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Performance of direct estradiol immunoassays with human male serum samples.

Accurate measurement of serum estradiol [([E.sub.2]).sup.5] is crucial to understanding reproductive development, physiology, and health as well as the origins of estrogen-dependent diseases. Whereas the androgens testosterone (T) and dihydrotestosterone exert dominant influence on male reproductive physiology and pathology, aromatization of T to [E.sub.2] has been recognized as an important local regulatory hormonal mechanism for certain tissues in men, notably brain (1) and bone (2). Consequently, the indications for serum [E.sub.2] measurements in men include clinical evaluation of gynecomastia; steroid-secreting testicular, adrenal, or hepatic tumors; or genetic aromatase excess, androgen insensitivity, or Peutz--Jeghers syndrome. In addition, serum [E.sub.2] measurement may be useful in monitoring anti-estrogen or aromatase inhibitor treatment for pubertal delay (with or without short stature), obesity, or male infertility and a threshold of circulating [E.sub.2] for maintenance of bone density in older men. Further clinical indications for serum [E.sub.2] measurement in men may have been overlooked due to the limitations of [E.sub.2] immunoassays for the low concentrations in male blood samples, notably in conditions of possible estrogen deficiency states or apparent estrogen excess, including hormone-sensitive cancers (3).

Thus, measurement of serum [E.sub.2] is as important for understanding numerous developmental and pathophysiological processes in men as it is in women. Yet although modern commercial [E.sub.2] immunoassays, which have been optimized for unextracted human serum ("direct"), may be adequate for measuring the high blood [E.sub.2] concentrations in women between menarche and menopause, and especially during ovarian stimulation, circulating [E.sub.2] concentrations are 10- to 100-fold lower in children, men, and postmenopausal or aromatase inhibitor--treated women and are challenging to measure. Awareness of problems with validity (analytical specificity, accuracy) of direct immunoassays, which was identified >25 years ago (4, 5), grew over the last decade following the recognition of limitations of T immunoassays, particularly at low circulating concentrations (6-8). Direct T immunoassays display assay-specific bias and analytical nonspecificity, most notably at low blood T concentrations in children, women, and androgen--deficient men (6-10). Therefore, aiming to measure the expected (picomolar) serum [E.sub.2] concentrations that render T immunoassays unreliable makes it unsurprising that direct [E.sub.2] immunoassays lack accuracy and validity for measurements of serum [E.sub.2] in not only men but also children (11) as well as postmenopausal (4, 12-14) or aromatase inhibitor-treated (15) women who have comparably low blood [E.sub.2] concentrations (16). Comparisons of direct [E.sub.2] immunoassays with mass spectrometry--based reference methods in men have previously been confined to single commercial [E.sub.2] immunoassays (17-19), whereas other studies focused on in-house [E.sub.2] immunoassays (15,20) or studied only women (4,12,13). Here we used a reference panel of sera to calibrate and compare the performance of widely used commercial direct [E.sub.2] immunoassays relative to the reference LC-MS method using a reference panel of healthy older men (8).

Materials and Methods


A reference panel of sera was formed from a nested cohort of participants in the Healthy Man Study (21) who were >40 years old and reported excellent or very good health with no health symptoms including sexual dysfunction or gynecomastia. Previously unthawed aliquots of serum (n = 101) collected during the study were distributed frozen to 5 laboratories, all holding accreditation to International Organization for Standardization (ISO) 15189 and participating in the national RCPAQAP (Royal College of Pathologists of Australasia Quality Assurance Programs) quality assurance program. The laboratories, selected to represent different commercial direct [E.sub.2] immunoassays, each ran the samples in duplicate in each assay.


The 5 commercial direct [E.sub.2] immunoassays were (A) Siemens ADVIA Enhanced Estradiol assay run on Centaur analyzer; (B) Siemens IMMULITE 2000 Estradiol assay run on IMMULITE 2000 analyzer; (C) Abbott ARCHITECT System Estradiol assay run on ARCHITECT analyzer; (D) Roche cobas Estradiol II assay run on Roche E170 analyzer; and (E) Beckman Coulter Access Estradiol assay run on DxI800 analyzer (these 5 immunoassays are listed in this article as A-E, respectively). All 5 methods provide reference intervals for [E.sub.2] in males, indicating that measurement of male sera is included in the intended use of the assays, and 4 of the 5 methods claim traceability to GC-MS. The samples were also measured by an LC-MS [E.sub.2] assay (22). To validate the serum [E.sub.2] measurements in the LC-MS assay, 3 certified reference materials (CRMs) for serum [E.sub.2] (CRM 576, CRM 577, and CRM 578) obtained from the European Commission's Institute for Reference Materials and Measurements (23) were run in duplicate on consecutive days in the LC-MS assay, including 1 set run together with the samples in this study. Serum T was measured in the same sample by the same LC-MS method (22) and serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and sex hormone--binding globulin (SHBG) by immunoassays as reported previously (21).


For each immunoassay, the distribution of values was determined by nonparametric methods, distributional deviation from gaussian by Shapiro--Wilk statistic, and bias (relative to MS) by Passing--Bablok regression and illustrated by deviance (modified Bland--Altman) and mountain plots (24) by use of NCSS, MedCalc, and Sigmaplot software. Samples with undetectable serum [E.sub.2] values were deleted from analysis of that immunoassay. We calculated body mass index (BMI) as weight (in kg) divided by the square of height (in meters) and body surface area (BSA) in [m.sup.2] by the Gehan--George formula (25). We calculated CV for an assay as the mean of the CVs for the 2 replicates for each sample without adjustment for serum [E.sub.2] concentration. Correlation was performed by linear least-squares analysis with a Bonferroni adjustment for multiple comparisons. Results are presented as mean and SE if gaussian, or as median and interquartile range (IQR) (25th, 75th centiles) otherwise.


The 101 participants had a median (IQR) age of 54 (48-62) years and BMI 25.9 (23.9-28.6) kg/[m.sup.2]. The group had a mean (SE) height 176 (0.7) cm, weight 81.2 (1.1) kg, and BSA 2.00 (0.01) [m.sup.2].

In terms of the assigned target serum [E.sup.2] concentrations, the CRM samples had a median accuracy of 100% [101% (6%)] at low concentrations (nominal certified concentration 31 pg/mL), 93% [93% (3%)] at medium concentrations (188 pg/mL), and 89% [89% (10%)] at high concentrations (365 pg/mL) without any significant between-day difference (P > 0.8).

The overall analysis of serum [E.sub.2] measurements (repeated-measures ANOVA) showed highly significant difference between assays ([F.sub.5995] = 8.84, P < [10.sup.-6]) and between men ([F.sub.95995] = 9.10, P < [10.sup.-6]) but not for intraassay replication (F1995 = 0.0, P = 1.0). Three immunoassays detected serum [E.sub.2] in all samples, whereas [E.sub.2] was detected in only 53% of samples in assay B and 72% in assay E. Table 1 lists the nonparametric centiles of distribution and pooled within-assay CVs for each immunoassay, and the immunoassays are compared in Table 2. Fig. 1 shows the overall distribution of serum [E.sub.2] concentrations displayed as notched box plots. For each immunoassay, the serum [E.sub.2] measurements had a nongaussian distribution in the natural scale (Table 1), and these were normalized by a logarithmic transformation for each assay except assay B, which had numerous undetectable samples (data not shown).

The bias parameters for each immunoassay relative to LC-MS are displayed as Passing--Bablok regressions (Fig. 2) and regression estimates (Table 1) as well as mountain plots (see Supplemental Fig. 1, which accompanies the online version of this article at and deviance plots (Fig. 3). The bias of each method was upward (positive) ranging from 12% to 53% (at mean) and 11% to 74% (at 25th centile), 6% to 55% (at median), and 11% to 48% (at 75th centile). Residual SD in the Passing--Bablok regressions were similar for 4 immunoassays and LC-MS but were significantly higher for assay B, which reflected its lower analytical sensitivity, lack of linearity, and greater deviation from regression slope of 1.0. The CVs were lower for 4 assays (A, B, C, and D) relative to LC-MS but higher in 1 (E).

To evaluate biological correlates of each assay, serum [E.sub.2] concentrations from the 5 [E.sub.2] immunoassays and LC-MS [E.sub.2] assay were correlated by linear regression with weight, BMI, and reproductive hormone variables (serum LH, FSH, T, SHBG) (Table 3). Serum [E.sub.2] correlated with serum LH and SHBG in the LC-MS assay but not with either gonadotropin in any immunoassays. Assay B results correlated negatively with weight and BMI, but this was not observed with any other assay. None of the assays generated serum [E.sub.2] results that correlated with age, height, or bone density at hip or spine (in either absolute bone density or T scores; data not shown).


In this study, we used a defined reference panel (8) comprising 101 serum samples from healthy older men, a nested cohort of the Healthy Man Study (21), to calibrate the performance of 5 commercial direct [E.sub.2] immunoassays as has been done previously for serum T immunoassays (8). On the basis of measurements of this reference panel, each immunoassay deviated significantly from the reference LC-MS method throughout the working range, with the magnitude of positive (upward) bias varying between assays from 6% to 74%. As the variability seen here is based on single versions of each assay, a significantly greater variability might be expected when interlaboratory variability is included (26). It is possible that the immunoassays are able to separate concentrations within reference intervals from clearly increased serum [E.sub.2] in men; however, method-dependent reference intervals would be required. This study did not address that question.

Our findings extend previous reports comparing direct [E.sub.2] immunoassays with MS-based methods in men but where only a single commercial immunoassay (17-19) or in-house immunoassays (15, 20) were examined. Our results are also consistent with the findings in postmenopausal or aromatase inhibitor--treated women with comparably low circulating [E.sub.2] concentrations (4,12,13,27). The strong, positive correlation of the LC-MS assay with serum T and SHBG was not matched by any immunoassay. The failure of serum [E.sub.2] concentration to correlate with BMI, proposed as a biological validity criterion for serum [E.sub.2] measurements in women (28), may reflect the biological differences in the range of circulating [E.sub.2] concentrations between sexes. The striking negative correlation of weight or BMI with serum [E.sub.2] concentration by assay B, but no other assay, may reflect the unknown cross-reacting steroids, responsible for the overestimation of serum [E.sub.2] measurements and poor correlation with LCMS, which may correlate inversely with weight or obesity. Because this assay produced fewer detectable values covering a narrower range of results, the weaker correlation with LC-MS is an expected finding.

The reasons for these discrepancies are related to the origins of steroid immunoassays, which were developed in 1969 (29) but delayed for a decade after the invention of peptide immunoassay because additional steps were required to adapt immunoassay methodology to valid measurement of nonimmunogenic small molecules such as steroids. Developing steroid-specific antibodies required conjugating steroids as haptens to larger immunogenic carrier proteins via small multivalent reactive bridge compounds. However, this made steroid antibodies that were epitope "blind" to the conjugation site, allowing for undesirable cross-reactivity with structurally related steroids (e.g., structurally related steroid precursors, metabolites, and conjugates produced by phase II metabolism). The original in-house steroid immunoassays developed in the 1970s used solvent extraction, chromatography, and structurally authentic ([H.sup.3], [C.sup.14]) tracers, a triplet of validity criteria for steroid immunoassay that removed structurally related steroids as well as nonspecific matrix interference in the immunoassay reactions. In the ensuing decades, [E.sub.2] immunoassays were commercialized primarily to monitor ovarian responses to gonadotropin stimulation, where excessive serum [E.sub.2] response was a risk indicator for dangerous overdosage effects such as high-order multiple pregnancies and/or life-threatening ovarian hyperstimulation syndrome. However, making a quantal distinction between dangerously high [E.sub.2] concentrations (>2000 pmol/L) and typical premenopausal concentrations (200-800 pmol/L) did not require quantitative accuracy at physiological [E.sub.2] concentrations. Subsequently, ultrasound monitoring of follicular growth has reduced the importance of this application of serum [E.sub.2] immunoassays. The subsequent growing demand for steroid immunoassays in clinical practice and research resulted in assay simplification to adapt steroids into semi-automated multiplex platforms and 1-plate/tube kit formats, preferred by routine pathology and research labs, respectively. This simplification eliminated preassay purification steps (extraction, chromatography) as well as [beta]-scintillation counting, with the latter forcing the replacement ofstructurally authentic steroid tracers with bulky conjugated steroids, which allowed for more convenient nonradioactive assay readouts. However, eliminating the triplet of validity criteria (preassay extraction and chromatography, authentic tracers) sacrificed the specificity of [E.sub.2] immunoassays and revealed the vulnerabilities of direct steroid immunoassay to artifacts from steroid cross-reactivity and matrix interference. Although the original validated steroid immunoassays are now confined to a few long-established laboratories, there is evidence that some but not all the limitations of direct [E.sub.2] immunoassays can be overcome by preassay solvent extraction (12, 28). However, since our study did not include any classical (indirect) estradiol immunoassays, it is not clear whether meeting the original triplet validity criteria is sufficient to overcome all limitations of direct [E.sub.2] immunoassays, and it remains possible that suboptimal antibody specificity could also contribute to the nonspecificity of steroid immunoassays.

The analytical sensitivity of 3 direct [E.sub.2] immunoassays was adequate to detect serum [E.sub.2] in all serum samples from healthy older men. This reflects generational improvement over less analytically sensitive previous [E.sub.2] immunoassays (13). Replicate imprecision was better with most platform [E.sub.2] immunoassays owing to the use of automated rather than manual pipetting; however, the most accurate direct [E.sub.2] immunoassay had replicate imprecision inferior to that of LC-MS, a long-recognized limitation of [E.sub.2] immunoassays (26). This presumably reflects the worse imprecision of immunoassays at the low end of their working range. On the other hand, the CV for the LC-MS assay in this study based on 2 replicates was higher than when a larger number of QC samples was pooled (22), although this does not exclude effects resulting from differences in internal standard: analyte ratios (30) or other deviations from ideal internal standard performance (31).

Although assay-dependent bias and analytical nonspecificity of direct [E.sub.2] immunoassays is established (11, 12, 14, 15, 27, 32-35), direct [E.sub.2] assays are often used in research involving tissue extracts and nonhuman serum samples, where their validity is doubtful (36, 37).

Overall, these findings indicate that direct [E.sub.2] immunoassays are suboptimal for use with human male serum where either absolute measurement is required, such as in clinical practice or research that hinges on interpreting [E.sub.2] measurements in single serum samples, or even in more self-contained research studies where small differences in measurements may be decisive, such as analytical epidemiology (16, 38, 39). These limitations underestimate the problems with direct [E.sub.2] immunoassays over time, since commercial kit or platform formats are subject to changes in proprietary components, notably (but not only) new antibodies, that have to be replaced. Hence direct [E.sub.2] immunoassays exhibit not only assay-dependent bias but are subject to unpredictable variability over time, all of which detract from assay performance and stability. By contrast, the advent of lower-cost, benchtop MS equipment that retains reference concentration specificity but now features analytical sensitivity matching the best steroid immunoassays renders MS-based [E.sub.2] assays more widely accessible. Although MS-based [E.sub.2] assays provide a durable chemical analysis free from method-dependent bias of immunoassays, like all biochemical measurements, MS-based assays maybe subject to different features such as those involving matrix effects, chromatography, and differences in monitored transitions, all of which require rigorous standardization and ongoing quality control (16, 28, 40), an essential but challenging process that remains to be completed.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: R.I. McLachlan, Eli Lilly Advisory Board.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: R.I. McLachlan, Australian NHMRC research support.

Expert Testimony: None declared.

Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: The authors are grateful to Dr. James Doery, Southern Cross Pathology Australia, Victoria; Dr. Chris Farrell, Laverty Pathology, NSW; Dr. Greg Ward, Sullivan and Nicholaides Pathology, Queensland; and Dr. Paul Willams, Sydney South West Pathology, NSW, as well as to Dr. Ken Sikaris, Melbourne Pathology, for encouraging the study.


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David J. Handelsman, [1] * Julie D. Newman, [2] Mark Jimenez, [1] Robert McLachlan, [3] Gideon Sartorius, [1] and Graham R.D. Jones [4]

[1] ANZAC Research Institute, Concord Hospital, University of Sydney, Sydney, NSW, Australia; [2] Southern Cross Pathology Australia, Clayton, VIC, Australia; [3] Prince Henry's Institute of Medical Research, Monash Medical Centre, Clayton, VIC, Australia; [4] SydPath, Department of Chemical Pathology, St Vincent's Hospital, Darlinghurst, NSW, Australia.

[5] Nonstandard abbreviations: [E.sub.2], estradiol; T, testosterone; ISO, International Organization for Standardization; RCPAQAP, Royal College of Pathologists of Australasia Quality Assurance Programs; CRM, certified reference material; LH, luteinizing hormone; FSH, follicle-stimulating hormone; SHBG, sex hormone--binding globulin; BMI, body mass index; BSA, body surface area; IQR, interquartile range.

* Address correspondence to this author at: ANZAC Research Institute, Concord Hospital, NSW 2139, Australia. E-mail

Received July 25, 2013; accepted December 3, 2013.

Previously published online at DOI: 10.1373/clinchem.2013.213363

Table 1. Serum [E.sub.2] distribution centiles
and Passing-Bablok analysis. (a)

                   LC-MS        A (b)       B (c)

Maximum          208          293          363
97.5th centile   147          195          287
75th centile      98          133          113
50th centile      82          118          87
25th centile      62          103          79
2.5th centile     36           76          73
Minimum           34           74          73
Detectable, %    100          100          53
Gaussiang          0.95 (h)     0.86 (h)   0.56 (h)
Mean (SD)         83 (28)     122 (30)     102 (43)
CV, %i            13.6          7.5        5.4
Correlation        1.0          0.71       0.55
  Slope          Reference      0.92       0.82
  Intercept      Reference     45 (k)      17
  Residual SD    Reference     16          28
  Linearity      Reference    [check]      x

                  C (d)      D (e)      E (f)

Maximum          285        344        323
97.5th centile   185        173        191
75th centile     145        120        115
50th centile     127        98         100
25th centile     108        81         83
2.5th centile    72         51         75
Minimum          68         43         75
Detectable, %    100        100        72
Gaussiang        0.92 (h)   0.81 (h)   0.67 (h)
Mean (SD)        127 (30)   104 (37)   106 (35)
CV, %i           10.8       10.9       23.7
Correlation      0.65       0.74       0.64
  Slope          1.02       1.07       0.82
  Intercept      45 (k)     15 (k)     28
  Residual SD    18         17         20
  Linearity      [check]    [check]    [check]

(a) Serum [E.sub.2] concentrations are in pmol/L.
To convert to pg/mL, divide by 3.67.

(b) A, Siemens ADVIA.

(c) B, Siemens IMMULITE 2000.

(d) C, Abbott ARCHITECT.

(e) D, Roche cobas.

(f) E, Beckman Coulter Access.

(g) Shapiro-Wilks W.

(h) Significant (P < 0.05) deviation from gaussian distribution.

(i) Mean coefficient of variation of duplicate measurements.

(j) Correlation with LC-MS.

(k) Significant (P < 0.05) difference from
slope = 1.0 or intercept = 0.0.

Table 2. Passing-Bablok regression and Pearson correlation of serum
[E.sub.2] concentrations measured by 5 immunoassays pair-wise. (a)

           A (b)          B (c)          C (d)

A (b)        1            0.660          0.699
B (c)   0.805, -5.44        1            0.646
C (d)   1.091, -5.75    1.230,13.5         1
D (e)   1.127, -36.0   1.154, -2.01   1.116, -43.6
E (f)   1.145, -43.8   1.442, -32.3   1.045, -41.7

           D (e)       E (f)

A (b)      0.802       0.692
B (c)      0.807       0.780
C (d)      0.728       0.671
D (e)        1         0.801
E (f)   0.927, -4.21   1

(a) Values above the diagonal indicate the Pearson correlation, and
below the diagonal the slope and intercept of the Passing--Bablok

(b) A, Siemens ADVIA.

(c) B, Siemens IMMULITE 2000.

(d) C, Abbott ARCHITECT.

(e) D, Roche cobas.

(f) E, Beckman Coulter Access.

Table 3. Correlation between serum [E.sub.2] concentrations
measured by 5 immunoassays and LC-MS assay and
weight, BMI, and reproductive hormones.

            MS       A (a)     B (b)     C (c)    D (d)   E (e)

Weight   -0.14       -0.17   -0.38 (f)   -0.20    -0.21   -0.21
BMI      -0.18       -0.09   -0.41 (f)   -0.27    -0.18   -0.24
LH        0.08        0.08    0.20        0.13     0.19   -0.07
FSH      -0.06       -0.03    0.03        0.02     0.03   -0.15
T         0.45 (f)    0.26    0.07        0.12     0.25    0.06
SHBG      0.34 (f)    0.17    0.24        0.05     0.26    0.03

(a) A, Siemens ADVIA.

(b) B, Siemens IMMULITE 2000.

(c) C, Abbott ARCHITECT.

(d) D, Roche cobas.

(e) E, Beckman Coulter Access.

(f) P < 0.001 for a nominal P < 0.05 with Bonferroni adjustment
for 36 pair-wise comparisons.
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Title Annotation:Endocrinology and Metabolism
Author:Handelsman, David J.; Newman, Julie D.; Jimenez, Mark; McLachlan, Robert; Sartorius, Gideon; Jones,
Publication:Clinical Chemistry
Article Type:Report
Date:Mar 1, 2014
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