ATTACHMENT 1 OF QN IMM 2014-10

Elecsys Estradiol III

Electrochemiluminescence immunoassay (ECLIA)
for the in vitro quantitative determination of
estradiol in human serum and plasma
Indication
Estrogens are responsible for the development of the secondary female sex characteristics and, together with endogenous
progestogens, e.g. progesterone, they control all the important female reproductive processes. The biologically most active
estrogen is 17-estradiol. Estrogens are produced primarily in the ovary during periods of non-pregnancy and within the
placenta during pregnancy, although small quantities are also formed in the testes and in the adrenal cortex. Measurement
of the estradiol concentration is used clinically in the elucidation of fertility disorders in the hypothalamus-pituitary-gonadal
axis, estrogen-producing ovarian and testicular tumors, gynecomastia, and hyperplasia of the adrenal cortex. Further clinical
indications are the monitoring of fertility therapy and determining the time of ovulation within the framework of in vitro
fertilization (IVF).13

Test principle: 2-step competitive assay

Two biotinylated monoclonal rabbit Streptavidin-coated
antibodies (IgGs) against Estradiol microparticles

Ru

+ + 9 min + 9 min
Detection
Ru

Estradiol in the sample Ru Ruthenylated

Estradiol

Step 1 (9 minutes): Step 2 (9 minutes): Step 3 (measurement):

25 L of the patient sample is incu-
bated with two estradiol (E2)-specific
biotinylated antibodies and mestero-
lone that liberates protein-bound
E2. The higher the E2-concentration
in the patients sample, the lower is
the amount of unbound biotinylated
antibody.
Unbound biotinylated antibody is now
saturated by adding estradiol conjugated
to a ruthenium complex. Simultaneously
added streptavidin-coated paramagnetic
microparticles bind all biotinylated
components in the reaction mixture.
The reaction mixture is aspirated into the
measuring cell where the microparticles are
fixed to the electrode surface by magnetic
action. The unbound substances are sub-
sequently removed. Chemiluminescence
is then induced by applying a voltage and
measured with a photomultiplier. The signal
yield is roughly reversely proportional to the
total E2 concentration in the sample.

Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a master
curve provided via the reagent barcode.
Elecsys technology
ECL (ElectroChemiLuminescence) is Roches technology for immunoassay detection. Based on this technology and combined
with well-designed, specific and sensitive immunoassays, Elecsys delivers reliable results. The development of ECL immunoassays
is based on the use of a ruthenium-complex and tripropylamine (TPA). The chemiluminescence reaction for the detection of
the reaction complex is initiated by applying a voltage to the sample solution resulting in a precisely controlled reaction. ECL
technology can accommodate many immunoassay principles while providing superior performance.

Elecsys Estradiol III test characteristics

Testing time 18 min
Test principle Competitive assay
Calibration 2 point
Traceability Standardized against CRM 6400a via ID-GC/MS (isotope dilution-gas chromatography/
mass spectrometry)4
Sample material Serum and Li heparin, K2-EDTA and K3-EDTA plasma
Sample volume 25 L
LoB, LoD, LoQ* (Specification) 11.0 pmol/L (3 pg/mL), 18.4 pmol/L (5 pg/mL), 91.7 pmol/L (25 pg/mL)
Measuring ranges 18.5 pmol/L 10.410 pmol/L, (5 3,000 pg/mL)
Intermediate imprecision Elecsys 2010, cobas e 411 analyzer: 2.312.3% (16410,716 pmol/L)
E170, cobas e 601, cobas e 602 modules: 2.212.8% (98.79,836 pmol/L)
Expected values

* LoB = Limit of Blank; LoD = Limit of Detection; LoQ = Limit of Quantitation (30% total error)

Test subjects N 5th percentile pmol/L Median pmol/L 95th percentile pmol/L
(90% CI*) (90% CI) (90% CI)
Healty men 146 99.4 (87.2101) 133 (129135) 192 (175223)
Healthy women
Follicular phase 110 98.1 (59.5107) 202 (186219) 571 (5061,148)
Ovulation phase 115 176.5 (151203) 484 (408544) 1,153 (1,0661,187)
Luteal phase 139 122 (40.0135) 370 (327419) 1,094 (8621,269)
Postmenopause 104 <18.4 (<18.4<18.4) 23.9 (<18.429.3) 183 (91.4533)
Healthy pregnant women
1st trimester 90 563 (467798) 2,580 (2,1153,550) 11,249 (8,35715,168)
2nd trimester 59 5,729 (5,4988,760) 25,224 (19,41432,090) 69,547 (61,14280,850)
3rd trimester 97 36,810 (31,2874 0,921) 67,785 (64,15272,152) >110,100 (<106,209110,100)
*CI = confidence interval

Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary
determine its own reference ranges.
References
Order information
1 Handelsman, D.J., Newman, J.D., Jimenez, M., McLachlan, R., Sartorius,
Elecsys Estradiol III 100 tests per rackpack 06656021 190 G., Jones, G.R. (2014). Performance of direct estradiol immunoassays
Elecsys Estradiol III 3 x 1 mL each of CalSet 06656048 190 with human male serum samples. Clin Chem 60(3):510-7.
CalSet Estradiol III Level 1 and 2 2 Rosner, W., Hankinson, S.E., Sluss, P.M., Vesper, H.W., Wierman, M.E.
PreciControl Universal 1 x 3 mL each of Preci 11731416 190 (2013). Challenges to the measurement of estradiol: an endocrine
Control Universal 1 and 2 (code 160 for US) society position statement. J Clin Endocrinol Metab 98(4):1376-87.
3 Melmed, S., Polonsky, K.S., Larsen, R.P., Kronenberg, H.M. (2012).
Diluent Multiassay 2 x 16 mL 05192943 190
Williams Textbook of Endocrinology. 12th edition. Philadelphia,
Saunders/Elsevier.
COBAS, COBAS E, LIFE NEEDS ANSWERS 4 Thienpont, L.M., Van Brussel, K.A., Verhseghe, P.G. et al (1988). Estradiol-
and ELECSYS are trademarks of Roche. 17-beta quantified in serum by isotope dilution-gas chromatography-mass
spectrometry. Clin Chem 34(10):2066-2069.
2014 Roche 5 Elecsys Estradiol III package insert (2014). Roche Diagnostics
Documentation, Basel.
Roche Diagnostics International Ltd
CH-6343 Rotkreuz
Switzerland
www.cobas.com