Beruflich Dokumente
Kultur Dokumente
APPROVED BY:
……….…………………………………..
DR WAN MOHD IZANI WAN MOHAMED
ENDOCRINE LABORATORY DIRECTOR
REGISTERED HOLDER
ENDOCRINOLOGY LABORATORY
TSH, FREE
T4,FREE T3
1. CONTENT
NO. CONTENT PAGE
1. Content 2
2. Record of Review 3
3. Record of Amendments 4
4. Objective, method, test principle 5-6
5. Clinical significance 7-8
6. Requirements 9-10
7. Procedure 11-14
8. Limitation / source of error 15
9. Limits and Ranges 15
10. References 15
1. RECORD OF REVIEW
1. RECORD OF AMMENDMENT
DATE VERSION DETAIL OF AMMENDMENT BY
NO
18 /03/2014 1 Standardize format for all STM’s
4.0 OBJECTIVE
Standard technical manual to determine in vitro quantitative of free triiodothyronine, free
thyroxine, and thyrotropin in human serum on an Automated Cobas e Immunoassay Analyzers.
4.1 METHOD
In vitro quantitative determination of free triiodothyronine, free thyroxine, and thyrotropin in
human serum are performed using Automated Cobas e Immunoassay Analyzers based on
electrochemiluminescent technology that is the using of ruthenium complex and the measuring
cell.
4.2TEST PRINCIPLE
FREE T3
Competition principle. Total duration of assay: 18 minutes.
• 1st incubation: 15 uL of sample and anti-T3-specific antibody labeled with a ruthenium complex.
• 2nd incubation: After addition of biotinylated T3 and streptavidin-coated microparticles, the still-
free binding sites of the labeled antibody become occupied, with formation of an antibody-hapten
complex. The entire complex is bound to the solid phase via interaction of biotin and streptavidin.
• The reaction mixture is aspirated into the measuring cell where the microparticles are
magnetically captured onto the surface of the electrode. Unbound substances are then removed
with ProCell. Application of a voltage to the electrode then induces chemiluminescent emission
which is measured by a photomultiplier.
• 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.
FREE T4
Competition principle. Total duration of assay: 18 minutes.
•
st
1 incubation: 15 uL of sample and a T4-specific antibody labeled with a ruthenium complex.
•
nd
2 incubation: After addition of biotinylated T4 and streptavidin-coated microparticles, the still-
free binding sites of the labeled antibody become occupied, with formation of an antibody-hapten
complex. The entire complex becomes bound to the solid phase via interaction of biotin and
streptavidin.
• The reaction mixture is aspirated into the measuring cell where the microparticles are
magnetically captured onto the surface of the electrode. Unbound substances are then removed
with ProCell. Application of a voltage to the electrode then induces chemiluminescent emission
which is measured by a photomultiplier.
• 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.
TSH
Sandwich principle. Total duration of assay: 18 minutes.
• 1st incubation: 50 uL of sample, a biotinylated monoclonal TSH-specific antibody and a
monoclonal TSH-specific antibody labeled with a ruthenium complex react to form a sandwich
complex.
• 2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes bound
to the solid phase via interaction of biotin and streptavidin.
• The reaction mixture is aspirated into the measuring cell where the microparticles are
magnetically captured onto the surface of the electrode. Unbound substances are then removed
with by a photomultiplier.
• 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.
FREE T4
The thyroid hormone thyroxine (T4) is physiologically part of the regulating circuit of the thyroid gland and
has an effect on general metabolism. The major fraction of the total thyroxine is bound to the transport
proteins (TBG, prealbumin, albumin). The free thyroxine (fT4) is the physiologically active thyroxine
component.
The determination of free thyroxine is an important element in clinical routine diagnostics. Free T4 is
measured together with TSH when thyroid function disorders are suspected. The determination of fT4 is
also suitable for monitoring thyrosuppressive therapy. The determination of free T4 has the advantage of
being independent of changes in the concentrations and binding properties of the binding proteins;
additional determination of a binding parameter (T-uptake, TBG) ' unnecessary.
A variety of methods are available for estimating the free thyroid hon direct measurement of fT4 and fT3
via equilibrium dialysis or ultrafiltration is mainly used as a reference method for standardizing the
immunological procedures generally used for routine diagnostic purposes. In the Elecsys FT4 test the
determination of free thyroxine is made with the aid of a specific anti- T4 antibody labeled with a
ruthenium complex.
The quantity of antibody used is so small (equivalent to approx. 1-2 % of the total T4 content of a normal
serum sample) that the equilibrium between bound and unbound T4 remains virtually unaffected.
TSH
Thyroid-stimulating hormone (TSH, thyrotropin) is a glycoprotein having a molecular weight of approx.
30000 daltons and consisting of two subunits. The beta-subunit carries the TSH-specific immunological
and biological information, whereas the alpha- chain carries species-specific information and has an
identical amino acid sequence to the alpha-chains of LH, FSH and hCG.
TSH is formed in specific basophil cells of the anterior pituitary and is subject to a circadian secretion
sequence. The hypophyseal release of TSH (thyrotropic hormone) is the central regulating mechanism for
the biological action of thyroid hormones. TSH has a stimulating action in all stages of thyroid hormone
formation and secretio; it also has a proliferative effect. The determination of TSH serves as the initial test
in thyroid diagnostics. Even very slight changes in the concentrations of the free thyroid hormones bring
about much greater opposite changes in the TSH level. Accordingly, TSH is a very sensitive and specific
parameter for assessing thyroid function and is particularly suitable for early detection or exclusion of
disorders in the central regulating circuit between the hypothalamus, pituitary and thyroid.
The Elecsys TSH assay employs monoclonal antibodies specifically directed against human TSH. The
antibodies labeled with ruthenium complex consist of a chimeric construct from human and mouse-
specific components.
As a result, interfering effects due to HAMA (human anti-mouse antibodies) are largely eliminated.
5. REQUIREMENTS
5.1 EQUIPMENT
Cobas e 411/601 Analyzer, Centrifuge, vortex mixer.
5.2 REAGENT
5.2.1 Free T3, Free T4, and TSH Elycsys from Roche Diagnostics
5.4 SPECIMEN
5.4.1 Serum collected - using standard sampling tubes or tubes containing separating gel:
Li-, Na-, NHVHEPARIN, K2-,K3-,Na2-EDTA, and sodium citrate plasma
*, gel tube.
Ensure the patients' samples, calibrators and controls are at ambient temperature (20 - 25 C) before
measurement because of possible evaporation effects, samples, calibrators and controls on the analyzers
should be measured within 2 hours.
6.0 PROCEDURE
NO ACTIVITY RESPONSIBILITY
6.1 CALIBRATOR
a. Two set of two levels is provided in separate kit. MLT/SO
b. Dissolve carefully the contents of one bottle by adding exactly 1.0 ml
of distilled water and allow to stand closed for 15 minutes to
reconstitute.
c. Mix carefully, avoiding the formation of foam.
d. Aliquot calibrators (250 uL aliquot volume) separately into microvials
and keep frozen at minus 20 or below for future use.
e. Keep frozen at minute 20 or below for future use.
Manual order
6.6 MAINTENANCE
MLT/SO
Weekly
a. Clean Incubator
b. Clean Aspiration Station
c. Clean S/R Probe
d. Clean Distilled Water Container
e. Clean Liquid Waste Container
f. Clean Sipper Probe
g. M.Cell Preparation (beginning of the week - 5 cycles)
MLT/SO
2 Weekly
a. Clean S/R Rinse Station
b. Clean Mixer Rinse Station
c. Clean Sipper Probe Rinse Station
d. Perform Liquid Flow Cleaning (1 cycle)
e. M.Cell Preparation (20 cycles)
f. Perform S/R Pipettor Prime (10 cycles)
g. Perform Sipper Pipettor Prime (10 cycles)
MLT/SO
2 Monthly
a. Clean Reagent Disk Compartment
b. Replace Pinch-valve Tubing
7. LIMITATION/SOURCES OF ERROR
7.1 In patients receiving therapy with high biotin doses (>5 mg/day), no sample should be taken until
at least 8 hours after the last biotin administration.
7.2 Patient with treatment lipid lowering agents containing D-T4 will affect the tes result.
7.3 Auto antibodies to thyroid hormones can interfere with this assay.
7.4 Extremely high titers of antibodies to analyte-specific antibodies, streptavidin or ruthenium can
occur and will affect Cortisol level.
9. REFERENCE
9.1 Cobas e 411/ 601 Analyzer Quick Guide Operator's Manual.
9.2 BIO-RAD Lyphochek Immunoassay Plus Control Levels 1, 2 and 3 (Package Insert)
9.3 Elecsys and cobas e analyzers reagents for FT3, FT4 and TSH Package Inserts.