ms_06437281190V2.

FT4 II
Free thyroxine

REF SYSTEM

Elecsys 2010
MODULAR ANALYTICS E170
06437281 190 200 cobas e 411
cobas e 601
cobas e 602

English R2 T4~biotin (black cap), 1 bottle, 18 mL:

Intended use Biotinylated T4 2.5 ng/mL; phosphate buffer 100 mmol/L, pH 7.0;
Immunoassay for the in vitro quantitative determination of free thyroxine in preservative.
human serum and plasma. Precautions and warnings
The electrochemiluminescence immunoassay “ECLIA” is intended for use For in vitro diagnostic use.
on Elecsys and cobas e immunoassay analyzers. Exercise the normal precautions required for handling all laboratory
Summary reagents.
Thyroxine (T4) is the main thyroid hormone secreted into the bloodstream Disposal of all waste material should be in accordance with local guidelines.
by the thyroid gland. Together with triiodothyronine (T3) it plays a vital role Safety data sheet available for professional user on request.
in regulating the body's metabolic rate, influences the cardiovascular Avoid foam formation in all reagents and sample types (specimens,
system, growth and bone metabolism, and is important for normal calibrators and controls).
development of gonadal functions and nervous system.1 Reagent handling
T4 circulates in the bloodstream as an equilibrium mixture of free and The reagents in the kit have been assembled into a ready‑for‑use unit that
serum bound hormone. Free T4 (fT4) is the unbound and biologically active cannot be separated.
form, which represents only 0.03 % of the total T4. The remaining T4 is
inactive and bound to serum proteins such as thyroxine binding globulin All information required for correct operation is read in from the respective
(75 %), pre-albumin (15 %), and albumin (10 %).2,3,4,5 reagent barcodes.
The determination of free T4 has the advantage of being independent of Storage and stability
changes in the concentrations and binding properties of these binding Store at 2‑8 °C.
proteins; additional determination of a binding parameter (T‑uptake, TBG) is Do not freeze.
therefore unnecessary. Therefore free T4 is a useful tool in clinical routine
diagnostics for the assessment of the thyroid status. It should be measured Store the Elecsys reagent kit upright in order to ensure complete
together with TSH if thyroid disorders are suspected and is also suitable for availability of the microparticles during automatic mixing prior to use.
monitoring thyrosuppressive therapy.1,6,7
Stability:
A variety of methods are available for estimating the free thyroid hormone
levels. The direct measurement of fT4 and fT3 via equilibrium dialysis or unopened at 2‑8 °C up to the stated expiration date
ultrafiltration is mainly used as a reference method for standardizing, the after opening at 2‑8 °C 84 days (12 weeks)
immunological procedures generally used for routine diagnostic purposes.6,7
In the Elecsys FT4 II assay a specific anti‑T4 antibody labeled with a on the analyzers 28 days (4 weeks) onboard
ruthenium complexa) is used to determine the free thyroxine. or
56 days (8 weeks) when stored
a) Tris(2,2'-bipyridyl)ruthenium(II)-complex (Ru(bpy) ) alternatively in the refrigerator and
Test principle on the analyzer, with the total time
Competition principle. Total duration of assay: 18 minutes. onboard on the analyzer not
exceeding 120 hours
▪ 1st incubation: 15 µL of sample and a T4‑specific antibody labeled with
a ruthenium complex. Specimen collection and preparation
▪ 2nd incubation: After addition of biotinylated T4 and streptavidin‑coated Only the specimens listed below were tested and found acceptable.
microparticles, the still‑free binding sites of the labeled antibody become Undiluted serum collected using standard sampling tubes or tubes
occupied, with formation of an antibody‑hapten complex. The entire containing separating gel.
complex becomes bound to the solid phase via interaction of biotin and Undiluted Li‑heparin, K2‑EDTA and K3‑EDTA plasma.
streptavidin.
Criterion: Recovery with a total deviation ≤ ± 0.6 pmol/L of initial value at
▪ The reaction mixture is aspirated into the measuring cell where the concentrations < 3 pmol/L; recovery within ± 10 % of initial value at
microparticles are magnetically captured onto the surface of the concentrations ≥ 3 pmol/L and slope 0.9‑1.1 + intercept within ≤ ± 2x Limit
electrode. Unbound substances are then removed with of Blank (LoB) + coefficient of correlation ≥ 0.95.
ProCell/ProCell M. Application of a voltage to the electrode then induces
chemiluminescent emission which is measured by a photomultiplier. Stable for 7 days at 2‑8 °C, 30 days at -20 °C.6 Freeze only once.
▪ Results are determined via a calibration curve which is instrument- The sample types listed were tested with a selection of sample collection
specifically generated by 2‑point calibration and a master curve provided tubes that were commercially available at the time of testing, i.e. not all
via the reagent barcode. available tubes of all manufacturers were tested. Sample collection systems
from various manufacturers may contain differing materials which could
Reagents - working solutions affect the test results in some cases. When processing samples in primary
The reagent rackpack is labeled as FT4 II. tubes (sample collection systems), follow the instructions of the tube
manufacturer.
M Streptavidin-coated microparticles (transparent cap), 1 bottle, 12 mL: Centrifuge samples containing precipitates before performing the assay.
Streptavidin-coated microparticles 0.72 mg/mL; preservative. Do not use heat‑inactivated samples.
R1 Anti‑T4-Ab~Ru(bpy) (gray cap), 1 bottle, 18 mL: Do not use samples and controls stabilized with azide.
Polyclonal anti‑T4‑antibody (sheep) labeled with ruthenium complex Ensure the samples, calibrators and controls are at 20‑25 °C prior to
75 ng/mL; phosphate buffer 100 mmol/L, pH 7.0; preservative. measurement.
Due to possible evaporation effects, samples, calibrators and controls on
the analyzers should be analyzed/measured within 2 hours.

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FT4 II
Free thyroxine

Materials provided Quality control

See “Reagents – working solutions” section for reagents. For quality control, use PreciControl Universal.
Materials required (but not provided) In addition, other suitable control material can be used.
▪ REF  06437290190, FT4 II CalSet, 4 x 1 mL Controls for the various concentration ranges should be run individually at
least once every 24 hours when the test is in use, once per reagent kit, and
▪ REF  11731416190, PreciControl Universal, for 2 x 3 mL each of following each calibration.
PreciControl Universal 1 and 2
The control intervals and limits should be adapted to each laboratory’s
▪ General laboratory equipment individual requirements. Values obtained should fall within the defined
▪ Elecsys 2010, MODULAR ANALYTICS E170 or cobas e analyzer limits. Each laboratory should establish corrective measures to be taken if
values fall outside the defined limits.
Accessories for Elecsys 2010 and cobas e 411 analyzers:
Follow the applicable government regulations and local guidelines for
▪ REF  11662988122, ProCell, 6 x 380 mL system buffer quality control.
▪ REF  11662970122, CleanCell, 6 x 380 mL measuring cell cleaning Calculation
solution
The analyzer automatically calculates the analyte concentration of each
▪ REF  11930346122, Elecsys SysWash, 1 x 500 mL washwater additive sample either in pmol/L, ng/dL or ng/L.
▪ REF  11933159001, Adapter for SysClean
Conversion factors: pmol/L x 0.077688 = ng/dL
▪ REF  11706802001, Elecsys 2010 AssayCup, 60 x 60 reaction vessels ng/dL x 12.872 = pmol/L
▪ REF  11706799001, Elecsys 2010 AssayTip, 30 x 120 pipette tips pmol/L x 0.77688 = ng/L
Accessories for MODULAR ANALYTICS E170, cobas e 601 and Limitations - interference
cobas e 602 analyzers: The assay is unaffected by icterus (bilirubin < 701 µmol/L or < 41 mg/dL),
▪ REF  04880340190, ProCell M, 2 x 2 L system buffer hemolysis (Hb < 0.621 mmol/L or < 1.0 g/dL), lipemia (Intralipid
< 2000 mg/dL), biotin (< 81.8 nmol/L or < 20 ng/mL), IgG < 7 g/dL, IgA
▪ REF  04880293190, CleanCell M, 2 x 2 L measuring cell cleaning
< 1.6 g/dL and IgM < 1 g/dL.
solution
Criterion: Recovery within ± 10 % of initial value.
▪ REF  03023141001, PC/CC‑Cups, 12 cups to prewarm ProCell M and
CleanCell M before use Samples should not be taken from patients receiving therapy with high
biotin doses (i.e. > 5 mg/day) until at least 8 hours following the last biotin
▪ REF  03005712190, ProbeWash M, 12 x 70 mL cleaning solution for run administration.
finalization and rinsing during reagent change
No interference was observed from rheumatoid factors up to a
▪ REF  03004899190, PreClean M, 5 x 600 mL detection cleaning solution concentration of 1200 IU/mL and samples from dialysis patients.
▪ REF  12102137001, AssayTip/AssayCup Combimagazine M, Any influence that might affect the binding behavior of the binding proteins
48 magazines x 84 reaction vessels or pipette tips, waste bags can alter the result of the fT4 tests (e.g. drugs, NTIs (Non‑Thyroid‑Illness) or
▪ REF  03023150001, WasteLiner, waste bags patients suffering from FDH (Familial Dysalbuminemic Hyperthyroxinemia)).
9,10
▪ REF  03027651001, SysClean Adapter M The test cannot be used in patients receiving treatment with lipid-lowering
Accessories for all analyzers: agents containing D‑T4. If the thyroid function is to be checked in such
▪ REF  11298500316, Elecsys SysClean, 5 x 100 mL system cleaning patients, the therapy should first be discontinued for 4‑6 weeks to allow the
solution physiological state to become re-established.11
Assay Autoantibodies to thyroid hormones can interfere with the assay.7
For optimum performance of the assay follow the directions given in this In vitro tests were performed on 17 commonly used pharmaceuticals. No
document for the analyzer concerned. Refer to the appropriate operator’s interference with the assay was found.
manual for analyzer‑specific assay instructions. The following special thyroid drugs were tested with concentrations shown
Resuspension of the microparticles takes place automatically prior to use. in the table below. No interference with the assay was found.
Read in the test‑specific parameters via the reagent barcode. If in Criterion: Recovery within ± 10 % of initial value.
exceptional cases the barcode cannot be read, enter the 15‑digit sequence
of numbers. Drug Concentration (µg/mL)
MODULAR ANALYTICS E170, cobas e 601 and cobas e 602 analyzers: Iodid 0.200
PreClean M solution is necessary.
Carbimazol 6
Bring the cooled reagents to approx. 20 °C and place on the reagent disk
(20 °C) of the analyzer. Avoid foam formation. The system automatically Thiamazol 80
regulates the temperature of the reagents and the opening/closing of the Propylthiouracil 300
bottles.
Perchlorat 2000
Calibration
Traceability: This method has been standardized against the Elecsys FT4 Propranolol 240
method. The Elecsys FT4 assay is traceable to the Enzymun‑Test which Amiodaron 200
was standardized using equilibrium dialysis.5,8
Every Elecsys reagent set has a barcoded label containing specific Prednisolon 100
information for calibration of the particular reagent lot. The predefined Hydrocortison 200
master curve is adapted to the analyzer using the relevant CalSet.
Fluocortolon 100
Calibration frequency: Calibration must be performed once per reagent lot
using fresh reagent (i.e. not more than 24 hours since the reagent kit was Octreotid 0.300
registered on the analyzer). Renewed calibration is recommended as
follows: In in vitro studies the drugs Furosemide and Levothyroxine caused elevated
fT4 findings at the daily therapeutic dosage level.
▪ after 1 month (28 days) when using the same reagent lot In rare cases, interference due to extremely high titers of antibodies to
▪ after 7 days (when using the same reagent kit on the analyzer) analyte‑specific antibodies, streptavidin or ruthenium can occur. These
effects are minimized by suitable test design.
▪ as required: e.g. quality control findings outside the defined limits

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FT4 II
Free thyroxine

For diagnostic purposes, the results should always be assessed in Elecsys 2010 and cobas e 411 analyzers
conjunction with the patient’s medical history, clinical examination and other
findings. Repeatability Intermediate
precision
Limits and ranges
Measuring range Sample Mean SD CV SD CV
pmol/L pmol/L % pmol/L %
0.3‑100 pmol/L (defined by the Limit of Blank and the maximum of the
master curve). Values below the Limit of Blank are reported as HS 2 13.3 0.169 1.3 0.301 2.3
< 0.3 pmol/L. Values above the measuring range are reported as
> 100 pmol/L. HS 3 24.5 0.307 1.3 0.518 2.1
Lower limits of measurement HS 4 63.5 1.06 1.7 2.10 3.3
Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantitation HS 5 91.2 1.63 1.8 4.11 4.5
(LoQ) PC Uc)1 15.7 0.139 0.9 0.279 1.8
Limit of Blank = 0.3 pmol/L
PC U2 40.0 0.417 1.0 1.15 2.9
Limit of Detection = 0.5 pmol/L
Limit of Quantitation = 3 pmol/L with a total allowable error of ≤ 30 % b) HS = human serum
c) PC = PreciControl Universal
The Limit of Blank, Limit of Detection and Limit of Quantitation were
determined in accordance with the CLSI (Clinical and Laboratory Standards MODULAR ANALYTICS E170, cobas e 601 and cobas e 602 analyzers
Institute) EP17‑A requirements.
The Limit of Blank is the 95th percentile value from n ≥ 60 measurements of Repeatability Intermediate
analyte‑free samples over several independent series. The Limit of Blank precision
corresponds to the concentration below which analyte‑free samples are Sample Mean SD CV SD CV
found with a probability of 95 %. pmol/L pmol/L % pmol/L %
The Limit of Detection is determined based on the Limit of Blank and the
standard deviation of low concentration samples. The Limit of Detection HS 1 1.58 0.080 5.0 0.100 6.3
corresponds to the lowest analyte concentration which can be detected HS 2 13.1 0.213 1.6 0.217 2.1
(value above the Limit of Blank with a probability of 95 %).
HS 3 24.1 0.381 1.6 0.516 2.1
The Limit of Quantitation is defined as the lowest amount of analyte in a
sample that can be accurately quantitated with a total allowable error of HS 4 61.9 1.26 2.0 2.03 3.3
≤ 30 %.
HS 5 87.8 2.33 2.7 3.40 3.9
Dilution
PC U1 15.6 0.278 1.8 0.293 1.9
Samples for fT4 determinations cannot be diluted, as T4 in the blood is
present in free and protein-bound forms which are in equilibrium. A change PC U2 39.3 0.646 1.6 0.913 2.3
in the concentration of the binding proteins alters this equilibrium.
Method comparison
Expected values A comparison of the Elecsys FT4 II assay (y) with the Elecsys FT4
Euthyroid: 12‑22 pmol/L (0.93‑1.7 ng/dL) assay (x) using clinical samples gave the following correlations:
These values correspond to the 2.5th and 97.5th percentile of results from a Number of samples measured: 170
total of 801 healthy test subjects studied.
Status: MCE Reference Range Thyroid, Status 1st quarter 1998. Passing/Bablok12 Linear regression
For detailed information about reference intervals in children, adolescents y = 0.978x - 0.142 y = 1.02x - 0.963
and pregnant women, refer to the brochure “Reference Intervals for
Children and Adults”, English: REF  04640292, German: REF  04625889. τ = 0.930 r = 0.996
This booklet also contains results of a detailed study about influencing The sample concentrations were between approx. 1.66 and 92.3 pmol/L.
factors on thyroid parameters in a well characterized reference group of Analytical specificity
adults.
The following cross-reactivities were found, tested with fT4 concentrations
Different inclusion and exclusion criteria were applied (e.g. sonographic of approx. 12.5 pmol/L (0.974 ng/dL) and 34.2 pmol/L (2.66 ng/dL):
results (thyroid volume and density) as well as criteria according to the
guidelines of the National Academy of Clinical Biochemistry - NACB). Cross-reactant Concentration tested Cross-reactivity
Each laboratory should investigate the transferability of the expected values ng/dL %
to its own patient population and if necessary determine its own reference
ranges. L‑T3 50000 0.005

Specific performance data D‑T3 50000 0.001

Representative performance data on the analyzers are given below. rT3 190000 0.003
Results obtained in individual laboratories may differ. 3‑iodo‑L‑tyrosine 10000000 0.000
Precision 3,5‑diiodo‑L‑tyrosine 10000000 0.000
Precision was determined using Elecsys reagents, samples and controls in
a protocol (EP5‑A2) of the CLSI (Clinical and Laboratory Standards 3,3’,5‑triiodothyroacetic acid 100000 0.0002
Institute): 2 runs per day in duplication each for 21 days (n = 84). The 3,3’,5,5’‑tetraiodothyroacetic 100000 0.001
following results were obtained: acid
Elecsys 2010 and cobas e 411 analyzers References
Repeatability Intermediate 1 Kronenberg HM, Melmed S, Polonsky KS, et al. Williams Textbook of
precision Endocrinology. Saunders Elsevier, Philadelphia, 12th edition, 2011.
Sample Mean SD CV SD CV 2 Robbins J, Rall JE. The interaction of thyroid hormones and protein in
biological fluids. Recent Prog Horm Res 1957;13:161-208.
pmol/L pmol/L % pmol/L %
HSb) 1 1.78 0.072 4.0 0.136 7.6

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FT4 II
Free thyroxine

3 Oppenheimer JH. Role of plasma proteins in the binding, distribution

and metabolism of the thyroid hormones. N Engl J Med
1968;278(21):1153-1162.
4 DeGroot LJ, Larsen PR, Hennemann G. Transport of thyroid hormone
and cell uptake. The thyroid and its diseases. Wiley and Sons, New
York, 1984:62-65.
5 Ekins RP. Measurement of free hormones in blood. Endocr Rev
1990;11(1):5-46.
6 Wu AHB. Tietz Clinical Guide To Laboratory Tests. Saunders Elsevier,
Philadelphia, 4th edition, 2006.
7 Brent GA. Thyroid Function Testing. Springer, Berlin, 1st edition, 2010.
8 Ekins RP, Ellis SM. The radioimmunoassay of free thyroid hormones in
serum. In Robbins J, Braverman LE (eds). Thyroid research,
Proceedings of the Seventh International Thyroid Conference, Boston.
Amsterdam, Excerpta Medica 1975:597.
9 Wada N, Chiba H, Shimizu C, et al. A novel missense mutation in
codon 218 of the albumin gene in a distinct phenotype of familial
dysalbuminemic hyperthyroxinemia in a Japanese kindred. J Clin
Endocrinol Metab 1997;82(10):3246-3250.
10 Arevalo G. Prevalence of familial dysalbuminemic hyperthyroxinemia in
serum samples received for thyroid testing. Clin Chem
1991;37(8):1430-1431.
11 Bantle JP, Hunninghake DB, Frantz ID, et al. Comparison of
effectiveness of thyrotropin-suppressive doses of D- and L-thyroxine in
treatment of hypercholesterolemia. Am J Med 1984;77(3):475-481.
12 Bablok W, Passing H, Bender R, et al. A general regression procedure
for method transformation. Application of linear regression procedures
for method comparison studies in clinical chemistry, Part III.
J Clin Chem Clin Biochem 1988 Nov;26(11):783-790.
For further information, please refer to the appropriate operator’s manual for
the analyzer concerned, the respective application sheets, the product
information and the Method Sheets of all necessary components (if
available in your country).
A point (period/stop) is always used in this Method Sheet as the decimal
separator to mark the border between the integral and the fractional parts of
a decimal numeral. Separators for thousands are not used.
Symbols
Roche Diagnostics uses the following symbols and signs in addition to
those listed in the ISO 15223‑1 standard.
CONTENT Contents of kit
SYSTEM Analyzers/Instruments on which reagents can be used
Reagent
Calibrator
Volume after reconstitution or mixing

COBAS, COBAS E, ELECSYS, MODULAR and PRECICONTROL are trademarks of Roche. INTRALIPID is a
trademark of Fresenius Kabi AB.
All other product names and trademarks are the property of their respective owners.
Significant additions or changes are indicated by a change bar in the margin.
© 2013, Roche Diagnostics

Roche Diagnostics GmbH, Sandhofer Strasse 116, D-68305 Mannheim

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