs creates a shared mental model amongst learners and programs, informs competency-based curriculum design, directs ad-hoc and formal entrustment decision-making, and provides standards for certifying bodies and boundaries for scope of practice. The framework intends to support curricular leaders looking to adopt new EPAs, or revise and define established EPAs for competency-based education.

Posaconazole-Induced Hypertension Masquerading as Congenital Adrenal Hyperplasia in a Child with Cystic Fibrosis.

BACKGROUND: Deficiency of 11ß-hydroxylase is the second most common cause of congenital adrenal hyperplasia (CAH), presenting with hypertension, hypokalaemia, precocious puberty, and adrenal insufficiency. We report the case of a 6-year-old boy with cystic fibrosis (CF) found to have hypertension and cortisol insufficiency, which were initially suspected to be due to CAH, but were subsequently identified as being secondary to posaconazole therapy. Case Presentation. A 6-year-old boy with CF was noted to have developed hypertension after administration of two doses of Orkambi™ (ivacaftor/lumacaftor), which was subsequently discontinued, but the hypertension persisted. Further investigations, including echocardiogram, abdominal Doppler, thyroid function, and urinary catecholamine levels, were normal. A urine steroid profile analysis raised the possibility of CAH due to 11ß-hydroxylase deficiency, and a standard short synacthen test (SST) revealed suboptimal cortisol response. Clinically, there were no features of androgen excess. Detailed evaluation of the medical history revealed exposure to posaconazole for more than 2 months, and the hypertension had been noted to develop two weeks after the initiation of posaconazole. Hence, posaconazole was discontinued, following which the blood pressure, cortisol response to the SST, and urine steroid profile were normalized. CONCLUSION: Posaconazole can induce a clinical and biochemical picture similar to CAH due to 11ß-hydroxylase deficiency, which is reversible. It is prudent to monitor patients on posaconazole for cortisol insufficiency, hypertension, and electrolyte abnormalities.

EQual Rubric Evaluation of the Association of American Medical Colleges' Core Entrustable Professional Activities for Entering Residency.

PURPOSE: To have subject matter experts evaluate the Core Entrustable Professional Activities for Entering Residency (Core EPAs) with the EQual rubric to determine if revisions were required and, if applicable, how to focus revision efforts. METHOD: Ten entrustable professional activity (EPA) experts were invited to evaluate the 13 Core EPAs. Experts had a 6-month window (December 2018-May 2019) to complete the evaluation, which contained the complete EQual rubric and 3 additional prompts, one of which-"Do you think this EPA requires revision?"-was limited to a "yes/no" response. Descriptive statistics for overall and domain-specific EQual rubric scores for each of the 13 Core EPAs were calculated. Free-text responses to why and/or how a Core EPA should be revised were summarized for any Core EPA that scored below a cutoff or for which the majority of experts recommended revision. RESULTS: Six experts completed the evaluation. Most Core EPAs' (9/13) overall score was above the cutoff, indicating that they align with the key domains of the EPA construct. The remaining 4 Core EPAs (2, 7, 9, and 13) scored below the overall cutoff, suggesting that they may require revision. A majority of experts felt that Core EPAs 6, 7, 9, and 13 required revision. With regard to domain-specific scores, Core EPAs 2, 3, 7, 9, and 13 were below the discrete units of work cutoff; Core EPAs 7, 9, and 13 were below the entrustable, essential, and important tasks of the profession of medicine cutoff; and Core EPA 9 was below the curricular role cutoff. CONCLUSIONS: The Core EPAs represent a promising initial framework of EPAs for undergraduate medical education. Some Core EPAs might benefit from revision. The process of improving the Core EPAs must continue if they are to standardize outcomes for medical school graduates.

Creating Entrustable Professional Activities to Assess Internal Medicine Residents in Training: A Mixed-Methods Approach.

Background: Competency-based medical education has not advanced residency training as much as many observers expected. Some medical educators now advocate reorienting competency-based approaches to focus on a resident's ability to do authentic clinical work. Objective: To develop descriptions of clinical work for which internal medicine residents must gain proficiency to deliver meaningful patient care (for example, "Admit and manage a medical inpatient with a new acute problem"). Design: A modified Delphi process involving clinical experts followed by a conference of educational experts. Setting: The Royal College of Physicians and Surgeons of Canada. Participants: In phase 1 of the project, members of the Specialty Committee for Internal Medicine participated in a modified Delphi process to identify activities in internal medicine that represent the scope of the specialty. In phase 2 of the project, 5 experts who were scholars and leaders in competency-based medical education reviewed the results. Measurements: Phase 1 identified important activities, revised descriptions to improve accuracy and avoid overlap, and assigned activities to stages of training. Phase 2 compared proposed activity descriptions with published guidelines for their development and application in medical education. Results: The project identified 29 activities that qualify as entrustable professional activities. The project also produced a detailed description of each activity and guidelines for using them to assess residents. Limitation: These activities reflect the practice patterns of the developers and may not fully represent internal medicine practice in Canada. Conclusion: Identification of these activities is expected to facilitate modification of training and assessment programs for medical residents so that programs focus less on isolated skills and more on integrated tasks. Primary Funding Source: Southeastern Ontario Academic Medical Organization Endowed Scholarship and Education Fund and Queen's University Department of Medicine Innovation Fund.

Modeling Congenital Adrenal Hyperplasia and Testing Interventions for Adrenal Insufficiency Using Donor-Specific Reprogrammed Cells.

Adrenal insufficiency is managed by hormone replacement therapy, which is far from optimal; the ability to generate functional steroidogenic cells would offer a unique opportunity for a curative approach to restoring the complex feedback regulation of the hypothalamic-pituitary-adrenal axis. Here, we generated human induced steroidogenic cells (hiSCs) from fibroblasts, blood-, and urine-derived cells through forced expression of steroidogenic factor-1 and activation of the PKA and LHRH pathways. hiSCs had ultrastructural features resembling steroid-secreting cells, expressed steroidogenic enzymes, and secreted steroid hormones in response to stimuli. hiSCs were viable when transplanted into the mouse kidney capsule and intra-adrenal. Importantly, the hypocortisolism of hiSCs derived from patients with adrenal insufficiency due to congenital adrenal hyperplasia was rescued by expressing the wild-type version of the defective disease-causing enzymes. Our study provides an effective tool with many potential applications for studying adrenal pathobiology in a personalized manner and opens venues for the development of precision therapies.

EQual, a Novel Rubric to Evaluate Entrustable Professional Activities for Quality and Structure.

PURPOSE: Entrustable professional activities (EPAs) have become a cornerstone of assessment in competency-based medical education (CBME). Increasingly, EPAs are being adopted that do not conform to EPA standards. This study aimed to develop and validate a scoring rubric to evaluate EPAs for alignment with their purpose, and to identify substandard EPAs. METHOD: The EQual rubric was developed and revised by a team of education scholars with expertise in EPAs. It was then applied by four residency program directors/CBME leads (PDs) and four nonclinician support staff to 31 stage-specific EPAs developed for internal medicine in the Royal College of Physicians and Surgeons of Canada's Competency by Design framework. Results were analyzed using a generalizability study to evaluate overall reliability, with the EPAs as the object of measurement. Item-level analysis was performed to determine reliability and discrimination value for each item. Scores from the PDs were also compared with decisions about revisions made independently by the education scholars group. RESULTS: The EQual rubric demonstrated high reliability in the G-study with a phi-coefficient of 0.84 when applied by the PDs, and moderate reliability when applied by the support staff at 0.67. Item-level analysis identified three items that performed poorly with low item discrimination and low interrater reliability indices. Scores from support staff only moderately correlated with PDs. Using the preestablished cut score, PDs identified 9 of 10 EPAs deemed to require major revision. CONCLUSIONS: EQual rubric scores reliably measured alignment of EPAs with literature-described standards. Further, its application accurately identified EPAs requiring major revisions.

A 13-Steroid Serum Panel Based on LC-MS/MS: Use in Detection of Adrenocortical Carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare malignancy, with an annual incidence of 1 or 2 cases per million. Biochemical diagnosis is challenging because up to two-thirds of the carcinomas are biochemically silent, resulting from de facto enzyme deficiencies in steroid hormone biosynthesis. Urine steroid profiling by GC-MS is an effective diagnostic test for ACC because of its capacity to detect and quantify the increased metabolites of steroid pathway synthetic intermediates. Corresponding serum assays for most steroid pathway intermediates are usually unavailable because of low demand or lack of immunoassay specificity. Serum steroid analysis by LC-MS/MS is increasingly replacing immunoassay, in particular for steroids most subject to cross-reaction. METHODS: We developed an LC-MS/MS method for the measurement of serum androstenedione, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, and testosterone. Assay value in discriminating ACC from other adrenal lesions (phaeochromocytoma/paraganglioma, cortisol-producing adenoma, and lesions demonstrating no hormonal excess) was then investigated. RESULTS: In ACC cases, between 4 and 7 steroids were increased (median = 6), and in the non-ACC groups, up to 2 steroids were increased. 11-Deoxycortisol was markedly increased in all cases of ACC. All steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC from non-ACC adrenal lesions. CONCLUSIONS: Serum steroid paneling by LC-MS/MS is useful for diagnosing ACC by combining the measurement of steroid hormones and their precursors in a single analysis.