A reference interval (RI)10 is a standard component of reporting a laboratory result and is important to transform a numerical value into clinically meaningful information. An RI is intended to inform the clinical care provider that laboratory values within the interval indicate a nondiseased condition. The most common approach is to base an RI on the central 95% of laboratory test values observed for a reference population that is free of diseases that influence that laboratory test result. Because many diseases are asymptomatic, it becomes difficult to qualify people for a nondiseased condition, thus biasing the selection of reference individuals. Furthermore, information on the full complement of disease conditions that influence a laboratory test may be unknown. Thus, RIs may be influenced by inappropriately selected reference populations.Another limitation in determining an RI is obtaining an adequate sample of a reference population to make an estimate of the central 95% of results with suitable uncertainty to be meaningful for interpreting a test result. The sample size requirement becomes even larger when partitioning by sex, age, ethnicity, menstrual cycle, and other parameters is necessary for meaningful RIs. CLSI Guideline EP28-A3c describes consensus approaches and some limitations for establishing and verifying RIs. However, some of the approaches in this guideline are statistically underpowered such that uncertainties in the RIs may not be appreciated.A particularly challenging situation is the requirement for laboratories to establish an RI for a laboratory developed measurement procedure (MP) or to verify the RIs proposed by the manufacturer of an in vitro diagnostic (IVD) MP. Identifying a suitable reference population for either requirement can be very challenging.Evolution in laboratory practice is needed to enable appropriate RIs to be adopted by laboratories. We asked experts with different perspectives to address issues we all face in establishing or …

Departments of Pathology and Medicine (L.M.D.), Pennsylvania State University School of Medicine,Hershey, Pennsylvania 17003; Division of Biostatistics and Epidemiology (S.E.H.), University ofMassachusetts, Amherst, Massachusetts 01003; Ann and Robert H. Lurie Children’s Hospital of Chicago(S.H.), Chicago, Illinois 60611; Nuffield Department of Population Health (T.K.), University of Oxford,Oxford OX3 7LF, United Kingdom; Department of Medicine (W.R.), Columbia University, College ofPhysicians and Surgeons, New York, New York 10032; Division of Endocrinology and Metabolism(R.J.S.), University of Virginia Health Sciences System, Charlottesville, Virginia 22903; Departments ofObstetrics/Gynecology and Preventive Medicine (F.Z.S.), University of Southern California Keck School ofMedicine, Los Angeles, California 90033; Division of Laboratory Sciences (H.W.V.), Centers for DiseaseControl and Prevention, Atlanta, Georgia 30333; and Division of Cancer Epidemiology and Genetics(R.G.Z.), National Cancer Institute, Bethesda, Maryland 20892

SHBG is a plasma protein that participates in the regulation of free estradiol and free testosterone in plasma. We discuss the concept of the nature of a free estradiol and how best to ascertain its value. It can be measured or calculated; the ways in which this can be done are explored along with the advantages and disadvantages of each.

Laboratory test results are used to inform decisions on the diagnosis and treatment of disease. Consistent and comparable results from different measurement procedures are important for developing clinical practice guidelines and for those guidelines to be applied to decisions about patient care. ISO document 17511:2003 ( In vitro diagnostic medical devices -- Measurement of quantities in biological samples -- Metrological traceability of values assigned to calibrators and control materials ) states that calibration of routine clinical laboratory measurement procedures be traceable to higher-order reference materials and reference measurement procedures. Such traceability can achieve consistent and comparable results that are sustainable over time and among different measurement procedures.The terms “standardized” and “harmonized” are frequently used interchangeably to refer to the condition in which results are consistent and comparable among different measurement procedures. “Standardized” refers to the condition in which calibration is traceable to a reference measurement procedure that is typically calibrated with an appropriate reference material. There are situations in which the reference measurement procedure defines the analyte without any primary reference material (e.g., enzyme activity). Standardization has the advantage of trustworthy reproducibility over time and location, because the reference measurement procedure provides a stable anchor for calibration traceability of routine clinical laboratory procedures. “Harmonized” is a more general term. It may include the standardized condition, but it also includes the condition in which results are consistent and comparable in the absence of a reference measurement procedure. The process of harmonization typically depends on the availability of a suitable reference material that can be used as a common calibrator among routine clinical laboratory measurement procedures.Despite the availability of reference measurement procedures and reference materials, results for many analytes are neither consistent nor comparable when measured with different clinical laboratory procedures. In this Q&A, experts in harmonization of test results offer their …

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.

In this issue of Clinical Chemistry , Botelho et al. present another candidate reference measurement procedure (RMP) for total testosterone in human serum (1). As the authors acknowledge, there are already 3 approved RMPs for testosterone in the literature (2–4). An RMP is a procedure that has, among other important requirements, negligible inaccuracy (a negligibly small deviation from the true value) compared with its reproducibility. The precise requisites for an RMP are set out by ISO15193 (International Organization for Standardization) (5), and candidate RMPs must be certified and approved by the Joint Committee for Traceability in Laboratory Medicine. RMPs are generally difficult to accomplish, time-consuming, and expensive; they are not intended for adoption by all laboratories conducting a test for a given measurand but rather are a standard against which other less complex, less expensive, and less time-consuming methods may be judged. The burden for developing an RMP is heavy—justifiably so. Nevertheless, for serum samples from both sexes, all of the stringent requirements set …

We examined the influence of numerous substances on the secretion of corticosteroid-binding globulin (CBG) and testosterone-estradiol binding globulin (TeBG) by a human hepatoma-derived cell line. Thyroxine, at physiologic concentrations, resulted in an increased secretion of TeBG but not CBG. Estrogens, antiestrogens, and androgens were without effect on either of these binding proteins.

IntroductionSex hormone-binding globulin (SHBG) is a sex steroid binding protein, originally described in humans as the major binding protein for estrogens and androgens in plasma (Anderson, 1974;Avvakumov, et al, 2010).By governing equilibrium conditions in plasma between bound and free sex steroids, SHBG regulates the availability of the latter to hormonally responsive tissues.Along with regulating free steroid concentrations in plasma, it is increasingly evident that SHBG also participates in other biological processes.These include, but are not limited to-activation of a rapid, membrane based steroid signaling pathway in tissues such as the prostate and breast (Rosner et al, 2010); spermatogenesis (Selva and Hammond, 2006); and a yet to be determined consequence of co-localization with oxytosin in brain cells (Caldwell et al, 2006).Plasma based SHBG is extensively studied, especially in the context of its regulation of free steroid concentrations and epidemiologic associations.The origin of plasma SHBG is, for all intents and purposes, the liver (Khan et al, 1981;Pugeat et al, 2010) (a differentially glycosylated isoform, androgen binding protein (ABP) is synthesized in the testis (Vigersky et al, 1976)).However, we now know that SHBG is also synthesized, albeit to a much lesser degree, in certain hormonally responsive tissues (Kahn et al, 2002).Early studies demonstrated immunoreactive SHBG in the prostate and breast (

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.

Sex hormone-binding globulin (SHBG) was initially described as a plasma protein synthesized in, and secreted by, the liver. It was discovered by its ability to bind certain androgens and estrogens and, for many years, was believed to serve as a transporter/reservoir for the steroids which it bound. Subsequently, it became clear that the cell membranes of selected tissues contained a receptor for SHBG (R(SHBG)). This review deals with what is known of that receptor - its anatomy, physiology and biochemistry.

INTRODUCTION:Endocrine changes during aging as well as endocrine disorders may either directly or indirectly modulate female sexual function by altering sex hormones, or by impacting on vascular, neurogenic, or psychologic factors.AIM:To review information on the impact of the hormonal changes associated with aging or those caused by endocrine disorders on female sexual function and current information on the risks and benefits of hormonal treatments.METHODS:Committee members outlined topics and reviewed the published literature on endocrine aspects of female sexual function over a 2-year period. Presentation of the recommendations were presented at the International Consultation on Sexual Medicine Paris, France 2009 and revised accordingly.MAIN OUTCOME MEASURES:Quality of data published in the literature and recommendations were based on the GRADES system.RESULTS:Recommendations and guidelines concerning the role of sex hormones and endocrine disorders in female sexual function were derived.CONCLUSIONS:Hormones are only one component of the many factors that contribute to normal sexual function in women. Further research is needed as to the impact of hormones and endocrine disorders on female sexual dysfunction and the benefits and risks of hormonal therapies.

Background: Human sex hormone-binding globulin (SHBG) regulates free sex steroid concentrations in plasma and modulates rapid, membrane based steroid signaling. SHBG is encoded by an eight exon-long transcript whose expression is regulated by a downstream promoter (P-L). The SHBG gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter (P-T), and two minor transcripts.Results: We report that transcriptional expression of the human SHBG gene is far more complex than previously described. P-L and P-T direct the expression of at least six independent transcripts each, resulting from alternative splicing of exons 4, 5, 6, and/or 7. We mapped two transcriptional start sites downstream of P-L and P-T, and present evidence for a third SHBG gene promoter (P-N) within the neighboring FXR2 gene; P-N regulates the expression of at least seven independent SHBG gene transcripts, each possessing a novel, 164-nt first exon (1N). Transcriptional expression patterns were generated for human prostate, breast, testis, liver, and brain, and the LNCaP, MCF-7, and HepG2 cell lines. Each expresses the SHBG transcript, albeit in varying abundance. Alternative splicing was more pronounced in the cancer cell lines. P-L- P-T- and P-N-derived transcripts were most abundant in liver, testis, and prostate, respectively. Initial findings reveal the existence of a smaller immunoreactive SHBG species in LNCaP, MCF-7, and HepG2 cells.Conclusion: These results extend our understanding of human SHBG gene transcription, and raise new and important questions regarding the role of novel alternatively spliced transcripts, their function in hormonally responsive tissues including the breast and prostate, and the role that aberrant SHBG gene expression may play in cancer.

1230 The role of androgens in prostate cancer is well established; yet not all facets of androgen signaling are well understood. Sex hormone-binding globulin (SHBG), which binds androgens in plasma, mediates a rapid androgen response in prostate cells through a specific membrane-based receptor (RSHBG) independent of the androgen receptor (AR). SHBG and its associated signaling pathways could provide potential therapeutic targets for prostate cancer. SHBG mRNA and protein is expressed by prostate cells, suggesting autocrine/paracrine properties. We hypothesize that SHBG regulates gene expression through RSHBG, and also acts as an intracellular buffer for androgens to modulate AR activation. The SHBG and p53 tumor suppressor genes colocalize on chromosome 17 to a region often deleted in tumors, implicating a role in prostate cancer for aberrant SHBG expression. In this study, we used microarray analysis to acertain the effects of SHBG on androgen signaling in the LNCaP prostate cell line. L5S2, a LNCaP-derived clonal cell line, reproducibly induces SHBG overexpression upon Ponasterone A (PonA) treatment. L5V4 is its noniducible isogenic sister vector control cell line. L5S2 and L5V4 cells were grown in androgen depleted medium, treated for 24hrs with PonA to induce SHBG or vehicle alone, then with DHT or vehicle alone for another 24 hours. Experiments were performed in triplicate. Cellular RNAs were isolated, labeled, and hybridized to Affymetrix Human Genome U133 Plus 2.0 Array chips. In response to SHBG overexpression and DHT, 1131 genes were significantly induced or repressed by conditions resulting in RSHBG activation and 187 DHT responsive genes were modulated by SHBG. Real-time PCR analysis was used to validate microarray results. Our results support a biologic role for RSHBG signaling in LNCaP cells. We also provide the first evidence that intracellular SHBG may regulate AR activity- decreased SHBG expression in prostate tumors may allow the AR to function under reduced intracellular androgen concentrations. Putative downstream targets of SHBG + DHT signaling include STEAP4, FKBP5, TIMP2, GPR89a, GPR30, and c-MYC, genes with important and provocative cellular functions with respect to androgen signaling and prostate cancer. If confirmed, we plan to identify intermediaries in SHBG-mediated androgen signaling, and address the functional role of dysregulated SHBG expression in prostate cancer.

Sex hormone-binding globulin (SHBG), a plasma protein that binds androgens and estrogens, also participates in the initial steps of a membrane-based steroid signaling pathway in human prostate and breast. We have recently shown that SHBG is expressed at the mRNA and protein levels in the prostate and breast. In this study, we addressed whether locally expressed SHBG: (1) Functions to regulate activation of membrane-based steroid signaling and (2) influences activation of the androgen (AR) and estrogen (ER) receptors. Using microarray analysis, we identified specific genes that are influenced by SHBG expression in LNCaP and MCF-7 cells in a manner consistent with each of these properties. These findings suggest that locally expressed SHBG can play a functional role in the steroid responsiveness of prostate and breast cells through multiple signaling pathways and that perturbations in local SHBG expression could contribute to prostate and breast cancer.

Objetivo: analizar el estado actrual de las evaluaciones clinicas de la testosterona libre y total. Resultados: la informacion se obtuvo de fuentes publicadas en bases de datos en linea del Colegio de Patologos Estadounidenses y de las experiencias de laboratorio y clinicas de de las sociedades participantes. Conclusiones: la prueba de competencia del laboratorio debe basarse no solo en la capacidad de medir con exactitud las muestras que contengan concentraciones conocidas de testosterona, sino en llegar a un acuerdo para utilizar el mismo metodo. Cuando se estandarice dicho metodo deben establecerse valores normativos para la testosterona libre y total para ambos generos y para los ninos

Sex hormone-binding globulin (SHBG), a protein that binds plasma androgens and estrogens, participates in the initial steps of a membrane-based steroid signaling pathway in human prostate cells that involves a specific receptor, RSHBG, and is independent of the androgen receptor (AR). We have recently shown that SHBG is locally expressed in prostate cells. We hypothesized that locally expressed SHBG has at least two functions in prostate cells, 1) to regulate membrane based steroid signalling, and 2) to bind androgens intracellularly, thereby regulating AR activity. Microarray analysis was used to identify genes whose expression is regulated by SHBG in a manner consistent with these two functions.

Sex hormone binding globulin (SHBG), a protein that binds plasma androgens and estrogens, participates in the initial steps of a membrane based steroid signaling pathway in human prostate cells. We have recently shown that SHBG is locally expressed in prostate cells. Here, we used microarray analysis of human LNCaP prostate cancer cells that are engineered to inducibly overexpress SHBG to investigate whether locally expressed SHBG regulates the activation of membrane based androgen signaling through the SHBG receptor, RSHBG, and/or intracellular activation of the androgen receptor (AR). We hypothesize that alterations of endogenous SHBG expression in prostate cancer, such as those presumed to be accompanied by deletion of the linked p53/SHBG gene locus on chromosome 17p13.1, can affect androgen signaling through RSHBG and the AR, thus contributing to cancer progression.