American Urological Association - Laboratory Diagnosis of Testosterone Deficiency

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Laboratory Diagnosis of Testosterone Deficiency

Published 2013

Panel Members

Darius A. Paduch, MD, PhD, Robert E. Brannigan, MD, Eugene F. Fuchs, MD, Edward D. Kim, MD, Joel L. Marmar, MD, Jay I. Sandlow, MD

Abstract

Evaluation and treatment of hypogonadal men has become an important part of urological practice. Fatigue, loss of libido and erectile dysfunction are commonly reported, but non-specific symptoms of low testosterone (T) and laboratory verification of low testosterone is an important part of evaluation in addition to a detailed history and physical exam. Significant intra-individual fluctuations in serum T levels, biological variation of T action on end organs, the wide range of T levels in human serum samples and technical limitations of currently available assays have led to poor reliability of T measurements in the clinical laboratory setting. There is no universally accepted threshold of T concentration that distinguishes eugonadal from hypogonadal men, thus laboratory results have to be interpreted in the appropriate clinical setting. This review focuses on clinical, biological and technological challenges that affect serum T measurements to educate clinicians regarding technological advances and limitations of currently available laboratory methods to diagnose hypogonadism. A collaborative effort led by the American Urological Association (AUA) between practicing clinicians, patient advocacy groups, government regulatory agencies, industry and professional societies is underway to provide optimized assay platforms and evidence based normal assay ranges to guide clinical decision making. Until such standardization is commonplace in clinical laboratories, the decision to treat should be based on the presence of signs and symptoms in addition to serum T measurements. Rigid interpretation of T ranges should not dictate clinical decision making or define coverage of treatment by third party payers.

Keywords

Testosterone
Bioavailable testosterone
Free testosterone
Sex-hormone binding globulin
Androgen deficiency
Hypogonadism
Assays

Introduction

The laboratory diagnosis of testosterone (T) deficiency is a challenge. Serum T levels are subject to temporal variation— diurnal, seasonal and age-related. Illness and certain medications, such as opiates and glucocorticoids, can temporarily affect T concentrations through central and peripheral effects. Total testosterone (TT) concentrations are affected by alterations in sex-hormone binding globulin (SHBG), which in turn can vary for a variety of reasons, including age, medications and medical comorbidities. There are several different assays for measurement of T levels, and performance characteristics, linearity, reproducibility, low level limits of detection and pre-analytical requirements differ among the assay platforms. The populations upon which the normal assay ranges are established differ between assays leading to a wide variety of normal ranges reported by different laboratories. Lastly, T circulates in the blood primarily bound specifically to SHBG or non-specifically to albumin, with only 2-3% of TT being free. Whether TT or free testosterone (FT) measurements most closely correlate with symptomatic androgen deficiency is a matter of debate. 

Androgen deficiency may become apparent at different ages within an individual or a population. T levels are affected by age; body mass index (BMI); and comorbidities, such as type 2 diabetes mellitus (T2DM), depression, anxiety, thyroid disorders, malnutrition, alcohol consumption and physical activity.  There is no large population-based study of T values from healthy, fertile men with normal sexual activity and reproductive function assessed by commonly accepted validation methods. The lack of these types of studies confuses clinical decision making and impairs comparison of assays on the same subject obtained in different laboratories. 

Because of the multitude of factors affecting the laboratory evaluation and interpretation of T levels, it is no surprise that a significant, universally accepted definition of T deficiency is lacking. The American Urological Association (AUA), together with the Endocrine Society and the Centers for Disease Control and Prevention (CDC), has been a leading force in addressing technical difficulties in T measurements, establishing clinically relevant normal assay ranges and harmonizing T assay performance across different platforms.1  Improving assay accuracy, sensitivity and reproducibility as well as advocating that laboratories disclose information about their methodologies with results and provide a standardized definition of normal populations used to establish normal assay ranges should help clinicians to deliver better care for their patients. 

Herein, we discuss the currently used assays for T measurement, their utility and limitations and the implications for clinical practice relevant to practice of urology and andrology.

Defining Testosterone Deficiency

There is no consensus among endocrinologists, urologists and clinical pathologists as to what defines a “low” T level. Published normal assay ranges for serum T are mostly based on studies in older men (>65 years old) and were not specifically designed to establish normal assay ranges in men with normal sexual and reproductive function. It is clear that T level correlates with overall health status and normal sexual function.  To best serve our patients, it is our belief that the normal ranges should be based on a predefined, healthy index population representing the demographic structure of the United States. The Endocrine Society recommends that total low T be defined using local, normal assay ranges in the presence of characteristic signs or symptoms diagnostic of hypogonadism.2 The Food and Drug Administration (FDA) uses a cut-off value of 300 ng/dL to define hypogonadism for clinical trial development and enrollment.  Meanwhile, a consensus statement from the International Society of Andrology (ISA), the International Society for Study of the Aging Male (ISSAM), the European Association of Urology (EAU), the European Association of Andrology (EAA) and the American Society of Andrology (ASA) recommended that TT levels above 350 ng/dL do not require treatment, and levels below 230 ng/dL (with symptoms) may require T replacement therapy.3 For levels between 230-350 ng/dL, the recommendation is to repeat the TT with SHBG for calculation of FT or direct measurement of FT by equilibrium dialysis.3 Similarly, it has been previously recommended that men with TT<200 ng/dL be treated as hypogonadal, those with TT>400 ng/dL be considered normal and those with TT 200-400 ng/dL be treated based on their clinical presentation if symptomatic.4

Considering that serum T level is used as a surrogate of target organ concentration of T and based on a review of the published literature and the best clinical judgment of the authors of this manuscript, this panel emphasizes that signs and symptoms suggestive of hypogonadism and laboratory measured T level are equally important indicators of hypogonadism and indicators for treatment until more research is done. We believe that rigid use of T cut-off (300 ng/dL) may lead to unnecessary treatment of asymptomatic men as well as under-treatment of men with persistent signs and symptoms.

Use of free or bioavailable T may aid in the biochemical diagnosis of hypogonadism, especially when results of the TT assay are equivocal or fail to reflect clinical presentation.3  There are no generally accepted lower limits of normal FT for the diagnosis of hypogonadism. According to expert opinion, a FT level below 65 pg/ml may provide supportive evidence for treatment.3 Corresponding values for bioavailable T depend on the method used and are not generally available for healthy young men.5 Calculated FT using measured TT and SHBG values is a feasible approach to include the SHBG variability in the interpretation of total T.6

It is no surprise that attempts to establish a uniform laboratory threshold that accurately distinguishes hypogonadal and eugonadal men among the broad range of subjects of different ages and ethnic background has been difficult given  lack of agreement on what constitutes the definition of  a “normal” subject, tendency to select men older than 65 years for published studies on hypogonadism, different inclusion and exclusion criteria among studies and technical differences in assays used. Mean age of subjects enrolled in the largest population studies on male hypogonadism was 75.4 for the MrOS arm in Sweden, 73.7 in the United States based arm of MrOS and 60 for the EMAS study.7-9 More studies focused on demographically matched healthy male populations are clearly needed to establish normal assay ranges for diverse age group of men. 

Prevalence Of Testosterone Deficiency

In a multi-ethnic, population-based observational study of 1,475 men aged 30-79 years in the United States, Araujo et al.10 observed the prevalence of symptomatic androgen deficiency in hypogonadal men (TT<300 ng/dL) to be 5.6% (95% CI, 3.6-8.6%). Symptomatic hypogonadism was defined as presence of low libido, erectile dysfunction, osteoporosis or fracture or two or more of following symptoms: sleep disturbance, depressed mood, lethargy or diminished physical performance. The prevalence of hypogonadism was lower in men less 70 years old (3.1-7.0%), but increased substantially with age to 18.4%. Men older than 50 with a T level below 300 ng/dL were more likely to have hypogonadism related symptoms (8.4%) as compared to younger hypogonadal men (4.2%).  Longitudinal population-based studies of aging men have also demonstrated that both TT and FT decline with age with a concomitant increase in SHBG levels.11-13 Given this evidence, it is estimated that by the year 2025, there will be approximately 6.5 million American men 30-80 years of age diagnosed with androgen deficiency.10 Mulligan et al. reported prevalence of hypogonadism (TT<300 ng/dL) to be 38.7% among men >45 years attending general clinical practice, but the mean age of subjects in this study was 60 years, thus limiting the conclusions of study.14 The ability to relate the symptoms of androgen deficiency to accurate and reliable laboratory values has obvious clinical implications.

Variability In Testosterone Concentrations

Serum T in men shows a wide range of variation, owing to episodic secretion, diurnal variation, glucose ingestion, week-to-week variation, seasonal variation and type of activities prior to blood draw.15 The amplitude and diurnal variations in free and bioavailable T levels are similar to those of TT.16, 17 One report has described a circadian pattern of SHBG levels as well, with peaks occurring in the early afternoon,17 but this finding has not been corroborated by other studies. Genetic variants in SHBG may affect TT.18 

Serum T levels peak in the early morning, followed by a progressive decline over the course of the day until they reach their nadir in the evening hours. Nadir values are approximately 15% lower than morning values, but they may even vary by as much as 50% in younger subjects.19 Therefore, sampling time is an important consideration when interpreting serum T. Historically it was recommended that samples be obtained in the morning, between 07:00 and 11:00 hours especially in younger men, but this approach has been challenged and is often not followed in clinical practice.20, 21 This diurnal pattern is blunted in older men, and time of measurement may be less important in this age group.16, 22 While it has been argued that morning T measurements are not necessary in older men due to blunting of the circadian rhythm, a substantial fraction of older men aged 65 to 80 years who have low serum T in the afternoon will have normal T concentrations in the morning. 20, 23 Week long night/day shift work does not seem to change T levels.24 However, in one study, low morning T in rapidly changing shifts was associated with significant changes in T levels.25 Thus, in men working at night, T should be checked during off days. 

Brambilla et al. showed that “biological” intra-individual variation in T levels of approximately 10% is observed when samples are collected from the same individual at the same time of the day over several days.23 Approximately 15% of healthy men may have a T level below the normal assay range in a 24-hour period. Furthermore, among men with an initial T concentration in the mildly hypogonadal range, approximately 30% will have a normal T upon repeat measurement.23 Day-to-day variations in T concentrations can be large enough to render a single T measurement inadequate to accurately characterize an individual’s levels. Repeated measurement of T may be obtained if an initial laboratory value does not align with a subject’s signs and symptoms.2 

Results of T measurements are affected by patient factors, such as glucose intake, triglyceride levels, medication taken and initial processing of a sample from blood draw point to analytical laboratory.26  Pre-analytical factors include various technical factors, such as types of collection tubes used to obtain samples, sample centrifugations, intermediate storage and environmental conditions of sample transport. For example, storage of serum or plasma in collection tubes following centrifugation can affect the results of measured T after processing; storage in ethylenediaminetetraacetic acid (EDTA) can adversely affect SHBG measurement and thereby affect the calculation of free hormones.26 To improve accuracy of testing, the reader is advised to discuss the type of collection tube and initial processing protocol with the laboratory they use. The time from blood draw to measurements is an additional factor to consider. Samples obtained in the morning are typically transported and processed the same day, but samples drawn in the afternoon may be inappropriately stored for processing on the following day thereby invalidating specimen and thus measurement results. Effects of the initial processing of a sample may differ between direct and indirect assays. 

Circulating T levels are influenced by a variety of medical conditions, including medications, acute illness, sexual activity and SHBG concentrations, among many others. These factors must be taken into consideration when ordering or interpreting any T assay.26, 27 The suppression of T is particularly profound in men on methadone maintenance therapy due to its long duration of action. Acute illness can also temporarily but significantly lower serum T. Thus diagnosis of androgen deficiency should not be made during an acute illness.2

Testosterone: Total, Free, And Bioavailable

T circulates in the body bound to either SHBG, albumin or corticosteroid binding globulin (CBG), or in an unbound form (free). SHBG-bound T represents approximately 44% of the TT, is tightly bound and unavailable to cells. However, albumin-bound T represents approximately 50% of the total concentration, is weakly bound and dissociates easily and rapidly. CBG-bound T represents 4% of the TT and, like albumin, is weakly bound and dissociates rapidly. FT represents only about 2-3% of the TT.28 The term “bioavailable” T refers to the sum of the CBG-bound, albumin-bound and free components and represents the T fraction that is available to cells. This term should not be confused with cellular and tissue biological availability of T to bind to androgen receptor and exert its androgenic action.

SHBG can vary considerably to affect TT levels. Because SHBG-bound T is not bioavailable, TT may be a poor indicator of the adequacy of circulating androgens available for target organs, but TT is the recommended initial test to diagnose hypogonadism. Conditions that increase SHBG, including aging, hyperthyroidism and hepatic cirrhosis, or that decrease SHBG, including obesity, diabetes mellitus and glucocorticoid use, affect bioavailability of T. (Table 1). The increase in SHBG with age means that older men may have a normal TT levels, even if they are hypogonadal, as they have low levels of free or bioavailable T. Conversely, obesity decreases SHBG and TT, even when the bioavailable fraction may be normal.4

Independent of SHBG levels, some of the features of metabolic syndrome, such as hypertension, dyslipidemia, insulin resistance and obesity, are commonly present in hypogonadal men.29, 30 Hypogonadotropic hypogonadism occurs frequently in men with T2DM and obesity.31 Measurement of T in men with metabolic syndrome and symptoms of T deficiency have been suggested, but effect of weight loss on diagnosis of hypogonadism and the utility of androgen replacement therapy in symptomatic men continues to be an active area of research.3 A recent longitudinal study by Haring et al. showed that low T increases risk of developing metabolic syndrome, thus underscoring importance of early diagnosis of hypogonadism even in young men.32           

Laboratory Measurement Of Testosterone

T assays and their interpretation pose several challenges. T concentrations in serum vary more than three orders of magnitude, depending on age, gender and the presence of disease, and an adequate assay must be able to maintain accuracy, sensitivity, specificity and linearity over a large range of concentrations. Thus, one assay may not fit all needs, and clinicians should be aware of performance characteristics of requested assays, especially in children and hypogonadal men. Other steroids in the circulation that are of similar structure and are present at high concentrations can lead to difficulties with assay interpretation due to the interference with T measurements and inaccurate test results. Age, ethnicity and gender-adjusted normal T concentration ranges using a standardized assay are lacking. Furthermore, there is no commonly agreed upon and universally used T-calibration laboratory standard in the United States.5 The US National Institute of Standards and Technology is offering a serum-based reference material (SRM 971) for calibration and calibration verification. The CDC is offering serum-based materials for calibration through its Hormone Standardization program, and the Australian National Metrology Institute is offering pure T as primary calibrator; in addition, state specific quality assurance programs exist in the United States.

Binding of T to SHBG, albumin and CBG has implications for accurate measurement of T concentration. Early T assays required T to be extracted or displaced from SHBG and albumin by dissolution into organic solvents, separated by column or thin layer chromatography and then measured by radioimmunoassay (RIA). This method offered several advantages, including the separation of interfering proteins and cross-reacting steroids to increase specificity, and capability to use large serum aliquots to increase sensitivity. With increased demand for economical and rapid alternatives, initial chromatography to separate different steroids was omitted and antibodies with better sensitivity and specificity allowed for the development of modern RIA, which continues to be used in many research and some clinical laboratories worldwide for the last 30 years.

Unfortunately, RIA is time intensive, expensive and creates environmental challenges due to the need for disposal of high volumes of radioactive materials. Over the last decade, the laboratory investigation of TT measurement has evolved from RIA, through automated enzyme-linked immunoassay testing available in most laboratories, to the new generation of liquid chromatography/mass spectrometry (LC-MS/MS) methods in reference laboratories and some hospitals. Correlation between these various methodologies can be poor, and national standardization is underway in this area where the goal is increased accuracy leading to “portability” and comparability of results obtained from the same patient.33, 34 From the technological aspects, improvement in T measurement accuracy, defined as closeness of agreement between a measured quantity value and a true quantity value of an analyte, is a critical component of the assay’s performance to establish uniform normal ranges. From a clinical aspect, the same result should be obtained if blood was drawn from the same patient, at the same time and sent to different laboratories, a scenario that is not common at present. Establishing national external quality control programs, such as those in Europe or offered through CDC in the United States, may improve the consistency of assay results across laboratories and help in clinical decision making. As even the most currently sensitive methods based on MS are prone to technical challenges, a continued laboratory quality control process has to be implemented.35 Otherwise, performance characteristics of preferred reference methods may drift down and match the level of unreliability of currently used assays.35                      

Radioimmunoassay (RIA)

RIAs are based on competitive binding of T to a T-specific antibody after sample pretreatment to displace T from bound fractions. The patient’s serum is mixed with a set amount of radioactively-labeled T tracer and a fixed amount of antibody against T. The amount of tracer displaced by the patient’s T is evaluated by measuring the radioactivity of the sample, and the patient’s T concentration is calculated (Figure 1).

T RIA requires a specific antibody with minimal cross-reactivity. T is a suboptimal antigen, structurally similar to other steroids, so the development of an antibody of high specificity for T is difficult.  For commercially available kits, different manufacturers use different sources and types of antibodies (monoclonal v. polyclonal), which may in turn have different binding affinities to T and varied cross-reactivities. This contributes to the variable results seen with different commercial kits.

Immunoassays (IAs)

Like RIAs, enzyme immunoassays (EIAs) are also based on the principle of competitive binding of T in serum and a non-radioactive tracer to a fixed amount of anti-T antibody. The tracer is T-bound to enzymes like horseradish peroxidase or acetylcholinesterase, which act on their respective substrates resulting in color change or emission of luminescence.  The EIAs for T are sometimes called enzyme linked immunosorbent assays (ELISAs); however, in both assays the tracer is T-conjugated to assay a specific enzyme or marker. IAs, similarly to RIAs, depend on high-quality antibodies, but an advantage of IAs is the elimination of problems with radioactive waste and ease of automatization.  EIAs and their modifications, like electrochemiluminescence, are the most commonly used methods to measure T in non-reference hospital and commercial laboratories. Commercial IA platforms have adequate accuracy for eugonal men but not TT levels less than 300 ng/dL (10.4 nm/L) as compared to LC-MS.36

Liquid Chromatography-Mass Spectrometry (LC-MS)

A direct method for qualitatively and quantitatively measuring T includes assessment of the differences in mass-to-charge ratio (m/z) using MS. T is first eluted from a biological sample matrix by gas chromatography (GC) or liquid chromatography (LC) and then captured, ionized, accelerated, deflected and detected by single or tandem MS. Addition of another MS in tandem (MS/MS) is used to quantify low levels of T in a complex matrix. LC-MS/MS combines automation with high precision, accuracy, specificity and wide linearity to achieve low column volume (CV) in normoganadal men, but with concentrations below 300 ng/dL (10.4 nmol/L), CV increases to 14 % even in highly experienced reference laboratories.37 Thientpont et al. showed that CV of measurements of the same standard reference sample varied from as low as 1% to as high as 8% in four national reference laboratories in the United States and Europe.35 Vesper et al. reported measured values between 198 ng/dL to 364 ng/dL (6.86-12.62 nmol/L) when a standard sample of 299 ng/dL (10.37 nmol/L) was measured by eight reference laboratories in the United States.37  The difference of -33% and +21% from standard exceeds intra-individual T variability of 9.3% and illustrates that even with LC-MS/MS men can be over or underdiagnosed.37 LC-MS/MS requires attention to calibration and between runs quality control. Current MS assays are developed, optimized and validated in-house using different procedures, instrumentation, reagents and calibrators. This can lead to differences in individual assay performances and limitations when comparing results.37 LC- MS/MS requires close attention to quality procedures, national standardization of procedures and calibrators and traceability to nationally recognized reference standard to sustain high level of accuracy and reproducibility.38

Laboratory Measurement Of Testosterone Fractions

The simplistic “free hormone hypothesis” assumes that only FT is available to the end-organs and biologically active at the tissue level,39 hence assays and formulas for various T fractions, including FT,  bioavailable (free and non-specifically bound) and androgen index, have also been developed to help with diagnosis.40-42

Some laboratories have directly measured bioavailable T by selective ammonium sulfate precipitation of SHBG bound T.43 Others measure FT by equilibrium dialysis or ultracentrifugation and advocate for this method as the most accurate assessment of physiologically active circulating hormone fraction.41 As direct methods to measure FT are technically difficult, calculation derived FT (cFT) and BT are sufficient in clinical practice as cFT levels correlate highly with FT measured using dialysis.44 Androgen index and analog RIA for FT (aFT), although recommended by Moreno et al., are considered poor indicators of androgenization by most authors.45-47 T treatment will affect cFT as shown by Ly et al.48

Free and bioavailable T are helpful in both screening and establishment of diagnosis of hypogonadism in men with equivocal TT results, but as they rely on accuracy and precision of T and SHBG, statistically they multiply error in measurement of each of assay used in calculation.

Accuracy, Precision, And Quality Controls Of Testosterone Assays

An accurate diagnosis of androgen deficiency requires availability of hormone assays with high levels of specificity, accuracy, precision and broad linearity, performed in clinical laboratories following internal and external quality procedures. Internal quality control implies that the assay has a high level of accuracy, precision and reproducibility based on repetitive assays of the same sample or set of samples within a clinical laboratory. External quality control of assay refers to performance of the assay when testing reference samples obtained from national programs like the College of American Pathologists (CAP) or the CDC Hormone Standardization Program. Calibration of an assay offers nationally available samples with known concentrations and isotope compositions—a critical step in initial optimization of an assay. The precision of an assay is independent of accuracy as it measures closeness in agreement between independent results of measurements obtained under stipulated conditions (each run in duplicate, with two runs per day over 20 days).49 Reproducibility refers to the closeness of agreement between results of successive measurements obtained under changed conditions (time, operators, calibrators, reagents and laboratory). Accuracy measures closeness to true value, and specificity addresses how well an assay detects only a specific substance (T) and does not detect closely related substances. Sensitivity represents the smallest amount of substance in a sample that can be accurately measured by an assay.50 Sensitivity is also called low limit of detection and is related to linearity of the assay–ability to predict unknown concentration from a set of standards.51

The above mentioned parameters of assays are defined by regulatory agencies and professional organizations in more or less general terms. A number of international quality standards (International Organization for Standardization (ISO) and Clinical Laboratory Standards Institute (CLSI)), accreditation agency guidelines (CAP, Joint Commission, U.K. Clinical Pathology Accreditation (CPA)), and regional laws (Clinical Laboratory Improvement Amendments of 1988 (CLIA'88)) exist describing the requirements for method verification and validation and other than as prescribed by applicable laws and regulations at state and federal levels. However, laboratory directors are not obligated to enroll in specific quality control programs or use calibrator that may be considered optimal by professional societies. This regulatory complexity brings additional levels of challenges to improve quality testing in T. T assays, like other reproductive hormone assays, were established in research laboratories that maintained their own internal quality controls. The increasing use of T assays in routine clinical practice has increased the demand for the T assays and created a shift to T assays performed in large-throughput laboratories that employ commercial, random-access, automated platform assays. 

The methodological changes required to make the transition to automation and high-throughput can be associated with a loss in accuracy and linearity for T assays regardless of methodology used. Widely differing reference ranges reported, both amongst the various automated assays and different laboratories using same methods, are as much the result of the performance of the assay as the selection of the index population. Salameh et al. reported a normal T range using LC-MS/MS in a population of 264 male normal volunteers aged 8-90 years enrolled by Quest Diagnostics Institute.44 In this study, subjects’ health was defined as “apparently healthy, ambulatory, non-medicated” without giving any specifics as to how samples were derived and criteria for enrollment. This study reported the normal T range to be between 250-1100 ng/dL for men 18-69 year old and 90-890 ng/dl for men 70 years old and above. Inter-assay CV was 11% with an accuracy of 102 % at 254 ng/dl. TT normal range for men age 18-39 years was 250-1200 ng/dL in this study. This is in striking contrast with data published by Bhasin et al., who also using LC-MS/MS reported a 95% confidence interval for healthy men between 18-40 years to be 405 – 1,124 ng/dL with inter-assay CV of 7.7% at 241 ng/dL.52 Assuming average CV of 10% using LC-MS/MS values of TT between 270 ng/dL and 330 ng/dL are consistent with hypogonadism using 300 ng/dL TT level cut-off point underscoring our statement that even with the best assays currently available, diagnosis has to be based on clinical picture and not T level alone. Sikaris et al. enrolled 124 fertile, healthy males (21-35) with normal semen analysis. Serum samples from young, fertile men were distributed among nationally recognized laboratories using immunoassays and compared to LC-MS/MS.  Using LC-MS/MS, mean TT was 18.2 nmol/L with Q1-Q3 (25%-75% confidence interval) between 14.6 nmol/L to 21.5 nmol/L. Normal range limits differed by 6% to 37% when the same serum samples were measured using immunoassays and compared to results obtained with LC-MS/MS. In this particular paper, authors showed that mathematical transformation of data and parametric v. non-parametric tests used to calculate normal ranges lead to different cut-off points.53 The three studies discussed above illustrate that both sample selection and assay performance lead to variance in reported normal ranges and underscores the need for standardization of selection of index population and assays used. 

There is no uniform standard of reporting the performance characteristics of assays used in studies, thus it is difficult to compare results and determine how much of variability between different reported normal ranges originate from the assay itself, especially in samples with low concentration of T. For example, Wang et al. compared serum T measurements from eugonadal and hypogonadal adult men with LC-MS versus manual RIAs and four commonly used commercially available immunoassay platforms.34 Using LC-MS/MS as the preferred method, they found that while some of the manual and automated assays could be used to assess T in eugonadal men, the majority were unacceptable for measurement of low T levels (i.e. in hypogonadal men), due to lack of precision and accuracy. Similar findings have been reported by Taieb et al., who found that immunoassay results varied as much as five-fold at TT concentrations below 230 ng/dL, and immunoassays generally overestimated T concentrations in subjects with T values in the low range.33 Validation studies of most T assays are performed using a standard sample with a T concentration of 400 ng/dL. The sensitivity and specificity of most assays is not measured at low T levels, the region where the accuracy and precision of the assay is worse regardless of platform used. At low T levels, as seen in women, children and early male puberty, serum T levels are comparable to those seen in castrated men. The low reliability and poor sensitivity of some of commercial T assays in these settings has led the authors to state that some commercially-available T assays were nearly useless for women and children, and in some cases, these assays were less accurate than guessing.54

Clinical laboratories employing automated immunoassays should validate the reference ranges, based on normal healthy men of different ages, rather than using manufacturer-supplied ranges.34 Reference ranges for commercial T assays are often based on small, convenient population samples not controlled for medical comorbidities or other factors that may affect sex hormone levels.55 Not surprisingly, therefore, reference ranges vary significantly between different commercial assays. Historically, the range of T in healthy young men using assays that utilize extraction and chromatography has approximated 275-1,000 ng/dL. In contrast, some commercial laboratories have reported the lower limit of the normal range to be as low as 84 ng/dL and the upper limit of the normal range as high as 1,727 ng/dL.55 An acceptable reference range for T assays should instead be based on a population of healthy men with verified normal sexual and reproductive function. A recent study by Bhasin et al. showed that enrolling healthy men is critical to establishment of normal ranges, but neither Bhasin et al. nor Sikaris et al. have evaluated both sexual and reproductive function using accepted and validated instruments.

To improve precision and accuracy, laboratories should undertake external quality control and proficiency testing programs through CAP, the CDC Hormone Standardization Program and others as dictated by federal, state and local regulations. Analysis of recent CAP proficiency survey data revealed that most currently available T assays missed these analytical performance goals of  imprecision of 5.3% and total error of 16.7%  by wide margins.38 What is interesting is that 2/4 LC-MS/MS assays failed to meet established quality criteria underscoring that it is not the technology but day-to-day good laboratory practices that are the critical factors for the future improvements in T testing.38 This magnitude of variability is of little value in clinical medicine. 

The laboratory diagnosis of T deficiency may, therefore, be confounded by limitations of both internal and external validation. An understanding of the assays employed by one’s specific laboratory, as well as an appreciation for population-specific reference ranges, is key for accurate clinical diagnosis.

Future Research And Areas Of Improvement

Reproducible T assays that reflect accurate serum concentrations and referenced to well-defined healthy population are important in order to correctly diagnose hypogonadism. Over the past 30 years, T assays have progressively become more economical, rapid and automated. However, scientific data suggests that most T measurements in typical clinical laboratories may be ≥30% different from the “true” serum T concentrations measured using the preferred reference method.  This leads to diagnostic and management dilemmas as insurance carriers will often require TT levels below 300 ng/dL to pay for T replacement therapy.

The poor reliability of T measurement is secondary to a combination of factors, including technical limitations of currently available assays, intra-individual variation and the wide range of T levels in human samples.

The AUA is a major stakeholder in efforts to create standardization of T assays. Widespread enrollment of clinical laboratories in accuracy-based programs, such as the CDC HoSt program, is a critical element to improve the quality. Use of standards and calibrators traceable to preferred reference methods is critical for “portability” of results in the same individual between different clinical laboratories.  The AUA is actively involved in studies aiming to define normal ranges using standardized tests and clinical signs and symptoms. Normal ranges need to be established in normal healthy patients of varying ages with normal sexual and reproductive function. Calibration of the methodology and population-based reference ranges for FT is also needed. Increased accuracy in the measurement of T and SHBG will lead to improvement in calculations of free- and bioavailable T.

The AUA and members of the panel suggest that the scientific community and manufacturers of assays should follow similar standards of reporting. Specifically, the studies should be performed using well defined, described and representative populations, and accepted and validated instruments to assess sexual function and fertility should be used. Uniform reporting of precision, accuracy and reproducibility across hypogonadal and normogonadal men (i.e., T levels <200 ng/dL, 200-299 ng/dL, 300-399 ng/dL, > 400 ng/dL) should be implemented.  Traceability of standards and calibrators used to assess accuracy and to adjust assays should be disclosed.  Description of the mathematical transformation of data and reporting using both parametric and non-parametric tests should be included in manuscripts and manufactures’ documentation. Such approaches will lead to better increased ability to compare results between different platforms and studies.

At this point, no specific assay can be recommended as superior, but each assay should be compared to minimum standards of accuracy based on biological variation. The CDC HoSt program reports that out of five assays that passed CDC standard for T testing, four are based on LC-MS/MS, and one is an immunoassay.38 This data support our decision to not assign superiority to a specific platform but to emphasize that each platform should meet similar standards of accuracy, precision and bias.38

Position statement: Based on the extensive review of published data and input from professional organizations, the members of this panel believe that, for now, diagnosis of hypogonadism should be based as much on the presence of signs and symptoms as on serum T measurement.  Based on overall poor quality of T testing in most clinical laboratories and age bias of published reference ranges, no patient should be denied coverage for treatment based solely on payer defined cut-off points if need for such treatment is established by a health professional. The AUA works closely with regulatory and professional agencies to improve assay performance and normal range, and as literature accumulates, this position will be reevaluated.

In summary, it is in our and our patients’ best interest to favor the advancement of the technological and clinical aspects of the biochemical diagnosis of hypogonadism to provide reliable, cost-effective and portable tools to aid clinicians in the diagnosis and treatment of this common condition.56  We encourage urologists and andrologists to discuss methodology and source of reference values with laboratory directors at their institutions to better understand limitations and advantages of local assays and to improve patient care.

References

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Table 1: Factors Affecting SHBG Levels 

Factors that increase SHBG

Factors that decrease SHBG

Aging
Hyperthyroidism
Estrogens
HIV disease
Anticonvulsants
Cirrhosis
Hepatitis

Obesity
Diabetes mellitus
Hypothyroidism
Glucocorticoids
Androgenic steroids
Nephrotic syndrome
Acromegaly

 Table 2: Assays for the Measurement of Total Testosterone, Free Testosterone, and Bioavailable Testosterone 

Total Testosterone

Radioimmunoassay (RIA)

Enzyme-linked Immunoassay (EIA)

Liquid Chromatography-Mass Spectroscopy (LC-MS)

Free Testosterone

Equilibrium Dialysis

Ultracentrifugation

Radioimmunoassay (RIA)

Calculated Free testosterone

Bioavailable Testosterone

Ammonium Sulfate Precipitation of SHBG

Calculated Bioavailable T

Figure 1

Figure 1

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