Julianne Cook Botelho

Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.

Standardization of testosterone measurements in humans.

Testosterone levels are used primarily for the diagnosis of hypogonadism in men and androgen excess in women. Current studies suggest that serum testosterone measurements may be indicated in a wide range of diseases and conditions. Translation of testosterone levels outside of the reference ranges into clinical treatment, appropriate cut offs for clinical guidelines and epidemiological studies with public health impact pose challenges due to the measurement variability among assays and in assay sensitivity. While introducing mass spectrometry technology can overcome some of these challenges and help to improve measurements, it faces variability issues similar to those observed with immunoassays that need to be addressed. To overcome these problems in testosterone testing, the Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences (CDC/NCEH/DLS) started a steroid hormone standardization project. Their objective was to create testosterone measurement results that are traceable to one accuracy basis, thus allowing measurements to be comparable across methods, time, and location. CDC/NCEH/DLS conducts activities to standardize and improve testosterone assays and laboratory measurements by establishing metrological traceability to a higher order reference method and material. In addition, the standardization effort includes pre- and post-analytical challenges, such as test selection, interpretation, and establishing reference ranges to improve the translation of standardized results into clinical guidelines and public health assessments. CDC is conducting these standardization activities in collaboration with the clinical, laboratory, and research communities.

Isotope-Dilution Liquid Chromatography/Tandem Mass Spectrometry Candidate Reference Method for Total Testosterone in Human Serum

BACKGROUND We developed and evaluated a candidate reference measurement procedure (RMP) to standardize testosterone measurements, provide highly accurate and precise value assignments for the CDC Hormone Standardization Program, and ensure accurate and comparable results across testing systems and laboratories. METHODS After 2 liquid/liquid extractions of serum with a combination of ethyl acetate and hexane, we quantified testosterone by isotope-dilution liquid chromatography-tandem mass spectrometry with electrospray ionization in the positive ion mode monitoring 289→97 m/z (testosterone) and 292→112 m/z ((3)C(13) testosterone). We used calibrator bracketing and gravimetric measurements to give higher specificity and accuracy to serum value assignments. The candidate RMP was evaluated for accuracy by use of NIST-certified reference material SRM971 and validated by split-sample comparison to established RMPs. We evaluated intraassay and interassay imprecision, measurement uncertainty, potential interferences, and matrix effects. RESULTS A weighted Deming regression comparison of the candidate RMP to established RMPs showed agreement with no statistical difference (slope 0.99, 95% CI 0.98-1.00, intercept 0.54, 95% CI -1.24 to 2.32) and a bias of ≤0.3% for NIST SRM971. The candidate RMP gave maximum intraassay, interassay, and total percent CVs of 1.5%, 1.4%, and 1.7% across the concentrations of testosterone typically found in healthy men and women. We tested structural analogs of testosterone and 125 serum samples and found no interferences with the measurement. CONCLUSIONS This RMP for testosterone can serve as a higher-order standard for measurement traceability and can be used to provide an accuracy base to which routine methods can be compared in the CDC Hormone Standardization Program.

CDC project on standardizing steroid hormone measurements

Estradiol and testosterone measurements are widely used to assess steroid hormone status and to monitor stimulative, suppressive, or replacement therapy among children and adults of both sexes. Despite their common application, these measurements - particularly at low concentrations - show only limited comparability among assays, such as those observed for testosterone among women or for estradiol among postmenopausal women. This shortcoming hampers progress in research and in research translation. To overcome this, the Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences (CDC/NCEH/DLS) initiated a project to standardize and to improve steroid hormone measurements. The project is a collaborative effort between institutions, organizations, and groups involved in estradiol and testosterone testing, test interpretation, and use. Specific activities are scheduled based on needs assessments conducted with the clinical, research, and public health communities. The initial focus of this standardization project is to improve analytical measurements through reference laboratory activities. As part of the projects translational activities, CDC/NCEH/DLS will work further with professional societies and organizations to improve pre- and postanalytical issues that affect results from these measurements and their interpretation.

Challenges and improvements in testosterone and estradiol testing.

Assays that measure steroid hormones in patient care, public health, and research need to be both accurate and precise, as these criteria help to ensure comparability across all clinical and research applications. This review addresses major issues relevant to assay variability and describes recent activities by the US Centers for Disease Control and Prevention (CDC) to improve assay performance. Currently, high degrees of accuracy and precision are not always met for testosterone and estradiol measurements; although technologies for steroid hormone measurement have advanced significantly, measurement variability within and across laboratories has not improved accordingly. Differences in calibration and specificity are discussed as sources of variability in measurement accuracy. Ultimately, a combination of factors appears to cause inaccuracy of steroid hormone measurements, with nonuniform assay calibration and lack of specificity being two major contributors to assay variability. Within-assay variability for current assays is generally high, especially at low analyte concentrations. The CDC Hormone Standardization (HoSt) Program is improving clinical assays, as evidenced by a 50% decline in mean absolute bias between mass spectrometry assays and the CDC reference method from 2007 to 2011. This program provides the measurement traceability to CDC reference methods and helps to minimize factors affecting measurement variability.

Performance Criteria for Testosterone Measurements Based on Biological Variation in Adult Males: Recommendations from the Partnership for the Accurate Testing of Hormones

BACKGROUND Testosterone measurements that are accurate, reliable, and comparable across methodologies are crucial to improving public health. Current US Food and Drug Administration-cleared testosterone assays have important limitations. We sought to develop assay performance requirements on the basis of biological variation that allow physiologic changes to be distinguished from assay analytical errors. METHODS From literature review, the technical advisory subcommittee of the Partnership for the Accurate Testing of Hormones compiled a database of articles regarding analytical and biological variability of testosterone. These data, mostly from direct immunoassay-based methodologies, were used to specify analytical performance goals derived from within- and between-person variability of testosterone. RESULTS The allowable limits of desirable imprecision and bias on the basis of currently available biological variation data were 5.3% and 6.4%, respectively. The total error goal was 16.7%. From recent College of American Pathologists proficiency survey data, most currently available testosterone assays missed these analytical performance goals by wide margins. Data from the recently established CDC Hormone Standardization program showed that although the overall mean bias of selected certified assays was within 6.4%, individual sample measurements could show large variability in terms of precision, bias, and total error. CONCLUSIONS Because accurate measurement of testosterone across a wide range of concentrations [approximately 2-2000 ng/dL (0.069-69.4 nmol/L)] is important, we recommend using available data on biological variation to calculate performance criteria across the full range of expected values. Additional studies should be conducted to obtain biological variation data on testosterone from women and children, and revisions should be made to the analytical goals for these patient populations.

Total testosterone quantitative measurement in serum by LC-MS/MS ☆

Reliable measurement of total testosterone is essential for the diagnosis, treatment and prevention of a number of hormone-related diseases affecting adults and children. A mass spectrometric method for testosterone determination in human serum was carefully developed and thoroughly validated. Total testosterone from 100 μL serum is released from proteins with acidic buffer and isolated by two serial liquid-liquid extraction steps. The first extraction step isolates the lipid fractions from an acidic buffer solution using ethyl acetate and hexane. The organic phase is dried down and reconstituted in a basic buffer solution. The second extraction step removes the phospholipids and other components by hexane extraction. Liquid chromatography-isotopic dilution tandem mass spectrometry is used to quantify the total testosterone. The sample preparation is automatically conducted in a liquid-handling system with 96-deepwell plates. The method limit of detection is 9.71 pmol/L (0.280 ng/dL) and the method average percent bias is not significantly different from reference methods. The performance of this method has proven to be consistent with the method precision over a 2-year period ranging from 3.7 to 4.8% for quality control pools at the concentrations 0.527, 7.90 and 30.7 nmol/L (15.2, 228, and 886 ng/dL), respectively. This method provides consistently high accuracy and excellent precision for testosterone determination in human serum across all clinical relevant concentrations.

High variability in serum estradiol measurements in men and women

To reduce the variability in estradiol (E2) testing and to assure better patient care, standardization of E2 measurements has been recommended. This study aims to assess the accuracy and variability of E2 measurements performed by 11 routine immunological methods and 6 mass spectrometry methods using single donor serum materials and to compare the results to a reference method. The contribution of calibration bias, specificity or matrix effects, and imprecision on the overall variability of individual assays was evaluated. This study showed substantial variability in serum E2 measurements in samples from men and pre- and post-menopausal women. The mean bias across all samples, for each participant, ranged between -2.4% and 235%, with 3 participants having a mean bias of over 100%. The data suggest that calibration bias is the major contributor to the overall variability for nine assays. The analytical performances of most assays measuring E2 concentrations do not meet current needs in research and patient care. Three out of 17 assays would meet performance criteria derived from biological variability of ±12.5% bias at concentrations ≥20 pg/mL, and a maximum allowable bias of ±2.5 pg/mL at concentrations <20 pg/mL. The sensitivity differs highly between assays. Most assays are not able to measure E2 levels below 10 pg/mL. Standardization, specifically calibration to a common standard by using panels of individual patient samples, can reduce the observed variability and improve the utility of E2 levels in clinical settings.

Serum Total Testosterone Concentrations in the US Household Population from the NHANES 2011–2012 Study Population

BACKGROUND Limited information is available about testosterone concentrations representative of the general US population, especially children, women, and non-Hispanic Asians. METHODS We obtained nationally representative data for total testosterone (totalT), measured with standardized LC-MS/MS, for the US population age 6 years and older from the 2011-2012 National Health and Nutrition Examination Survey (NHANES). We analyzed 6746 serum samples and calculated the geometric means, distribution percentiles, and covariate-adjusted geometric means by age, sex, and race/ethnicity. RESULTS The 10th-90th percentiles of totalT values in adults (≥20 years) was 150-698 ng/dL (5.20-24.2 nmol/L) in men, 7.1-49.8 ng/dL (0.25-1.73 nmol/L) in women, and 1.0-9.5 ng/dL (0.04-0.33 nmol/L) in children (6-10 years old). Differences among race/ethnic groups existed in children and men: covariate-adjusted totalT values in non-Hispanic Asians were highest among children (58% compared to non-Hispanic black children) and lowest among men (12% compared to Mexican-American men). Covariate-adjusted totalT values in men were higher at age 55-60 years compared to ages 35 and 80 years, a pattern different from that observed in previous NHANES cycles. CONCLUSIONS TotalT patterns were different among age groups in men compared with previous NHANES cycles. Covariate-adjusted totalT values peaked at age 55-60 years in men, which appeared to be consistent with the increased use of exogenous testosterone. Differences among race/ethnic groups existed and appeared more pronounced in children than adults.

Normal Pubertal Development in Daughters of Women with PCOS: A Controlled Study

Context Daughters of women with polycystic ovary syndrome (PCOS) are thought to be at increased risk for developing stigmata of the syndrome, but the ontogeny during puberty is uncertain. Objective We phenotyped daughters (n = 76) of mothers with PCOS and daughters (n = 80) from control mothers for reproductive and metabolic parameters characteristic of PCOS. Design, Setting, and Participants We performed a matched case/control study at Penn State Hershey Medical Center that included non-Hispanic, white girls 4 to 17 years old. Intervention We obtained birth history, biometric, ovarian ultrasounds, whole-body dual-energy X-ray absorptiometry scan for body composition, 2-hour glucose challenged salivary insulin levels, and two timed urinary collections (12 hours overnight and 3 hours in the morning) for gonadotropins and sex steroids. Main Outcome Measures We measured integrated urinary levels of adrenal (dehydroepiandrosterone sulfate) and ovarian [testosterone (TT)] steroids. Other endpoints included integrated salivary insulin levels and urinary luteinizing hormone levels. Results There were no differences in detection rates or mean levels for gonadotropins and sex steroids in timed urinary collections between PCOS daughters and control daughters, nor were there differences in integrated salivary insulin levels. Results showed that 69% of Tanner 4/5 PCOS daughters vs 31% of control daughters had hirsutism defined as a Ferriman-Gallwey score >8 (P = 0.04). There were no differences in body composition as determined by dual-energy X-ray absorptiometry between groups in the three major body contents (i.e., bone, lean body mass, and fat) or in ovarian volume between groups. Conclusions Matched for pubertal stage, PCOS daughters have similar levels of urinary androgens and gonadotropins as well as glucose-challenged salivary insulin levels.