Clinical Chemistry 50:1
160 –165 (2004)
Endocrinology and
Metabolism
Immunoassay of Estradiol: Unanticipated
Suppression by Unconjugated Estriol
Zhimin (Tim) Cao,1* Thomas A. Swift,2 Clint A. West,1 Thomas G. Rosano,2 and
Robert Rej1,3
Background: Accurate measurement of estradiol is important in clinical settings. The quality of laboratory
estimations of estradiol may be assessed through external quality-assurance surveys.
Methods: Estradiol was measured by microparticle enzyme immunoassay (MEIA) and other immunoassays.
Proficiency testing of medical laboratories was conducted using samples prepared from normal male human serum supplemented with exogenous estradiol and
other steroid and nonsteroid hormones, and participant
laboratories measured estradiol by a variety of commonly used immunoassay techniques.
Results: The imprecision (CV) for measurement of estradiol [100 –300 ng/L (367–1102 pmol/L)] was <22% for
most analytical techniques. Greater imprecision, as high
as 40% for the same concentration range, was observed
for the (AxSYM) MEIA method in the proficiency testing event of September 2001. Results from this method
were bimodal in distribution. We found that unconjugated estriol at concentrations >1.5 ␮g/L (>5.2 nmol/L)
interfered with the MEIA method, leading to decreased
recovery of added estradiol by up to 50%. This suppression in estradiol measurement was prevented by dilution of the specimen before measurement. Addition of
unconjugated estriol gave a positive bias in some other
immunoassay methods for estradiol. Poor comparability
among the immunoassay methods for measurement of
estradiol at clinically relevant concentrations [⬃60 ng/L
(220 pmol/L)] was revealed.
Conclusions: A negative interference of unconjugated
estriol with the MEIA method is a source of error for
estradiol measurement. Lack of specificity and lack of
comparability among immunoassay methods for estradiol may have detrimental effects on medical practice.
© 2004 American Association for Clinical Chemistry
The steroid hormone estradiol is the most potent among
the estrogens. Accurate determination of estradiol concentrations in human serum and plasma is important in
many clinical settings (1– 4 ). In most clinical laboratories,
estradiol is measured by immunoassay methods in conjunction with sensitive detection technologies and automated instrumentation. Such measurements are usually
robust, economical, and precise (5 ). Estradiol is classified
as a “type A” analyte by the WHO because it is a
chemically well-defined compound (6 ). Although its measurement should be method-independent, performance
quality for estradiol assay has been questioned for more
than a decade (7–11 ). Difficulties in measurement of
estradiol may include interfering substances, low endogenous concentration in most patient samples, a lack of
analytical specificity, errors in calibration, and lack of
traceability.
In this study, we demonstrate that the presence of
unconjugated estriol interferes with the measurement of
estradiol in the AxSYM microparticle enzyme immunoassay (MEIA) method; we also study the nature of the
interference, determine whether such interference occurs
in other immunoassay methods, and examine the comparability among methods used in clinical practice for
estradiol measurement.
1
Laboratory of Molecular Diagnostics, Wadsworth Center for Laboratories
and Research, New York State Department of Health, Albany, NY.
2
Department of Pathology and Laboratory Medicine, Albany Medical
Center Hospital and College, Albany, NY.
3
Department of Biomedical Sciences, School of Public Health, State
University of New York, Albany, NY.
*Address correspondence to this author at: Laboratory of Molecular
Diagnostics, Wadsworth Center, New York State Department of Health,
Albany, NY 12201-0509. Fax 518-474-9185; e-mail [email protected].
Received June 18, 2003; accepted October 3, 2003.
Previously published online at DOI: 10.1373/clinchem.2003.023325
Materials and Methods
Blood specimens were collected, without additives, from
patients and from a healthy volunteer, and serum was
prepared by leaving the specimens at room temperature
for 30 min, followed by centrifugation at 2000g for 20 min.
Serum was either used immediately or stored for ⬍14
days at or below ⫺40 °C. This study was approved by the
New York State Department of Health Institutional Re-
160
161
Clinical Chemistry 50, No. 1, 2004
view Board. Processed male human serum used for the
preparation of proficiency testing samples was from
Bioresource Technology, Inc. All steroids, from Sigma
Aldrich Co., were dissolved in methanol before addition
to serum. The immunoassay instruments and associated
reagents that were used for measurements in our laboratories were as follows: AxSYM and IMx (Abbott Laboratories), and Elecsys 2010 (Roche Diagnostics Co.). Other
instruments and methods involved in the study included
Architect (Abbott Laboratories); Immuno 1, ACS:180, and
ADVIA Centaur Estradiol-6 assay (Bayer Corp.); Coat-ACount, Immulite, and Immulite 2000 (Diagnostic Products
Corp.); Access (Beckman Coulter Inc.); Vitros ECi (Ortho
Clinical Diagnostics); and VIDAS (bioMérieux, Inc.).
The AxSYM method is a heterogeneous immunoassay
in which estradiol from the specimen binds to rabbit
polyclonal anti-estradiol antibodies that are linked to
microparticles. On removal of unbound materials, estradiol–alkaline phosphatase conjugate is added and binds
to available sites. After washing, 4-methylumbelliferyl
phosphate is added, and the fluorescent product is measured; the intensity of the signal is inversely proportional
to the estradiol concentration in specimens (12 ).
Results
We prepared proficiency testing samples with estradiol
concentrations ranging from ⬃20 to 600 ng/L.4 Specimens
were distributed to 430 laboratories enrolled in the endocrinology proficiency testing program; 159 of these submitted results for estradiol testing. The mean CV for all
methods was 8.4% at estradiol concentrations ⬎100 ng/L.
Greater imprecision (CV ⫽ 33– 40%; n ⫽ 16) at estradiol
concentrations ranging from ⬃150 to 400 ng/L was observed for the (AxSYM) MEIA method in the proficiency
testing event of September 2001. Results from this method
were bimodal in distribution, with apparent centers at
⬃120 and 240 ng/L.5
We examined the possibility that components of the
proficiency testing specimens interfered with the AxSYM
method. Steroid components of the endocrinology proficiency testing specimens are listed in Table 1. We examined their potential to interfere with the AxSYM method
by adding estradiol to processed human serum (proficiency testing serum matrix) to a final concentration of
⬃1000 ng/L, together with one of the steroids listed in
Table 1 at either of two concentrations (one within and
one above the reference intervals). Estradiol in each
sample was measured by the AxSYM method in an
undiluted sample and a 1:5 automated dilution. (Abbott
Laboratories has instructed users of the AxSYM instrument to perform a 1:5 automatic dilution of samples for
some specimens.) Of the steroids tested, only unconjugated
4
The conversion factors to molar units are: estradiol, ng/L ⫻ 3.67 ⫽
pmol/L; unconjugated estriol, ␮g/L ⫻ 3.47 ⫽ nmol/L.
5
Details regarding this proficiency test, as well as others, are available at
http://www.wadsworth.org/chemheme.
Table 1. Effect of other steroids on measurement of
estradiol by the AxSYM (MEIA) method.
Measured estradiol, ng/L
Steroid added
Unconjugated estriol
5.0 ␮g/L
10.0 ␮g/L
Cortisol
108 ␮g/L
308 ␮g/L
Estriol 3-sulfate
5.0 ␮g/L
10.0 ␮g/L
Testosterone
4.2 ␮g/L
5.2 ␮g/L
Progesterone
7.4 ␮g/L
15.4 ␮g/L
Undiluted
1:5 dilution
Relative recovery
(undiluted/1:5
dilution),a %
729
547
918
873
79
63
993
⬎1000
1033
1047
96
⬎96
984
⬎1000
1112
1084
88
⬎92
⬎1000
⬎1000
1038
1198
⬎96
⬎83
946
919
967
780
98
118
a
A range of 86 –96% was obtained with use of three fresh patient sera without
addition of exogenous estradiol and unconjugated estriol.
estriol showed an inhibitory effect on estradiol measurements, in the undiluted specimen. The magnitude of this
effect was substantially reduced when the assay was performed with the 1:5 automated dilution protocol.
To confirm the results described above and to eliminate the possibility of a matrix effect in the pooled human
serum, we prepared two series of specimens by adding
various amounts of unconjugated estriol and a constant
concentration of estradiol (200 ng/L) to fresh human
serum and to the proficiency serum matrix. The concentrations for unconjugated estriol corresponded to the
range of concentrations found in nonpregnant and pregnant women (first 28 weeks), and the estradiol concentration of 200 ng/L was characteristic of late follicular and
mid-luteal phases (13 ). Estradiol was measured by the
AxSYM method in both an undiluted sample and in a 1:5
automatic dilution. The measured estradiol concentrations in the specimens of both series were substantially
decreased, by up to 50%, in the presence of unconjugated
estriol at 20 ␮g/L (Fig. 1, solid lines). However, the
inhibitory effect of unconjugated estriol could be attenuated, from the original 50% down to ⬃10%, by use of the
1:5 automated dilution (Fig. 1, dashed lines).
We also examined the effect of exogenous estriol on the
measurement of estradiol with the IMx, an instrument
that also uses the MEIA technique, at four different
concentrations of estradiol (Fig. 2). At a serum estradiol
concentration of 750 ng/L, unconjugated estriol at 15
␮g/L suppressed the measured result by ⬃20%, and the
effect on the IMx assay (Fig. 2) was generally similar to
that found for the AxSYM (Fig. 1).
To determine whether an increase in the estradiol
concentration could reduce or eliminate the inhibitory
effect of unconjugated estriol, we prepared two sets of
162
Cao et al.: Estriol Interference in Estradiol Immunoassays
Fig. 1. Effect of unconjugated estriol on the measurement of estradiol
by the AxSYM instrument.
Unconjugated estriol was added at the concentrations shown on the abscissa to
a specimen containing estradiol at 200 ng/L. Specimens were proficiency test
serum (F) and patient serum (f). Estradiol concentrations were determined by
the AxSYM method, in either undiluted samples (solid lines) or in a 1:5 automatic
dilution (dashed lines). The measured estradiol concentration at 1 ␮g/L of
unconjugated estriol was considered 100%. A separate experiment (not shown)
showed no significant difference in recovery between the samples containing
unconjugated estriol at 1 ␮g/L and those without exogenous estriol. Data
represent results of four measurements (error bars, SD).
specimens using both fresh human serum and proficiency
serum matrix. To each set of samples, unconjugated
estriol was added at a concentration of 10 ␮g/L and
estradiol was added at concentrations ranging from 100 to
1000 ng/L. Estradiol was then measured by the AxSYM
method without dilution. Increasing estradiol concentra-
Fig. 2. Effect of unconjugated estriol on the measurement of estradiol
by the IMx instrument.
Unconjugated estriol was added at the concentrations shown on the abscissa to
proficiency test serum containing estradiol at 360 (⽧), 750 (f), 1200 (F), or
1400 (Œ) ng/L. Estradiol concentrations were determined by the IMx method
with no dilution. Data represent results of four measurements (error bars, SD).
tions did not increase the percentage recovery of estradiol,
nor did they reduce the inhibitory effect of unconjugated
estriol (Fig. 3).
To determine the relationship between the concentrations of estradiol and unconjugated estriol in association
with the observed suppression, we prepared a series of 24
samples, using six fresh patient sera, with various concentrations of endogenous and/or added estradiol (20 –
980 ng/L). These sera were aliquoted into four subspecimens, and unconjugated estriol (up to 30 ␮g/L) was
added. Estradiol was measured on the AxSYM instrument
without dilution. We observed three different types of
effect depending on the amount of estradiol in the specimens: a positive interference for estradiol concentrations
of ⬍100 ng/L; no apparent interference for estradiol at
⬃100 ng/L; and a negative interference for estradiol ⬎150
ng/L (Fig. 4).
We examined the effect of unconjugated estriol on
other immunoassay methods for estradiol by preparing
two proficiency samples: one containing unconjugated
estriol (at 4 ␮g/L), and the other with no such addition.
These samples were analyzed by participant laboratories
using various immunoassay methods in the May 2001 and
January 2002 proficiency testing events conducted by our
laboratory. The results, summarized in Table 2, show a
positive interference of unconjugated estriol in all of these
methods, with one exception; however, the effect seen in
this latter method was inconclusive because of the low
number of participants and relatively high imprecision.
The increase in the measured estradiol concentration
attributable to addition of unconjugated estriol ranged
from 21% to 389%. We repeated this study using patient
serum, rather than proficiency serum matrix, and similar
results were obtained (data not shown).
Fig. 3. Effect of estradiol concentration on the estradiol recovery
decreased by the presence of unconjugated estriol.
Specimens were proficiency test serum matrix (circles) and fresh patient serum
(squares) containing estradiol (100 –1000 ng/L) and unconjugated estriol at 10
␮g/L. Estradiol concentrations were measured by the AxSYM method without
dilution. Closed symbols represent the mean of four measurements (error bars,
SD). Open symbols indicate single measurements.
163
Clinical Chemistry 50, No. 1, 2004
Table 2. Effect of unconjugated estriol on immunoassays
for estradiol measurement.
Estradiol measured, ng/L (CV)a
Method
Fig. 4. Pattern of unconjugated estriol interference with measurements
of estradiol.
Unconjugated estriol (0 –30 ␮g/L) and estradiol (20 –980 ng/L) were added to
fresh patient sera, and estradiol concentrations were measured on an AxSYM
instrument without dilution. The value of each point represents the mean of four
measurements (error bars, SD). The measured estradiol concentrations on the
ordinate are expressed in logarithmic scale.
Further examination of Table 2 shows a high variability
among the methods used to measure estradiol in the
absence of unconjugated estriol. The overall method mean
was 40.4 ng/L with a range of 26.6 – 64.6 ng/L; the relative
difference for each method is presented in Fig. 5. To rule
out the possibility of a matrix effect, we also performed
this experiment with serum from a nonpregnant female
and obtained results similar to those shown in Fig. 5 (data
not shown).
Discussion
Although estriol is the most abundant of the estrogens, it
is less potent than estradiol. In pregnancy, the concentration of estriol can be increased several hundredfold.
Interference attributable to unconjugated estriol in immunoassays has been described and is usually the result of
positive cross-reactivity with the antibodies used for
estradiol measurement (14 ). Unconjugated estriol is usually present at concentrations far exceeding the concentration of estradiol (15 ). However, a negative interference
of unconjugated estriol in estradiol immunoassays has not
been reported previously. We found that unconjugated
estriol lowers the recovery of estradiol in the AxSYM
(MEIA) method at several concentrations of the two steroids
and in both patient specimens containing exogenous
estriol and estradiol and in proficiency testing specimens.
Our study revealed three types of effects of unconjugated estriol on estradiol measurements; the type of effect
varied according to the concentration of estradiol in the
specimen (Fig. 4). At estradiol concentrations ⬍100 ng/L,
the effect of estriol resembled that of a cross-reacting
interferent. At estradiol concentrations ⬎200 ng/L, the
effect of estriol was to suppress the measured concentration of estradiol. In the range of ⬃100 –200 ng/L estradiol,
there was little apparent effect, presumably because of a
Architect
(n ⫽ 3)b
AxSYM
(n ⫽ 16)
IMx
(n ⫽ 3)
VIDAS
(n ⫽ 4)
ACS:180
(n ⫽ 14)
ADVIA Centaur
(n ⫽ 30)
Access
(n ⫽ 9)
Immuno 1
(n ⫽ 5)
Immulite
(n ⫽ 32)
Immulite 2000
(n ⫽ 15)
Coat-A-Count
(n ⫽ 13)
Vitros ECi
(n ⫽ 5)
Elecsys 2010
(n ⫽ 6)
Overall mean
(CV)
No unconjugated
estriol added
Unconjugated
estriol added
(4 ␮g/L)
Change attributable
to addition of
unconjugated
estriol, %
48.8 (18%)
39.3 (31%)
⫺20
29.7 (17%)
48.0 (13%)
62
40.9 (46%)
74.0 (23%)
81
31.8 (25%)
99.3 (12%)
212
63.4 (16%)
111.0 (14%)
75
64.6 (20%)
110.0 (9%)
70
32.5 (24%)
159.0 (9%)
389
37.4 (10%)
45.1 (7%)
21
40.6 (24%)
71.1 (13%)
75
31.6 (23%)
65.0 (12%)
106
46.4 (11%)
61.5 (20%)
33
26.6 (13%)
69.0 (25%)
159
30.5 (25%)
69.0 (33%)
126
40.4 (31%)
78.5 (42%)
94
a
Results from laboratories participating in proficiency testing over the period
May 2001 to January 2002; mean and interlaboratory CV (%).
b
Number of participants.
cancellation effect between the two phenomena. Although
we did not investigate the mechanism of these interferences, a suggestion put forth by Valdes and Jortani (12 )
sheds light on the possible mechanism of the negative
type of effect. They proposed that interfering substances
bind to antibodies during the initial reaction and then
dissociate during the wash step. As a consequence, relatively more binding sites become available for phosphatase-labeled conjugate to occupy, giving an increased
amount of fluorescent product and a decreased analytical
result. Negative interference with the MEIA technique,
similar to the one we present here, has been reported for
the measurement of digoxin (16 –20 ). Further investigations would be required to confirm whether this may be a
general problem of the assay design in MEIA technology.
The accuracy of measurement of specimens containing
estradiol ⬎200 ng/L and an increased concentration of
estriol was improved by dilution. Abbott Laboratories,
through the AxSYM estradiol package insert, currently
has explicit instructions to users that “patient specimens
with an estradiol assay value greater than or equal to 250
pg/mL (ng/L) must be diluted, retested, and the diluted
164
Cao et al.: Estriol Interference in Estradiol Immunoassays
Fig. 5. Comparison of immunoassay methods measuring estradiol.
Aliquots of pooled male sera without added estradiol and unconjugated estriol
were measured for estradiol concentrations using the following methods: A,
Architect; B, AxSYM; C, IMx; D, VIDAS; E, ACS:180; F, ADVIA Centaur; G, Immuno
1; H, Access; I, Immulite; J, Immulite 2000; K, Coat-A-Count; L, Vitros ECi; M,
Elecsys 2010. The mean of all estradiol measurements (40.4 ng/L) was
considered as 0%.
result reported”. However, at various time points this
concentration was either 150 or 250 ng/L. This procedure
was to minimize “issues associated with depressed results
for fertility management patients”. The cause of interference was not known, and we here identify unconjugated
estriol as the likely source of error. Results from our
proficiency surveys indicate that laboratories using the
AxSYM method do not uniformly follow the manufacturer’s guidance. We therefore observed a bimodal distribution of results from this method, with apparent centers of
⬃120 and 240 ng/L. Participant laboratories in the former
group reported results from 99 to 140 ng/L and likely
failed to dilute the specimen. Laboratories in the latter
group reported results from 201 to 275 ng/L and likely
did perform the dilution that minimized the interference
of estriol. No laboratories reported results in the range
141–200 ng/L. The use of differing protocols among
participant laboratories was likely the origin of the large
CV obtained for the AxSYM method in the proficiency
surveys.
The important role of proficiency testing in the unmasking of laboratory errors is apparent from this study.
Those proficiency samples that were found to have associated with them an increased imprecision for measurement of estradiol contained unconjugated estriol at concentrations ⬎3 ␮g/L. Concentrations far exceeding 3
␮g/L may be found during pregnancy, and even some
nonpregnant females have been shown to have serum
concentrations up to 2.4 ␮g/L (15 ). Any concentration of
estriol exceeding 1.5 ␮g/L was found to depress the
measured concentration of estradiol when estradiol exceeded 200 ng/L. The concentration range of estradiol
that we examined also mimicked concentrations characteristic of pregnancy (⬎200 ng/L) and of the early follicular phase (⬍100 ng/L). Using patient specimens with
added estriol and estradiol, we were able to reproduce all
of the effects found with pooled serum or proficiency test
samples. We therefore believe that the samples used in
this study were commutable (21 ), both for analyte concentration and for the matrix used. Definitive confirmation of our findings would require obtaining clinical
specimens with endogenous concentrations of estradiol
and estriol similar to those that we prepared by adding
exogenous steroids.
Although the error attributable to the interference of
estriol in the MEIA procedure may approach 50%, the
clinical impact of this inhibitory effect may not be widespread because of the typically low concentration of
unconjugated estriol in healthy nonpregnant women and
because of the use of the recommended 1:5 dilution of
patient samples by a proportion of laboratories. However,
for certain medical conditions in which the unconjugated
estriol concentration increases, e.g., pregnancy and obesity (22 ), results of estradiol measurements using the
MEIA method on an AxSYM or IMx instrument may need
to be confirmed by another technology.
Poor agreement among methods was also revealed by
proficiency testing (Table 2 and Fig. 5). We also found
similar poor agreement when patient specimens were
tested. This lack of agreement among methods may be
related to issues of calibration and/or antibody specificity. A strategy for improving intermethod agreement has
emerged from an experiment involving recalibration of all
methods, using a standardized reference material (23 ).
However, the continued existence of poor method agreement (Table 2 and Fig. 5) indicates that the latter model
has yet to be fully adopted or that unstandardized reference methods are used by manufacturers within the in
vitro diagnostics industry. The existing “reference” methods vary in their internal standards, derivatives, extraction procedures, and gas chromatography–mass spectrometry conditions (24 –29 ). Any of these variables can
potentially influence measurements (29 ). For example,
two different derivatization techniques to determine estradiol produced significantly different results (26 ); perhaps, therefore, standardization of the so-called reference
methods for estradiol measurements may be required.
Accurate measurement of estradiol is particularly important in an in vitro fertilization setting. Estradiol concentration is often used for dose adjustment during gonadotropin treatment and as an estimate of ovarian
reserve, ovarian stimulation response, and pregnancy
outcome (1– 4 ). The discrepancies in the literature (30 –32 )
regarding the recommendations for decisions based on
serum estradiol concentration may well be attributable to
the differential effect of estriol on different analytical
methods or to the differences among the methods that we
describe here (Table 2 and Figs. 1–5). For the specimen
without exogenous estriol, the reported concentrations of
estradiol ranged from 26.6 to 64.6 ng/L depending on the
method used (Table 2); individual laboratory results
ranged from ⬍20 to 191 ng/L. For the specimen with
exogenous estriol at 4 ␮g/L, the reported concentrations
of estradiol ranged from 39 to 159 ng/L, depending on the
Clinical Chemistry 50, No. 1, 2004
method used (Table 2); individual laboratory results
ranged from 23 to 196 ng/L. These data show that the
current state of practice for measurement of estradiol does
not meet clinical needs, at least for certain applications
(31 ). The presence of unconjugated estriol in specimens
further compromises reliability (Table 2).
In summary, an external quality-assurance/proficiency
testing program revealed a source of error in the estimation of estradiol in serum. Deficiencies identified in this
study occurred in three areas: (a) unconjugated estriol
interferes with the measurement of estradiol in the
AxSYM (MEIA) method; (b) inconsistency of participant
laboratories in compliance with the manufacturer-recommended dilution protocol for the AxSYM instrument
leads to poor interlaboratory agreement; and (c) there is a
lack of agreement in results among immunoassay techniques for measurement of estradiol.
14.
15.
16.
17.
18.
19.
Abbott Laboratories (Abbott Park, IL) provided reagents
supporting portions of this project.
References
1. Evers JL, Slaats P, Land JA, Dumoulin JC, Dunselman GA. Elevated
levels of basal estradiol-17␤ predict poor response in patients
with normal basal levels of follicle-stimulating hormone undergoing in vitro fertilization. Fertil Steril 1998;69:1010 – 4.
2. Mikkelsen AL, Andersson AM, Skakkebaek NE, Lindenberg S.
Basal concentrations of oestradiol may predict the outcome of
in-vitro maturation in regularly menstruating women. Hum Reprod
2001;16:862–7.
3. Licciardi FL, Liu HC, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and
pregnancy outcome in patients undergoing in vitro fertilization.
Fertil Steril 1995;64:991– 4.
4. Phelps JY, Levine AS, Hickman TN, Zacur HA, Wallach EE, Hinton
EL. Day 4 estradiol levels predict pregnancy success in women
undergoing controlled ovarian hyperstimulation for IVF. Fertil Steril
1998;69:1015–9.
5. Ross JW, Fraser MD, Moore TD. Analytic clinical laboratory precision–state of the art for thirty-one analysis. Am J Clin Pathol
1980;74(4 Suppl):521–30.
6. World Health Organization. WHO Consultation on International
Biological Standards for In Vitro Diagnostic Procedures, September 14 –15, 2000, Geneva, Switzerland. http://www.who.int/
biologicals/Meeting-Reports/Doc/BIVD00Sep14.pdf (Accessed
May 2003).
7. Taieb J, Benattar C, Birr AS, Lindenbaum A. Limitations of steroid
determination by direct immunoassay. Clin Chem 2002;48:583–5.
8. Middle J. Standardization of steroid hormone assays. Ann Clin
Biochem 1998;35:354 – 63.
9. Diver MJ, Nisbet JA. Warning on plasma oestradiol measurement.
Lancet 1987;2:1097.
10. Sinicco A, Raiteri R, Rossati A, Savarino A, Perri GD. Efavirenz
interference in estradiol ELISA assay. Clin Chem 2000;46:734 –5.
11. Mikkelsen AL, Borggaard B, Lebech PE. Results of serial measurement of estradiol in serum with six different methods during
ovarian stimulation. Gynecol Obstet Invest 1996;41:35– 40.
12. Valdes R Jr, Jortani S. Unexpected suppression of immunoassay
results by cross-reactivity: now a demonstrated cause for concern.
Clin Chem 2002;48:405– 6.
13. Carr B. Disorders of the ovaries and female reproductive tract. In:
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
165
Wilson JD, Foster DW, Kronenberg HM, Larse PR, et al. eds.
Williams textbook of endocrinology, 9th ed. Philadelphia: WB
Saunders, 1998: 751– 817.
Kim JB, Barnard GJ, Collins WP, Kohen F, Lindner HR, Eshhar Z.
Measurement of plasma estradiol-17␤ by solid-phase chemiluminescence immunoassay. Clin Chem 1982;28:1120 – 4.
Wright JV, Schliesman B, Robinson L. Comparative measurements of serum estriol, estradiol, and estrone in non-pregnant,
premenopausal women; a preliminary investigation. Altern Med
Rev 1999;4:266 –70.
Steimer W, Muller C, Eber B, Emmanuilidis K. Intoxication due to
negative canrenone interference in digoxin drug monitoring. Lancet 1999;354:1176 –7.
Steimer W, Müller C, Eber B. Digoxin assays: frequent, substantial, and potentially dangerous interference by spironolactone,
canrenone, and other steroids. Clin Chem 2002;48:507–16.
Dasgupta A, Trejo O. Suppression of total digoxin concentrations
by digoxin-like immunoreactive substances in the MEIA digoxin
assay. Elimination of negative interference by monitoring free
digoxin concentrations. Am J Clin Pathol 1999;111:406 –10.
Dasgupta A, Biddle DA, Wells A, Datta P. Positive and negative
interference of the Chinese medicine Chan Su in serum digoxin
measurement. Elimination of interference by using a monoclonal
chemiluminescent digoxin assay or monitoring free digoxin concentration. Am J Clin Pathol 2000;114:174 –9.
Datta P, Dasgupta A. Bidirectional (positive/negative) interference
in a digoxin immunoassay: importance of antibody specificity. Ther
Drug Monit 1998;20:352–7.
Rej R, Proficiency testing, matrix effects, and method evaluation
[Editorial]. Clin Chem 1994;40:345– 6.
Fishman J, Boyar RM, Hellman L. Influence of body weight on
estradiol metabolism in young women. J Clin Endocrinol Metab
1975;41:989 –91.
Thienpont LM, De Leenheer AP. Efforts by industry toward standardization of serum estradiol-17␤ measurements. Clin Chem
1998;44:671– 4.
Siekmann L. Determination of oestradiol-17␤ in human serum by
isotope dilution-mass spectrometry. Definitive methods in clinical
chemistry, II. J Clin Chem Clin Biochem 1984;22:551–7.
Thienpont LM, Verhaeghe PG, Van Brussel KA, and De Leenheer
AP. Estradiol-17␤ quantified in serum by isotope dilution-gas
chromatography-mass spectrometry: reversed-phase C18 highperformance liquid chromatography compared with immuno-affinity chromatography for sample pretreatment. Clin Chem 1988;34:
2066 –9.
Dehennin L. Estradiol-17␤ determined in plasma by gas chromatography-mass spectrometry with selected ion monitoring of mixed
silyl ether-perfluoroacyl ester derivatives and use of various stableisotope-labeled internal standards. Clin Chem 1989;35:532– 6.
Gaskell SJ, Brownsey BG. Immunoadsorption to improve gas
chromatography/high-resolution mass spectrometry of estradiol17␤ in plasma. Clin Chem 1983;29:677– 80.
Wu H, Ramsay C, Ozaeta P, Liu L, Aboleneen H. Serum estradiol
quantified by isotope dilution– gas chromatography/mass spectrometry. Clin Chem 2002;48:364 – 6.
Wolthers BG, Kraan GP. Clinical applications of gas chromatography and gas chromatography-mass spectrometry of steroids.
J Chromatogr A 1999;843:247–74.
Ravhon A, Lavery S, Trew G, Margara R, Winston R. Predictive
value of serum estradiol levels for IVF pregnancy outcome after
gonadotropin stimulation? Fertil Steril 1999;71:183– 4.
Phophong P, Ranieri DM, Khadum I, Meo F, Serhal P. Basal
17␤-estradiol did not correlate with ovarian response and in vitro
fertilization treatment outcome. Fertil Steril 2000;74:1133– 6.
Check JH. Low and high responders—at what levels of serum
estradiol do things start to get fuzzy? Fertil Steril 1999;71:582–3.