Estradiol Serum Levels are Crucial to Understand Physiological/Clinical Setting in Both Sexes: Limits of Measurement of Low Estradiol and Evaluation of a Sensitive Immunoassay

Background: Measure of serum 17β-Estradiol is essen��� to understand physiology, development and health of repr����e processes in both sexes. Commercially immunoassays are not enough ����e and accurate to assess low concentra��� of E2. Purpose of this study was to compare a new ����e immunoassay for estradiol measurement with respect to method current in our laboratory and to evaluate the performance of the new method. Methods: Four pools of pa��t sera with E2 concentra�� close to clinical decision values were prepared. To test the repeatability of new method the 5x5 protocol was used and CVs were calculated. To evaluate the performance of new method, 50 samples with E2 concentra���covering the whole measurable interval were selected and assayed. Linearity ����� LoB (Limit of Blank) and LoD (Limit of Det����ere determined. Results: The new assay showed good total repeatability demonstrated by low CV values, and good linear rela��ship with respect to current method (R=0.9926) as demonstrated by linear regression. Non-parametric regression showed for the new method a slight constant and pr������ systema��error that, however, resulted not ���� ant from ��erence plot analysis, with a general tendency to overes��te results for the current method. Performances of the new method resulted acceptable within the maximum admissible error derived from the literature, and a good linearity over a wide range of ����� was showed. LoD value con��������� a ������ ement of low estradiol. Conclusion: In conclusion, ����e assay is suitable to replace the method used in our laboratory with ���� ant improvement in the measurement of low serum estradiol levels.


Introduction
17β-Estradiol (E2) is the predominant steroid hormone belonging to estrogens according to the receptor type to which they bind. It is the primary female sex hormone produced mainly by ovary, but it is also secreted by the adrenal gland and placenta during pregnancy, and controls the development and maintenance of female sexual characteris��� E2 is also synthesized at low levels in males because some peripheral target ���� express the enzyme aromatase and this elicits the conversion of circula��

E2 Measurement by Access Estradiol and Access Sensitive Estradiol assays
Estradiol measurements were performed by using Access Estradiol assay and Access ���� e Estradiol assay on Beckman Coulter UniCel DxI 600 automated pla� orms (Beckman Coulter Diagnos��� Brea, CA, USA) according to the manufacturer's ins�����

Repeatability of Access Sensitive Estradiol
To test the repeatability of the new method, four pools of pa�� t sera with E2 concentra��� close to clinical decision values were prepared. Level 1 at E2 concentra�� about 20 pg/ mL, Level 2 at E2 concentra�� about 300-400 pg/mL, Level 3 at E2 concentra�� about 1000-2000 pg/mL, and Level 4 at E2 concentra�� about 3500-4500 pg/mL. For each level once a day for 5 days, 5 independent replicates of the same sample were analyzed by using the Access ���� e Estradiol assay (5x5 protocol) on one automated pla� orm. In each session the analy� al quality was assessed by using control charts, and results, expressed as pg/mL, were sta�V � ally analyzed as described below.

Performance of Access Sensitive Estradiol
To evaluate the performance of the new method, 65 serum samples previously measured with Access Estradiol assay, and having E2 concentra��� covering the whole measurable interval and the range of values clinically observable, were selected. Samples in duplicate were assayed over a period of 9 days by using the Access ���� e Estradiol assay and the obtained results were subjected to graphical and sta�V � al analysis as described below.

Statistical and graphical analysis
Sta�V � al analysis of results was carried out using the MedCalc sta�V � al �� are for biomedical research (www.medcalc.org). For the repeatability test the daily mean, standard devia�� and variance were calculated. General mean, variance within days, variance between days and total variance within the laboratory were also calculated in order to obtain total repeatability expressed as coe��� t of varia����� testosterone to E2 and androstenedione to estrone. In males, E2 plays a ��� al role in sexual ���� being that it is essen�� for modula�� spermatogenesis, libido and er��� ���on. Therefore, the accurate measure of serum estradiol is essen�� to understand physiology, development and health of repr��� ve processes in both sexes as well as the cause of diseases related to estrogens.
Beckman Coulter recently put on the market the new Access ���� e Estradiol assay � ering improved measurement of low levels of E2, such as those typically found in men, pediatric popula��� and post-menopausal women. The aims of the present study are (i) The comparison between the new method Beckman Coulter Access ���� e Estradiol assay and the current method Beckman Coulter Access Estradiol assay, (ii) The evalua�� of performance of the new method in terms of repeatability and acceptability. Acceptability of method is evaluated on the basis of the maximum admissible analy� al total error described in the literature (www.westgard.com).

Analytical sensitivity: Limit of Blank (LoB) and Limit of Detection (LoD)
To detect analy� al sensibility of the new method, LoB and LoD were determined. LoB is the highest apparent analyte concentra�� expected to be found when replicates of a blank sample containing no analyte are tested: LoB=mean blank +1.645 (SD blank ). LoD is the lowest analyte concentra�� likely to be reliably dis�� ished from the LoB and at which det��� is feasible. LoD is determined by ����� both the measured LoB and test replicates of a sample known to contain a low concentra�� of analyte: LoD=LoB+1.645 (SD low concentra����� ) [17,18].

Ethics
Having performed the research on pre-exis�� serum samples, anonymized and deiden��� prior to start the study, referred for r��� E2 determina��� no ins����� review board approval was necessary. Authors had not access to any private health informa�� from the pa�� ts involved. However, informed consent was obtained by all subjects enrolled in the study. The research has been carried out in accordance with The Code of Ethics of the World Medical Associa�� (Declara�� of Helsinki) for experiments involving humans.

Repeatability of Access Sensitive Estradiol
Since the new method was already validated by the manufacturer following Approved Guideline from Clinical and Laboratory Standard Ins��W e [19][20][21][22][23][24], a rapid protocol was carried out to verify what was declared. Following sugges��� in recent literature we adopted 5x5 protocol in order to obtain a more realis��� ������ epeatability [25].
The 5×5 protocol indicated a very good total repeatability as shown by the low values of coe��� t of varia�� ranging from 1.5% to 7.3% for the �� erent levels of E2 concentra�� ( Table  1). The highest CV value, whilst s�� within 10% of variability, was observed, as expected, at the lower E2 concentra��� vels.

Performance of Access Sensitive Estradiol
From linear regression analysis the compared methods showed a good linear rela���� with a Pearson correla�� coe��� t R of 0.9926 ( Figure 1). However, simple linear regression assumes that the current method is free of error (reference method) and that the error of the new method is normally distributed and constant at all studied concentra��� but these assump��� are rarely met in pr���� For this reason alterna� e regression models are recommended, such as the non-parametric method [13,14]. In fact, non-parametric Passing-Bablok regression ( Figure  2) highlighted a slight constant and pr����� systema�� error for the new method (value 0 not included in CI 95% for intercept and value 1 not included in CI 95% for slope). Cusum test for linearity, only tes�� the applicability of the Passing-Bablok method, indicated no ���� ant devia�� from linearity (p=0.83), and the residual plot represen�� the dis����� of �� erences around the �� ed regression line, showed that residuals are randomly distributed above and below the regression line, with a greater dispersion at E2 concentra��� higher than about 300 pg/mL measured with the method current (Supplemental Figure  1A-B).
Another useful graphical analysis is that of Bland-Altman, especially when the measuring interval is large as in the case of E2 [15]. The diagram allows the highligh�� of systema�� �� erences between the two methods and de��� them in a quan�W a� e way. The Bland-Altman graphical analysis in which the zero value is included in CI 95% con��� a slight systema�� but not ���� ant error (Figure 3). Moreover we also observed for the current method a general tendency to overes��te results with respect to the new method that is more evident at high concentra������� te (Figure 3).
Acceptability of the new method, based on the maximum acceptable error obtained from literature, was determined by the method evalua�� decision chart, MEDx chart [16] for judging method performance. The imprecision and the systema�� devia�� or bias are ��W ed r���� ely on the abscissas and on the ordinates, represen�� as a point the performance of the new method. To construct the MEDx chart, the value corresponding to the maximum acceptable error for E2 (26.86%), obtained from Desirable Biological Varia�� Database ���� a��� at www. westgard.com, was reported on the y axis, while on the x axis were reported � e values (maximum error divided for 2, 3, 4, 5 and 6). This corresponds to acceptability or quality criteria, allowing the division of the graph into six areas (from right to le� in the graph: unacceptable, poor, marginal, good, excellent, world-class). The coordinates of the new method's performance point uses the imprecision calculated from the repeatability test for the abscissa, and the bias obtained using the equa�� of the non-parametric regression line applied to a certain level of E2 concentra��� or the ordinate.
The new method for Level 1 of concentra�� showed a performance located in the area of unacceptable quality, whereas for Level 2 performance, this was located in the area of good-excellent quality, for Level 3 in the area of marginal-poor quality and for Level 4 in the area of marginal quality ( Figure 4A-C). Although for the Level 1 sample, the new assay is ����� by MEDx chart as unacceptable ( Figure 4A), a CV of 7.3% (Table 1) was obtained at this concentra�� (20 pg/mL), which represents a very good imprecision. What is more, for the current Access Estradiol assay the manufacturer declared a CV of 21% for concentra�� of 50 pg/mL, and in our lab for concentra�� 17.8 pg/mL of EQA Immunocheck (Qualimedlab s.r.l., www. qualimedlab.it), a CV value of about 50% was obtained, documen�� a very high imprecision for low E2 levels measured with the current method.
In the ���� linearity test, up to 1:128 ����� the obtained concentra�� values corrected for ���� factor were between 89% and 117% of the whole sample. On the other hand, further ����� of the sample provided results greater than 120% or not determinable when compared to the non-diluted sample ( Figure 5). The new method, showing a good linearity over a wide range of ����� also provides � xibility and reliability to measure samples with �� erent concentra��� of E2, in ����� samples with high levels of analyte can be diluted

Figure 3
Bland-Altman plot. The �� erences between observa�� pairs are ��W ed against their mean and the average of the �� erences (bias) and its 95% con���� limit lines are drawn on the same plot. to measure r���� when using direct immunometric assays, including chemiluminescent and enzyma��immunoassays, without a preceding ��� a�� step [26][27][28]. In fact, the increasing demand for steroid hormones dosage led to laboratory assay ���� a�� to allow the use of unextracted serum on highly automated instrumenta�� pla� orms. This introduced bias and lack of ������ mainly at low concentra��� [1] such as those typically found in men, pediatric popula��� and post-menopausal women. As men��� previously, GC-MS/MS and LC-MS/MS represent the two reference methods but these methods are not very suitable for r��������� In this study we compare a new ���� e immunoassay for E2 measurement with respect to the method current in our laboratory and evaluate the performance of the new method in terms of repeatability and acceptability. The 5×5 protocol indicated a good total repeatability as demonstrated by low values of CV for the �� erent levels of concentra��� From linear regression analysis the compared methods showed a good linear rela����� however, non-parametric Passing-Bablok regression highlighted a slight constant and pr����� nega� e bias for the new method. The Bland-Altman graphical analysis con��� this slight systema�� but not ���� ant bias with a general tendency to overes��te results using the current method. In ���� to good precision at the low E2 concentra��� performances of the new method resulted acceptable within the maximum admissible error derived from the literature as demonstrated by the method decision chart (performance located in the areas of acceptability).
���� linearity tests showed good linearity over a wide range of ��� ns and, ���X , the LoD value demonstrated an improvement for measurement of low estradiol concentra�� s when compared to the current method.
There is no doubt that the Access ���� e Estradiol assay provides improved precision and accuracy at the low estradiol levels. For low E2 levels, literature reports elevated CV values as in the paper by Hendelsman et al. [1] where a CV of 23.7% and a several ��� to ensure that its values fall within the standard curve range without a� ������ acy and precision.

Analytical sensitivity of Access Sensitive Estradiol
The LoB value, determined by using four blank samples as previously explained, was 8.63 pg/mL, with a range of observa�� from 0.00 to 13.08 pg/mL ( Figure 6A). From the bar graph of frequency dis����� of E2 levels expressed as Rela� e Luminescence Units (RLU) can be visually evaluated that data are symmetrically distributed (normal or gaussian dis���� ), as demonstrated by normal dis����� curve superimposed over the histogram (with mean and standard devia�� showed) (Supplemental Figure 2).
The manufacturer reported for LoB a ��� al value ≤ 10 pg/mL E2 with a range of observed results between 5.0 and 7.5 pg/ mL. For LoD the declared ��� al value was ≤ 15 pg/mL E2 with a range of 9.4-12.4 pg/mL. Therefore, results obtained in analy� al ������ determina�� in terms of LoB and LoD were in perfect agreement with the manufacturer's declara�� on the product data sheet.
In ����� , the LoD value of 13.99 pg/mL calculated from our data set not only con��� what was declared by the manufacturer, but also allowed to state that Access ���� e Estradiol assay � ers improved measurement of low levels of analyte.

Discussion
Although the determina�� of estradiol levels in the blood is crucial for understanding the physiological and clinical se��� in both sexes, low concentra��� of estradiol (<30 pg/mL) are ��� lt   In the human body, estradiol is metabolized to more than 100 conjugated and unconjugated metabolites and many of them may cross-react with an���� used in immunoassays, producing an overes���� of results [29]. An extr��� step in direct assays may remove poten��� interfering substances, in ����� water soluble cross-r���� steroids, rendering E2 results much more similar to that obtained with indirect assays including the extr��� phase [27]. Direct assays have several advantages, in ����� for r��� use and to perform large epidemiological studies; they require less quan�� of sample and are less laborious, having characteris�� that conform to the high automa�� of the assay. However, they are less accurate for the non����� binding of interfering molecules or for unclear matrix e� ects. In fact, matrix �� erences between serum samples and pure ��� ons of estradiol used to generate the standard curve in a direct immunoassay may also a� ect validity of results.
In ����� , hemolyzed and lipemic samples may interfere with the binding of an� en to an������ The ������ encountered when measuring E2 concentra�� in serum samples are more or less the same as those encountered when measuring all steroid hormones, and are related to the adapta�� of immunoassay to valid measurement of nonimmunogenic small molecules such as steroids [1]. Steroids should be conjugated to big immunogenic proteins to develop ���� an��T ies, but this allows for unwelcome cross-r����� with structurally related molecules such as precursors, metabolites and conjugates. As previously men���� steroid immunoassays have been used in the original methods employing solvent extr��� of samples, chromatography separa�� and structurally authen��tracers to remove interferences from similar molecules and matrix. The growing demand for E2 measurement, especially to monitor ovarian response to gonadotropin s��� �� in the emergent pr��� of medically assisted procrea��� has led to the marke�� of highly automated immunoassays without extr���� chromatography and authen�� tracers, suitable for r��� purposes but much less ���� and accurate. These assays are ������ aimed at measuring physiological (up to 500 pg/mL) or dangerously high (more than 2000 pg/mL) concentra��� of E2. High ����� y and accuracy are, however, necessary to measure low levels of E2 such as those that occur in men, postmenopausal women, children and aromatase inhibitor treated pa�� ts. To date, mass spectrometry is the reference method for measuring sex hormone levels in male and female [10,30]. Furthermore, a recombinant cell ultr���� e estrogen bioassay which correlates well with GC-MS/MS data was described [31]. Though modern immunoassays for estradiol are reasonably well suited for the diagnosis and management of inf���� (despite imprecision and �� erences between methods), the very low concentra��� crucial in non-repr��� e ���� are s�� ����� to measure [7,8]. With lowend precision (LoD ≤ 15 pg/mL) and state-of-the-art ����� y (LoQ ≤ 19 pg/mL), the new Access ���� e Estradiol assay may help laboratories to deliver more accurate results for pa�� ts seeking answers to repr��� e health ques��� and its use is ������ indicated for the measurement of very low levels of estradiol to assess ����� clinical c����� such as inborn errors of sex-steroid metabolism, disorders of puberty, estrogen de���� in men, ther���� drug monitoring during lowdose female hormone replacement therapy and an�� trogen treatment.
In conclusion, the new method has a very good total repeatability as shown by low CV values and, even in the presence of a minimum propo���� systema�� bias compared to the current method, is acceptable within the maximum admissible error obtained from literature. It also demonstrated a good linearity over a wide range of ����� and is � xible and reliable for analyzing samples with high levels of analyte a� er ���� . Finally, from LoD value it is possible to state that this assay also � ers improved measurement of low levels of serum estradiol.