Clinical Chemistry 43: 1165-1171, 1997;
(Clinical Chemistry. 1997;43:1165-1171.)
© 1997 American Association for Clinical Chemistry, Inc.
Analytical and clinical evaluation of the Immulite® estradiol assay in serum from patients undergoing in vitro fertilization: estradiol increase in mature follicles
François Dancoine1,a,
Gisèle Couplet1,
Jacques Buvat2,
Catherine Guittard3,
Gérard Marcolin3 and
Jean-Claude Fourlinnie1
1
Laboratoire de Biologie Médicale Drs Leduc et Ass, 17 Rue de la Digue, F-59020;
2
49 Rue de la Bassée, F 59000;
3
145 Ave. de Dunkerque, F 59000 Lille, France
a Author for correspondence. Fax +33 3 20 40 42 64.
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Abstract
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We examined an immunoassay for estradiol (E2) on the
Immulite®—an automated, random access chemiluminescent
immunoassay system—to determine its accuracy and precision required
for in vitro fertilization (IVF) studies. The assay, which has a
reportable range from 73 to 7300 pmol/L, demonstrated good linearity
under dilution, a detection limit of 44 pmol/L, and interassay CVs of
12.6% and 7.6% at 466 and 6164 pmol/L, respectively. In a
retrospective analysis of 545 serum samples, the assay showed adequate
agreement with an antibody-coated-tube RIA. The two E2 assays showed
good agreement, even on samples from patients receiving a variety of
different estrogen replacement therapies. Longitudinal studies of
individual IVF cycles showed good parallelism between the automated
system and the RIA, and results by the automated assay correlated well
with the total number of follicles.
Key Words: indexing terms: chemiluminescence • radioimmunoassay • follicular growth • embryo transfer
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Introduction
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Estradiol (E2) measurements and transvaginal ultrasound are used
to monitor the follicular development in controlled ovarian
hyperstimulation.1
The goal of the stimulation with exogenous
gonadotropins, combined with the administration of gonadoliberin
analogs (GnRHa) in patients undergoing in vitro fertilization and
embryo transfer (IVF-ET), either with clomiphene citrate or with
gonadotropins alone, is to induce the ovaries to produce multiple
retrievable oocytes to raise pregnancy rates (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The
prognostic value of E2 and follitropin (FSH) concentrations measured at
day 3 of IVF cycles is associated with the number of follicles and with
pregnancy rates (15)(16). The combination of
E2 concentrations and the number of oocytes can be predictive for
ovarian hyperstimulation (17). Therefore, the measurement
of E2 remains an essential diagnostic test in evaluating the progress
of stimulation, in making dosage adjustments for each patient, and in
predicting follicular maturity. Many IVF centers offer patients
cryopreserved embryo replacement cycles, ovum donation, and
surrogacy—procedures that require the use of hormone replacement
therapy (HRT) in which an artificial cycle is created by giving E2 in
combination with progesterone. IVF testing requires quick turnaround of
results and accurate E2 determinations with antibodies showing low
cross-reactivity with other steroids characteristic of HRT cycles
(18).
The aim of this study was to compare a manual solid-phase E2 RIA
procedure (E2 Coatria-II) with an automated chemiluminescent method on
the Immulite® system (Immulite E2 assay) in patients
undergoing IVF-ET.
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Materials and Methods
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instrumentation
Immulite (Diagnostic Products Corp., Los Angeles, CA) is an
automated, random-access immunoassay analyzer with a solid-phase
washing process (19) and a chemiluminescent detection
system (20)(21).
Immulite E2 is a solid-phase chemiluminescent immunoassay. The solid
phase, a polystyrene bead enclosed within the Immulite test unit, is
coated with a polyclonal rabbit antibody specific for E2. The
patient's serum sample and alkaline phosphatase-conjugated E2 are
simultaneously introduced into the test unit. During a 60-min
incubation at 37 °C with intermittent shaking, E2 in the serum
sample competes with the enzyme-labeled E2 for a limited number of
antibody binding sites on the bead. Unbound enzyme conjugate is removed
by the patented five-spin-wash technique. Chemiluminescent substrate, a
phosphate ester of adamantyl dioxetane, is added and the test unit is
incubated for 10 min. The substrate is hydrolyzed by alkaline
phosphatase to an unstable anion. The decomposition of the anion yields
a sustained emission of light. The bound complex, corresponding to the
photon output, is inversely proportional to the concentration of E2 in
the sample. A single determination involves 25 µL of serum, and the
stated working range is 73 to 7300 pmol/L. The antibodies used in the
Immulite E2 assay are derived from coupling the E2 molecule at the
specificity-enhancing sixth position. The enhanced specificity of this
17 ß-E2 antiserum makes measurement of E2 concentrations more
accurate (18).
The E2 Coatria-II (Biomérieux, Charbonnières-les-Bains,
France) RIA methodology is a traditional immunoassay with
antibody-coated tubes. After an incubation of 90 min the tubes are
aspirated, washed with water, and then counted in a gamma counter. The
E2 calibrators in the Coatria-II range from 73 to 18 355 pmol/L.
The E2 assay on the Ciba-Corning (Boston, MA) ACS-180 instrument was
used to compare and demonstrate the cross-reactivity of the E2 assays
in samples from six patients treated with estrogen replacement therapy.
analytical evaluation
A comparison study between the E2 Immulite and the Coatria-II
assays was performed on 534 serum samples.
The specificity of the Immulite E2 antibodies was checked by assaying
the serum of four patients receiving 0.75 mg of Oestrogel®
(natural E2; Besins-Iscovesco, Paris, France) applied twice a day, and
of two patients with Estraderm® patches (natural E2;
Ciba-Geigy, Bâle, Switzerland). The same serum samples were
assayed with the Coatria-II and Ciba-Corning assays. A further
comparison was made between the Immulite E2 assay and the Coatria-II
assay in five patients receiving 2 mg of Divina® (E2
valerate; Innothera, Arcueil, France), in one patient receiving 2 mg of
oral Progynova® (E2 valerate; Schering, Lys-les-Lannoy,
France), in 23 patients with a cream application of Oestrogel, and in
11 patients with Estraderm patches.
The analytical intraassay imprecision of the Immulite E2 assay was
determined by assaying two serum samples 21 and 17 times, respectively.
The interassay imprecision was established with two serum samples
assayed in 85 and 60 lots, respectively, with different kit lots on the
Immulite analyzer. The E2 Coatria II interassay imprecision was
calculated on two serum samples in 113 batches with different kit lots.
The recoveries of high E2 concentrations (>7300 pmol/L) on the
Immulite analyzer were verified and compared with the E2 Coatria II
concentrations in 71 serum samples. The serum samples were diluted 1:2
with the appropriate E2 sample diluent for the Immulite and were
measured undiluted with the Coatria-II assay.
Linearity under dilution was verified in three serum samples after
serial dilution of samples with E2 concentrations of 6773, 5558, and
918 pmol/L.
clinical evaluation
Human blood samples collected in our study were obtained
in accordance with the standards of the Ethical Committee of our
institution.
E2 concentrations were measured in 178 serum samples from 41 patients
undergoing ovarian stimulation. GnRHa treatment
(Decapeptyl®, DTRP6, Triptoréline®;
Ipsen/Biotech, Paris, France) to suppress hormone activity during the 2
weeks before gonadotropins were administered to two-thirds of all the
patients was implemented, and then depending on the ovarian response, a
further 10- to 14-day treatment followed. The remaining one-third of
the patients had a short protocol (10 days). In all, five different
gonadotropins were given to stimulate the growth of the follicles: 8
patients with human menopausal gonadotropin, Inductor®
(Pharmagyne, Paris, France); 7 patients with a human postmenopausal
gonadotropin, Neopergonal® (Serono, Boulogne, France); 15
patients with the menotropin Humegon® (Organon, Fresnes,
France); 4 patients with urinary purified FSH, Fertiline®
(Pharmagyne); and 7 patients with highly purified urinary human FSH,
Urofollitropin® (Metrodine; Serono).
All sera were stored at -22 °C and assayed retrospectively with the
Immulite E2 assay. All the results were compared with those of a
traditional E2 RIA methodology (Estradiol Coatria-II).
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Results and Discussion
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analytical evaluation
Comparison between the E2 Immulite and the Coatria-II
concentrations.
Fig. 1
shows a linear regression (±SD) obtained in 534 serum samples:
y (Immulite) = 0.80 (± 0.006)x (Coatria-II) -
9.4 (± 31) pmol/L; Sy|x = 556,
r = 0.985.
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Figure 1. Correlation of E2 concentrations (pmol/L) obtained by the
Immulite (y) and the Coatria-II (x) assays on 534
serum samples.
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Detection limit.
The lower limit of detection, defined
as the concentration corresponding to the mean luminescence of the zero
minus 2 SD, was 44 pmol/L for the Immulite assay. The Coatria II assay,
defined as the concentration corresponding to the mean counts of the
zero minus 2 SD, was 26 pmol/L.
Assay precision.
The Immulite intraassay CVs were 8.2%
and 5.2% at 925 and 5910 pmol/L, respectively; the interassay CVs were
12.6% and 7.6% at 466 and 6164 pmol/L, respectively. The E2 Coatria
II interassay CVs were 6.5% and 6.2% at 1194 and 7840 pmol/L,
respectively.
Specificity.
Fig. 2
represents the Immulite E2 results obtained for 40 specimens
from patients representing four different estrogen replacement
therapies, showing in general good agreement with matching Coatria II
E2 results. These results suggest that the two assays have similar (but
not identical) specificities. The following linear regressions (± SD)
were obtained (y = Immulite, x =
Coatria II) under Divina: y = 0.64 (±
0.089)x + 70.3 (± 54.5),
Sy|x = 82; under Oestrogel:
y = 0.79 (± 0.014)x + 40.0 (± 11.7),
Sy|x = 51; and under
Estraderm therapy: y = 1.20 (± 0.055)x
- 43.2 (± 23.9), Sy|x = 66. Only
one patient was treated with Progynova, and the E2 results were 1000
and 1350 pmol/L with Immulite and Coatria II, respectively.
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Figure 2. Correlation of E2 concentrations (pmol/L) obtained by the
Immulite and the Coatria-II assays in patients with three different
estrogen replacement therapies.
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Factors underlying the several exceptions to good agreement between
matched results have not been further characterized and are most likely
caused by unidentified cross-reactants or matrix effects.
In contrast, ACS-180 yielded results of 154, 158, 257, 808, 881, and
1061 pmol/L for six specimens, which were all consistently higher than
matching Immulite results of 44, 73, 92, 147, 143, and 257 pmol/L,
respectively, and matching Coatria-II results of 44, 37, 103, 158, 143,
and 393 pmol/L, respectively. The much higher ACS-180 E2 results
appeared to reflect a susceptibility to cross-reacting estrogen
metabolites, and we therefore discontinued comparisons with this assay.
Recovery of E2 concentrations >3700 pmol/L on the Immulite
in comparison with RIA.
A linear regression of y
(Immulite) = 0.82 (± 0.0016)x (Coatria-II) - 248 (± 889)
pmol/L; Sy|x = 556,
r = 0.97 was obtained in 182 serum samples with high E2
concentrations.
Dilution recovery.
Fig. 3
shows the dilution recovery of three serum samples with E2
concentrations falling within the calibration range. As shown by the
statistical approach indicated in the graphs, good and acceptable
dilution recovery was obtained.
clinical evaluation
Downregulation by GnRHa; long protocol.
Before ovarian
stimulation started, downregulation was obtained in all the patients.
Fig. 4
indicates the linear regression of the E2 concentrations,
obtained with the Immulite and the Coatria-II E2 assays, at the
beginning of gonadotropin stimulation; mean E2 concentrations of 87.7
(range: 44–228) and 79.2 (range: 40–176) pmol/L, respectively, were
obtained with y (Immulite) = 0.65 (± 0.19)x
(Coatria-II) + 36.3 (± 15.8); Sy|x
= 37, r = 0.49.
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Figure 4. E2 concentrations (pmol/L) obtained by the Immulite and
Coatria-II in 41 patients after downregulation with GnRHa.
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E2 concentrations with different types of ovarian
hyperstimulation.
Table 1
gives an overview of the mean number of days of stimulation,
the mean of the total number of ampoules of FSH used during the
stimulation, the mean of the final E2 concentrations before oocyte
retrieval, and the mean of the daily percent E2 increase. A mean (±
SD) of 11.6 (± 2.8) FSH stimulation days were obtained in 41 patients,
with five different pharmacological FSH preparations. An average (±
SD) of 30.9 (± 16.3) FSH ampoules was used per patient, and a mean (±
SD) E2 concentration of 4906 (± 2707) pmol/L was obtained with the
Immulite E2 assay, just before oocyte retrieval, corresponding to an
increase of 54.7%(± 49.9). The mean E2 concentration in these 41
patients as measured with the RIA was 6079 pmol/L, resulting in a
80.5% increase. During the follow-up in patients with ovarian
stimulation, good parallelism between the two E2 methodologies could be
observed, although slightly lower E2 concentrations were obtained with
the Coatria-II in the lower range, and higher E2 concentrations with
the RIA technique were seen at concentrations >3650 pmol/L. Fig. 5
and
Fig. 6
indicate a correlation (r = 0.50) between the
measured E2 concentrations and the number of days of ovarian
stimulation, and a poor, nonsignificative correlation
(r = 0.09) between the E2 concentrations and the number
of ampoules, respectively. These observations reinforce the need to
individualize each ovulation induction protocol by regular measurement
of E2 serum in combination with transvaginal ultrasound.
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Table 1. Overview of number of days and number of ampoules used
during ovarian stimulation, and the corresponding Immulite E2 (pmol/L)
concentrations at the last day of
stimulation.
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Figure 5. Correlation between the Immulite E2 concentrations
(pmol/L) and the number of days of ovarian stimulation (all
therapies).
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Figure 6. Correlation between the Immulite E2 concentrations
(pmol/L) and the number of FSH ampoules used for ovarian stimulation.
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Relation between E2, total number of follicles, total number
of follicles with a diameter >16 mm, and total number of oocytes.
In 28 of 41 patients, embryo transfer was achieved; the results are
listed in Table 2
. In total, 178 serum samples were obtained from 41 patients,
averaging 4.3 serum E2 measurements per patient. Only the 28 patients
with embryo transfer were taken into account; mean final E2
concentrations in those 28 patients at the day of oocyte retrieval were
7001 pmol/L (RIA) and 5462 pmol/L (Immulite). The average number of
follicles per patient (tF) obtained was 15, with an average of 5
follicles of diameter 
16 mm. A mean of 9 oocytes was retrieved, of
which 7 (on average) could be fertilized. Mean E2 concentrations (only
the number of follicles with a diameter >16 mm) just before oocyte
retrieval were measured by RIA and Immulite, and yielded concentrations
of 1400 and 1092 pmol/L, respectively. When mean concentrations of E2
were compared with the tF, the number of retrieved oocytes (rO), and
the number of fertilized oocytes (fO), 364, 607, and 780 pmol/L,
respectively, were obtained with the Immulite E2 assay. Linear
regressions and correlation coefficients between E2 and the number of
follicles with a diameter >16 mm (F16), tF, rO, and fO are shown in
Table 3
. Correlation factors (r) were found between the E2
concentrations and tF, rO, and fO of 0.64, 0.57, and 0.51,
respectively. A correlation coefficient r = 0.46
between the F16 and the E2 concentrations was found.
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Table 3. Linear regression and correlation factor between Immulite
E2 concentrations (pmol/L) and the F16, the tF, the rO, and the fO at
the day of oocyte
retrieval.
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In conclusion, the comparison data exhibit reasonably good
agreement between RIA and Immulite up to 7300 pmol/L, although a manual
dilution step is required on the Immulite analyzer. E2 measurements
will still be used for ovulation monitoring in combination with
transvaginal ultrasound. The data in this study show that the E2
concentration, as measured in a patient's serum during ovulation
induction monitoring, is reflected in the tF (mean E2 concentration per
follicle = 364 pmol/L). The mean E2 concentration obtained as
calculated per follicle with a diameter of 16 mm was 1092 pmol/L. An
acceptable correlation coefficient between the F16 and the E2 was
obtained; however, better correlation was obtained between E2 and the
tF or the rO. It is important for each laboratory to establish a
reference range for E2 concentrations to avoid ovarian hyperstimulation
syndrome. Our results demonstrate that the E2 measurements on the
automated Immulite system reflect excellent performance by combining
highly specific antibodies with a high degree of automation, resulting
in a quick turnaround time. The mean E2 concentration (1092 pmol/L)
calculated per F16, as measured on the Immulite analyzer, corresponds
to the expected E2 concentration range per mature follicle at the day
of oocyte retrieval.
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Footnotes
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1 Nonstandard abbreviations: E2, estradiol; GnRHa, gonadoliberin agonist; IVF, in vitro fertilization; ET, embryo transfer; FSH, follitropin; HRT, hormone replacement therapy; tF, total number of follicles; rO, retrieved oocytes; fO, fertilized oocytes; and F16, number of follicles with a diameter >16 mm. 
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Abstract
Introduction
