Hubert W. Vesper

Reference Ranges for Testosterone in Men Generated Using Liquid Chromatography Tandem Mass Spectrometry in a Community-Based Sample of Healthy Nonobese Young Men in the Framingham Heart Study and Applied to Three Geographically Distinct Cohorts

CONTEXT Reference ranges are essential for partitioning testosterone levels into low or normal and making the diagnosis of androgen deficiency. We established reference ranges for total testosterone (TT) and free testosterone (FT) in a community-based sample of men. METHODS TT was measured using liquid chromatography tandem mass spectrometry in nonobese healthy men, 19-40 yr old, in the Framingham Heart Study Generation 3; FT was calculated. Values below the 2.5th percentile of reference sample were deemed low. We determined the association of low TT and FT with physical dysfunction, sexual symptoms [European Male Aging Study (EMAS) only], and diabetes mellitus in three cohorts: Framingham Heart Study generations 2 and 3, EMAS, and the Osteoporotic Fractures in Men Study. RESULTS In a reference sample of 456 men, mean (sd), median (quartile), and 2.5th percentile values were 723.8 (221.1), 698.7 (296.5), and 348.3 ng/dl for TT and 141. 8 (45.0), 134.0 (60.0), and 70.0 pg/ml for FT, respectively. In all three samples, men with low TT and FT were more likely to have slow walking speed, difficulty climbing stairs, or frailty and diabetes than those with normal levels. In EMAS, men with low TT and FT were more likely to report sexual symptoms than men with normal levels. Men with low TT and FT were more likely to have at least one of the following: sexual symptoms (EMAS only), physical dysfunction, or diabetes. CONCLUSION Reference ranges generated in a community-based sample of men provide a rational basis for categorizing testosterone levels as low or normal. Men with low TT or FT by these criteria had higher prevalence of physical dysfunction, sexual dysfunction, and diabetes. These reference limits should be validated prospectively in relation to incident outcomes and in randomized trials.

Vitamin D status as an international issue: National surveys and the problem of standardization

Abstract Wide spread variation in measurement results of total 25-hydroxyvitamin D (25(OH)D) confounds international efforts to develop evidence-based clinical guidelines. Accordingly, NIH Office of Dietary Supplements (ODS) in collaboration with CDC National Center for Environmental Health (NCEH), National Institute of Standards and Technology (NIST) and Ghent University established the Vitamin D Standardization Program (VDSP) in November 2010. VDSP objectives include: (1) standardize 25(OH)D concentration measurements in national health surveys around the world, (2) evaluate survey differences, (3) extend standardization efforts to assay manufacturers, and to clinical, commercial, and research laboratories, (4) promote standardization of emerging metabolites of vitamin D status, and (5) enable the use of standardized data in patient care and public health. An interlaboratory comparison study is being conducted to assess measurement variability among current assays. Participants include national health surveys from Australia, Canada, Germany, Ireland, Mexico, South Korea, UK and USA, 15 assay manufacturers, and two external quality assurance programs. CDC will implement a formal laboratory certification program. Standardization activities will use single-donor, fresh-frozen serum collected using the CLSI C37 protocol. Initial assay performance criteria, based on biological variability data, are ≤10 % imprecision and ≤5 % bias in relation to the reference values. An ancillary study on commutability of NIST SRM 972a, external quality assurance testing materials is included. To increase the comparability of existing data from different national surveys, master equations will be developed to facilitate the conversion of already existing national survey data to the NIST-Ghent University reference measurement procedures.

Challenges to the Measurement of Estradiol: An Endocrine Society Position Statement

OBJECTIVE The objective of the study was to evaluate the current state of clinical assays for estradiol in the context of their applications. PARTICIPANTS The participants were appointed by the Council of The Endocrine Society and charged with attaining the objective using published data and expert opinion. EVIDENCE Data were gathered from published sources via online databases (principally PubMed, Ovid MEDLINE, Google Scholar), and the clinical and laboratory experience of the participants. CONSENSUS PROCESS The statement was an effort of the committee and was reviewed by each member. The Clinical Affairs Committee, the Council of The Endocrine Society, and JCEM reviewers reviewed the manuscript and made recommendations. CONCLUSIONS The measurement of estradiol in biological fluids is important in human biology from cradle to grave. In addition to its centrality in sexual development, it has significant effects on skin, blood vessels, bone, muscle, coagulation, hepatic cells, adipose tissue, the kidney, the gastrointestinal tract, brain, lung, and pancreas. Alterations in its plasma concentration have been implicated in coronary artery disease, stroke, and breast cancer. Although modern immunoassays and liquid chromatography/tandem mass spectrometry-based methods for estradiol are reasonably well suited to the diagnosis and management of infertility (nonetheless, imprecision and method-to-method differences remain problematic), the very low concentrations that appear to be crucial in nonreproductive tissues are a separate and more difficult issue. Such levels of estradiol are too low to be routinely measured accurately or precisely, and further evolution of analytical methods and the way in which estradiol is standardized is needed.

Toward Excellence in Testosterone Testing: A Consensus Statement

BACKGROUND Testosterone assays are widely used. However, deficiencies in these assays limit their broad and effective implementation and threaten the health of those patients whose medical care relies upon its accurate measurement. Furthermore, the translation of research findings into information useful for patient care, such as new evidence-based clinical guidelines, is not possible unless both research and clinical assays are held to higher standards than are currently required. A group of concerned stakeholders was convened to address this problem. METHODS Representatives of multiple professional societies, government, and industry, having a stake in ensuring that testosterone levels are measured accurately and reliably, met to identify goals, objectives, and actions necessary to bring about the standardization of assays for testosterone. RESULTS To ensure highly accurate testosterone testing that will result in improved diagnosis, treatment, and prevention of disease through the use of standardized assays, a series of recommendations were agreed upon. The recommendations included the following: technical improvements for assay standardization; education of health care providers, patients, and all others concerned with testosterone testing; plans to encourage all concerned journals, government agencies, and health insurance companies to support this effort; and encouragement to manufacturers to develop better and more cost effective assays. CONCLUSION A preliminary timeline was set out to implement the recommendations of the Group.

Traceability in Laboratory Medicine

BACKGROUND In patient and population samples, generation of analytical results that are comparable and independent of the measurement system, time, and location is essential for the utility of laboratory information supplied in healthcare. Obtaining analytical measurement results with such characteristics is the aim of traceability in laboratory medicine. As awareness of the benefits of having traceable measurement results has increased, associated efforts have been directed toward making traceability a regulatory requirement and developing approaches to enable and facilitate the implementation of traceability. Although traceability has been a main focus of many laboratory standardization activities in the past, discussions are still ongoing with regard to traceability and its implementation. CONTENT This review provides information about the traceability concept and what needs can be fulfilled and benefits achieved by the availability of traceable measurement results. Special emphasis is given to the new metrological terminology introduced with this concept. The review addresses and describes approaches for technical implementation of traceable methods as well as the associated challenges. Traceability is also discussed in the context of other activities to improve the overall measurement process. SUMMARY Establishing metrological traceability of measurement results satisfies basic clinical and public health needs, thus improving patient care and disease control and prevention. Large advances have been made to facilitate the implementation of traceability. However, details in the implementation process, such as lack of available commutable reference materials and insufficient resources to develop new reference measurement systems continue to challenge the laboratory medicine community.

Interlaboratory comparison study of serum total testoserone measurements performed by mass spectrometry methods

BACKGROUND Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays. METHODS Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)-tandem mass spectrometry (HPLC-MS/MS, GC-MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis. RESULTS The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid-liquid extraction for sample preparation or GC-MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range. CONCLUSIONS The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.

Evaluation of Vitamin D Standardization Program protocols for standardizing serum 25-hydroxyvitamin D data: a case study of the program's potential for national nutrition and health surveys

BACKGROUND The Vitamin D Standardization Program (VDSP) has developed protocols for standardizing procedures of 25-hydroxyvitamin D [25(OH)D] measurement in National Health/Nutrition Surveys to promote 25(OH)D measurements that are accurate and comparable over time, location, and laboratory procedure to improve public health practice. OBJECTIVE We applied VDSP protocols to existing ELISA-derived serum 25(OH)D data from the Irish National Adult Nutrition Survey (NANS) as a case-study survey and evaluated their effectiveness by comparison of the protocol-projected estimates with those from a reanalysis of survey serums by using liquid chromatography-tandem mass spectrometry (LC-tandem MS). DESIGN The VDSP reference system and protocols were applied to ELISA-based serum 25(OH)D data from the representative NANS sample (n = 1118). A reanalysis of 99 stored serums by using standardized LC-tandem MS and resulting regression equations yielded predicted standardized serum 25(OH)D values, which were then compared with LC-tandem MS reanalyzed values for all serums. RESULTS Year-round prevalence rates for serum 25(OH)D concentrations <30, <40, and <50 nmol/L were 6.5%, 21.9%, and 40.0%, respectively, via original ELISA measurements and 11.4%, 25.3%, and 43.7%, respectively, when VDSP protocols were applied. Differences in estimates at <30- and <40-nmol/L thresholds, but not at the <50-nmol/L threshold, were significant (P < 0.05). A reanalysis of all serums by using LC-tandem MS confirmed prevalence estimates as 11.2%, 27.2%, and 45.0%, respectively. Prevalences of serum 25(OH)D concentrations >125 nmol/L were 1.2%, 0.3%, and 0.6% by means of ELISA, VDSP protocols, and LC-tandem MS, respectively. CONCLUSION VDSP protocols hold a major potential for national nutrition and health surveys in terms of the standardization of serum 25(OH)D data.

Levels of Plasma trans-Fatty Acids in Non-Hispanic White Adults in the United States in 2000 and 2009

Levels of trans-fatty acids (TFAs) in blood come from natural sources, such as milk, and industrial sources, such as partially hydrogenated vegetable oils. Dietary intake of TFAs increases low-density lipoprotein cholesterol (LDL-C) and has other adverse metabolic effects.1 Changing to a diet low in TFAs may lower the LDL-C level and decrease the risk for cardiovascular disease. To assist consumers, the Food and Drug Administration amended its regulations in 2003 to require that TFA content be declared on the nutrition label of foods and dietary supplements.2 Some community and state health departments have required restaurants to limit TFAs and reductions have been shown in supermarket and restaurant products.

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.

Standardization of Immunoassays for Measurement of High-Sensitivity C-reactive Protein. Phase I: Evaluation of Secondary Reference Materials

BACKGROUND Inflammation contributes to the development and progression of atherosclerosis, and C-reactive protein (CRP) can be used as a marker to assess risk for cardiovascular diseases. As variability among existing high-sensitivity CRP (hsCRP) assays can lead to misclassification of patients and hamper implementation of population-based medical decision points, standardization of hsCRP assays is needed. METHODS We evaluated five proposed secondary reference materials, including two diluted preparations of Certified Reference Material 470 (CRM470), two preparations of a serum-based material with recombinant CRP added, and one serum-based material with isolated CRP added. Twenty-one manufacturers participated in the comparison with 28 different assays. We examined imprecision, linearity, and parallelism with these materials and with fresh serum. RESULTS All materials had similar imprecision; CVs for the undiluted materials were 2.1-3.7%. None of the materials was linear across all assays. Each had between one and three cases of nonlinearity, with one preparation of CRM470 having the fewest cases of nonlinearity. Although none of the materials was parallel across all assays, the differences in slope from fresh serum were similar across all assays. CONCLUSIONS All materials performed similarly with regard to imprecision, linearity, and parallelism. As one preparation of CRM470 had slightly better characteristics than the other materials and because CRM470 had been certified previously as a reference material for the acute-phase reactant range, it will be used in the next phase to standardize hsCRP assays.