ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.
Free T3, Free T4 and TSH HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
APPROVED BY:
……….………………………………….. DR WAN MOHD IZANI WAN MOHAMED ENDOCRINE LABORATORY DIRECTOR
CONTROLLED COPY NO: 0
REGISTERED HOLDER ENDOCRINOLOGY LABORATORY
TSH, FREE T4,FREE T3 PREPARED BY DESIGNATION
: MUHAMMAD AMALI BIN KAMARUDIN : QUALITY MANAGER / SCIENCE OFFICER : AINUN IZZATI RAZALI :DOCUMENT CONTROLLER :AFIQAH IYLIA AB HALIM :DEPUTY DOCUMENT CONTROLLER
CHECKED BY DESIGNATION
: MUHAMMAD AMALI BIN KAMARUDIN : ENDOCRINE LABORATORY DEPUTY DIRECTOR/QUALITY MANAGER
AUTHORISED BY DESIGNATION
: DR WAN MOHD IZANI BIN WAN MOHAMED : ENDOCRINE LABORATORY DIRECTOR
Page 1 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO. 1.
HUSM/ENDO/STM 2
VERSION NO.
2
VERSION DATE.
17.09.2014
CONTENT NO.
CONTENT
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
Page 2 of 15
PAGE
15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
1. RECORD OF REVIEW
No semakan
Tarikh
Nama Penyemak
semakan
1
18/03/2013 Muhammad Amali b Kamarudin
2
08/09/2014 Ainun Izzati Razali
Page 3 of 15
Tandatangan pengesahan
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO.
2
VERSION DATE.
17.09.2014
1. RECORD OF AMMENDMENT DATE
VERSION
DETAIL OF AMMENDMENT
BY
NO 18 /03/2014
1
Standardize format for all STM’s Specimen storage information Separate record of amendment from front/ cover page and add page for record of review Add STM objective
17 /09/2014
2
6.12 Procedure dispatch of results Remove: a.original request forms and printed will be not dispatched accept for STAT sample will be manually dispatched via Endocrine laboratory pigeonhole or direct to wad/ clinic. b.Carbonized copy of request form and its printed results for the day is compiles the next day and kept for 3 years. c.Results are also kept in LIS database for traceability.
Page 4 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
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 4.2.1 PRINCIPLE OF THE TEST METHOD 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 stillfree 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 2point calibration and a master curve provided via the reagent barcode.
Page 5 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
FREE T4 Competition principle. Total duration of assay: 18 minutes. •
1 incubation: 15 uL of sample and a T4-specific antibody labeled with a ruthenium complex.
•
2
st
nd
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 2point 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 2point calibration and a master curve provided via the reagent barcode.
Page 6 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.
Free T3, Free T4 and TSH HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
4.2.2 CLINICAL SIGNIFICANCE FREE T3 Triiodothyronine is one of the thyroid hormones present in serum which regulate metabolism. Determination of this hormone concentration is important for the diagnostic differentiation of euthyroid, hyperthyroid and hypothyroid states. The major fraction of total triiodothyronine is bound to the transport proteins (TBG, prealbumin, albumin). Free triiodothyronine (fT3) is the physiologically active form of the thyroid hormone triiodothyronine (T3). The determination of free T3 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) is therefore unnecessary. The sequential testing procedure and the use of a labeled antibody reduces the possibility of interference due to altered binding properties of the serum, as can occur with assays employing labeled antigen (analog method). A variety of methods are available for estimating the free thyroid hormone levels. The 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 FT3 test the determination of free triiodothyronine is made with the aid of a specific anti- T3 antibody labeled with a ruthenium complex.
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
Page 7 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.
Free T3, Free T4 and TSH HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
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 mousespecific components. As a result, interfering effects due to HAMA (human anti-mouse antibodies) are largely eliminated.
Page 8 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
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 5.2.2 SHELF LIFE (days) - Free T3
Unopened at 2-8 °C
Up to the stated expiration date
After opening at 2-8 °C
12 weeks
On the analyzer
6 weeks
5.2.3 SHELF LIFE (days) - Free T4 Unopened at 2-8 °C
Up to the stated expiration date
After opening at 2-8 °C
12 weeks
On the analyzer
4 weeks
5.2.4 SHELF LIFE (days) - TSH Unopened at 2-8 °C
Up to the stated expiration date
After opening at 2-8 °C
12 weeks
On the analyzer e 601
6 weeks
On the analyzer e 411
8 weeks
5.3 QUALITY CONTROL MATERIAL
QC NAME Level 1 Lyphochek Immunoassay Plus Control Level 2 Level 3
Page 9 of 15
CAPACITY PER VIAL 12 X 5 mL
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE :
Free T3, Free T4 and TSH
PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
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.
5.4.2 Sample preparation a. Draw 3 ml blood from the patient and transfer it into plain glass / plastic container. b. Leave it at room temperature not less than half and hour. c.
Centrifuge at 3000 - 3500 rpm for 5 minutes.
d. Transfer the supernatant (serum) as much as possible into 5 ml polypropylene tube. e. Run the test or keep it in the fridge or freezer. (Stability: 5 days at 2 - 8 C, 3 month at -20 C)
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.
Page 10 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO.
2
VERSION DATE.
17.09.2014
6.0 PROCEDURE NO
ACTIVITY
RESPONSIBILITY
6.1 CALIBRATOR MLT/SO
a. Two set of two levels is provided in separate kit. 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.
6.2 CONTROLS (Lyphochek Immunoassay Plus Control Levels 1, 2 and 3) a. Reconstitute each vial with 5.0 mL of distilled or deionized water.
MLT/SO
b. Allow the control to stand for 15 minutes.
c. Aliquot controls (250 uL aliquot volume) separately into hitachi cup for use.
6.3 BLOOD SAMPLES MLT/SO
a. Collect 2 - 3 ml blood into plain tube. b. Centrifuge at 3000 to 3500 rpm for 5 minutes.
c. Transfer supernatant (serum) as much as possible into 5 mL plastic (polypropylene) tube.
6.4 DAILY START-UP a. Check status of reagents in System Overview.
MLT/SO
b. Check levels of PC2/CC2 & PC1/CC1. c.
Empty liquid waste. Check solid waste less than 1100.
d. Fill up water reservoir (1 L dH20 : 10 mL Syswash). e. Replace empty trays of Cup/Tip. f.
Select Reagent Scan.
g. Go to Maintenance > Finalization Maintenance
h. Click Select > START
Page 11 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
6.5 ORDER CONTROL With barcoded controls
MLT/SO
a. Place the relevant barcoded controls in the sample rack. b. Press START. Manual order a. Go QC > Control. Select control, then click Position Assignment. b. Select control, select rack position. Press Assign > OK c.
Proceed to QC > Status. Highlight tests, then click Select.
d. Click Save e. Press START
6.6 ORDER SAMPLE a.
Go to Workplace > Test Selection
MLT/SO
b. Enter rack number, position and sample ID. c.
Select test/profile. Click Save
d. Load samples on rack. e. Press START
6.7 ORDER STAT SAMPLE (During Operation Sample) a. Go to Workplace > Test Selection
MLT/SO
b. Enter rack number, position and sample ID. c. Select test/profile. Click Save d. Load samples on rack.
e. Choose STAT Rack (global button)
6.8 END OF THE DAY a. Go to Maintenance > Finalization Maintenance
MLT/SO
b. Click Select > START
Page 12 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
6.6 MAINTENANCE Weekly
MLT/SO
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)
2 Weekly
MLT/SO
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) 2 Monthly
MLT/SO
a. Clean Reagent Disk Compartment
b. Replace Pinch-valve Tubing
6.10 REPORT AND INTERPRETATION FT3 : 3.9 – 6.7 pmol/L
MLT/SO
FT4 : 12-22 pmol/L TSH : 0.27 – 4.2 mIU/L
Page 13 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
6.11 INTERNAL QUALITY CONTROL a. Run the Lyphochek Immunoassay Plus Control Tri- Level immediately
MLT/SO
after Daily Start-up before analyzing the patients' samples. b. If the results of the controls lie outside the control ranges, repeat the determination.
c. Note any corrective actions taken
6.12 DISPATH RESULS a. Release the results in LIS, put comment if required.
MLT/SO
b. Results are also kept in LIS database for traceability.
Page 14 of 15
STANDARD TECHNICAL MANUAL
ENDOCRINE LABORATORY, HOSPITAL UNIVERSITI SAINS MALAYSIA
Free T3, Free T4 and TSH
TITLE : PROCEDURE NO.
HUSM/ENDO/STM 2
VERSION NO. VERSION DATE.
2 17.09.2014
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.
8. LIMITS AND RANGES Free T3 0.4 - 50.0 pmol/L
Free T4 0.3 -100.0 pmol/L
TSH 0.005-100.0 mlU/L
3.1 - 6.8 pmol/L
12.0-22.0 pmol/L
0.27 - 4.20 mlU/L
Measuring Range Reference Range
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.
Page 15 of 15
STANDARD TECHNICAL MANUAL
