Phadia 250
EliA anti-TPO Test-Specific Reagents
EliA anti-TPO Well (Art. No. 14-5641-01)
anti-TPO Well;
short name: tpo
anti-TPO FLUOROENZYMEIMMUNOASSAY FOR THYROID PEROXIDASE ANTIBODIES
FOR IN VITRO DIAGNOSTIC USE
Human serum in
PBS containing BSA,
detergent and sodium azide (0.095 %);
symbol: pos
EliA uses a modular reagent system. All information needed to understand the use of the
EliA tests can be found in the analyte specific DfU and the corresponding EliA Control DfU.
INTENDED USE
EliA anti-TPO is intended for the in vitro quantitative measurement of IgG antibodies directed
to thyroid peroxidase (TPO) in human serum and plasma (Li-heparin, EDTA) as an aid in the
clinical diagnosis of autoimmune thyroiditis and Graves' disease in conjunction with other
laboratory and clinical findings. EliA anti-TPO uses the EliA IgG method on the instrument
Phadia 250.
ready for use; store
dry at 2-8 °C until
expiration date
Control containing
IgG antibodies to TPO
and TG
6 single-use vials
(0.3 ml each);
sufficient for 2 determinations per vial
Ready for use; store
at 2-8 °C until expiration date
EliA Thyroid Positive Control 250 is prepared from selected pooled human sera.
EliA IgG/IgM/IgA Negative Control 250 (Art. No 83-1037-01)
Human serum in
PBS containing BSA,
detergent and sodium azide (0.095 %);
symbol: neg
SUMMARY AND EXPLANATION OF THE TEST
Thyroid autoimmunity comprises a number of distinct but pathogenically related autoimmune
disorders of the thyroid gland, such as Graves’ disease or Hashimoto’s thyroiditis. Among other
things, these diseases are characterized by the presence of autoantibodies that are directed
against thyroid antigens, such as TSH receptor, thyroglobulin (TG) or thyroid peroxidase (TPO,
formerly known as the thyroid microsomal antigen).
Thyroid peroxidase (TPO) is an integral membrane hemoprotein (100 kDa) which catalyzes the
iodination of thyroglobulin (TG), as well as the coupling of two di-iodotyrosine residues in the
TG molecule to form thyroxine.1
TPO antibodies are present in the serum of most patients with Hashimoto's thyroiditis, in more
than 70% of those with Graves' disease, and, to a variable degree, in patients with non-thyroid
autoimmune diseases and some normal subjects.2 The titers do not correlate with thyroid
functional status. Individual patients with either disorder may have decreases in TPO antibody
titers during therapy, but others have no change.3 On the other hand, there is a good correlation
between the degree of lymphocytic infiltration of the thyroid gland and the presence of TPO
antibodies.2
For the development of an immunoassay it is necessary to obtain reproducible qualities and
amounts of a highly purified antigen. The TPO in the EliA anti-TPO assay is synthesized in the
eukaryotic Baculovirus/Sf9-cell system to achieve maximum antigen purity and quality.4
Multiparameter control containing normal
sera from healthy
donors
6 single-use vials
(0.3 ml each);
sufficient for 2 determinations per vial
ready for use; store
at 2-8 °C until expiration date
EliA IgG/IgM/IgA Negative Control 250 is prepared from selected pooled human sera.
EliA Method-Specific Reagents (Phadia 250)
EliA Sample Diluent (Art. No 83-1023-01)
Sample Diluent (yellow
colored); PBS containing
BSA, detergent and sodium
azide (0.095 %)
6 bottles (48 ml each);
sufficient for ≥ 6 x 180 dilutions
ready for use; store at 2-8 °C
until expiration date
EliA IgG Conjugate 50 (Art. No 83-1017-01)
IgG Conjugate (blue colored);
ß-Galactosidase anti-IgG
(mouse monoclonal antibodies) in PBS containing
BSA and sodium azide
(0.06 %); symbol: El-G
PRINCIPLES OF THE PROCEDURE
6 wedge shaped bottles
(5 ml each); sufficient
for 6 x 50 determinations
ready for use; store at 2-8 °C
until expiration date
DO NOT FREEZE
DO NOT REUSE
EliA IgG Conjugate 200 (Art. No 83-1018-01)
The EliA anti-TPO Wells are coated with human recombinant TPO antigen. If present in the patient's
specimen, antibodies to TPO bind to their specific antigen. After washing away non-bound antibodies, enzyme-labeled antibodies against human IgG antibodies (EliA IgG Conjugate) are added
to form an antibody-conjugate complex. After incubation, non-bound conjugate is washed away
and the bound complex is incubated with a Development Solution. After stopping the reaction, the
fluorescence in the reaction mixture is measured. The higher the response value, the higher the
amount of antibody bound and detected in the sample tested. To evaluate test results, the response
for patient samples is compared directly to the response for calibrators.
IgG Conjugate (blue colored);
ß-Galactosidase anti-IgG
(mouse monoclonal antibodies) in PBS containing
BSA and sodium azide
(0.06 %); symbol: El-G
6 wedge shaped bottles
(19 ml each); sufficient
for 6 x 200 determinations
ready for use; store at 2-8 °C
until expiration date
DO NOT FREEZE
DO NOT REUSE
EliA IgG Calibrator Strips (Art. No 83-1015-01)
human IgG (0, 4, 10, 20, 100,
600 μg/l); in PBS containing
BSA, detergent and sodium
azide (0.095 %)
REAGENTS / MATERIAL
EliA reagents are available as modular packages, each purchased separately. All packages
except for the EliA Thyroid Positive Control 250 and the EliA IgG/IgM/IgA Negative Control 250
are required to carry out an EliA anti-TPO test.
The EliA anti-TPO Wells are packed in carriers which are stored in sealed aluminium foil bags
containing a desiccant.
Issued March 2016
4 carriers (16 wells
each); sufficient for
64 determinations
EliA Thyroid Positive Control 250 (Art. No 83-1113-01)
DIRECTIONS FOR USE
CONTENTS
250-5641- 020 / US
coated with human
recombinant thyroid
peroxidase antigen
5 strips
6 single-use vials per strip
(0.3 ml each); sufficient for
one calibration curve
(double determination)
ready for use; store at 2-8 °C
until expiration date
Manufactured from human sera.
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WARNING! Reagents contain sodium azide (NaN3) as a preservative. NaN3 may be toxic
if ingested or absorbed by skin or eyes. NaN3 may react with lead and copper plumbing
to form highly explosive metal azides. On disposal, flush with a large volume of water to
prevent azide build-up. Please refer to decontamination procedures as outlined by CDC
or other local and national guidelines. Waste Bottle and ImmunoCAP/EliA Well Waste
Container may be contaminated by potentially infectious material. Use appropriate safety
measures and wear gloves.
EliA IgG Curve Control Strips (Art. No 83-1016-01)
human IgG (20 μg/l); in PBS
containing BSA, detergent
and sodium azide (0.095 %)
symbol: CC-1
5 strips
Each strip contains 6 x 0.3 ml
CC-1 (double determination)
ready for use; store at 2-8 °C
until expiration date
Manufactured from human sera.
EliA IgG Calibrator Well (Art. No 14-5509-01)
IgG Calibrator Well coated
with mouse monoclonal antibodies; short name: Gcal
4 carriers (12 wells each); sufficient for 48 determinations
ready for use; store dry at
2-8 °C until expiration date
Indication of Instability
Phadia 250 Instrument Software has built-in acceptance limits for the calibration curve
and the curve control. EliA Wells are moisture sensitive. An activity loss that might
occur due to inappropriate handling can be detected using the appropriate EliA Control.
For more information see Phadia 250 User's Guide/Reference Manual.
Phadia 250 General Reagents
Development Solution (Art. No. 10-9440-01)
Development Solution 0.01 %
4-Methylumbelliferyl-β-D-galactoside, <0.0010% preservative*
6 bottles (17 ml each);
sufficient for 6 x >170 determinations
ready for use; store at 2-8 °C
until expiration date
DO NOT FREEZE
INSTRUMENT
The Phadia 250 Instrument processes all steps of the test. For further information
regarding test set-up, instrumentation and software etc. see Phadia 250 User's Guide/
Reference Manual.
Development Solution (Art. No. 10-9441-01)
Development Solution 0.01 %
4-Methylumbelliferyl-β-D-galactoside, <0.0010% preservative*
6 bottles (11 ml each);
sufficient for 6 x >110 determinations
ready for use; store at 2-8 °C
until expiration date
DO NOT FREEZE
SPECIMEN COLLECTION, HANDLING AND PREPARATION
The procedure can be performed with serum or plasma specimens. Lipemic, hemolyzed or microbially contaminated samples may give poor results and should not be used.
CLSI-Document H18-A4 recommends the following storage conditions for samples:
• Separated serum/plasma should remain at room temperature for no longer than eight hours.
• If assays will not be completed within eight hours, serum/plasma should be refrigerated (2 to 8°C).
• If assays are not completed within 48 hours, or the separated serum/plasma will be stored beyond
48 hours, serum/plasma should be frozen at or below -20°C.
Avoid repeated freezing and thawing.
Note: It is the responsibility of the individual laboratory to use all available references and/or its
own studies to determine specific stability criteria for its laboratory. In general, laboratories should
perform validation studies before implementing a change in specimen acceptance criteria.
Stop Solution (Art. No. 10-9442-01)
Stop Solution 4 % Sodium
Carbonate
6 bottles (119 ml each); sufficient for 6 x >560 determinations
ready for use; store at
2-32 °C until expiration date
Stop Solution (Art. No. 10-9479-01)
Stop Solution 4 % Sodium
Carbonate
6 bottles (65 ml each); sufficient
for 6 x >292 determinations
ready for use; store at
2-32 °C until expiration date
Dilution Plates (Art. No. 12-3907-08)
MicroWell ™ plates with 96
wells, 0.5 ml each; Polypropylene
100 plates per package;
sufficient for 100 x 96 samples
ready for use
DO NOT REUSE
* Preservative: mixture of 5-chloro-2-methyl-2H-isothiazol-3-one [EC no. 247-500-7] and 2-methyl-2H-isothiazol-3-one [EC no. 220-239-6] (3:1).
Washing Solution (Art. No. 10-9422-01/10-9202-01)
For information see separate Washing Solution package insert.
The expiration date for each of the complete packages is stated on the outer label. However,
each component is stable until the date stated on the respective vial label.
Sample Dilution
Samples must be diluted with EliA Sample Diluent. A 1:200 dilution of the samples is
required for the EliA anti-TPO test. Samples can be diluted manually, but instrument dilution is recommended and is a default setting in Phadia 250 Software.
Material required but not provided by Phadia AB:
• Measuring cylinder 1000 ml
• Clinical laboratory reagent water (CLRW, may be used in place of Type I and Type II water) as
defined by CLSI Standards Document, C3-A4: Preparation and Testing of reagent Water in the
Clinical Laboratory; Approved Guideline-Fourth Edition
PROCEDURE
Handling of EliA anti-TPO Well
In the Phadia 250 storage chamber, carriers are stable for up to 28 days. If you are not
expecting to use them up within this time, the carriers should be loaded via the Phadia 250
Loading Tray and, for stability reasons, must be put back into the desiccant-containing
foil bag directly after the run. Because it is important to store the wells in dry conditions at
2-8°C, the bag must be properly resealed. If stored under these conditions, the shelf-life
from the date of first opening is 9 months, if not limited by the expiry date stated on the
carrier and foil bag.
WARNINGS AND PRECAUTIONS
• For in vitro diagnostic use.
• Do not use reagents beyond their expiration dates.
• We do not recommend to pool reagents.
• Some of the reagents are manufactured from human blood components. The source materials have been tested by FDA approved immunoassay for hepatitis B surface antigen, for
antibodies to HIV1, HIV2 and hepatitis C virus and found negative. Nevertheless, all recommended precautions for the handling of blood derivatives should be observed. Please refer to
Human Health Service (HHS) Publication No. (CDC) 93-8395 or local and national guidelines
on laboratory safety procedures.
• Caution: Federal Law restricts this device to sale by or on the order of a licensed practitioner.
250-5641- 020 / US
Issued March 2016
Lot specific barcode
Use the built-in barcode reader to enter the lot specific information of EliA anti-TPO Well,
EliA IgG Calibrator Well and EliA IgG Conjugate. In case of manual handling make sure to
enter the characters below the barcode.
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Published 2016-04-15
anti-TPO
On-board stability of reagents
CALIBRATION AND REFERENCE MATERIAL
• EliA Wells
EliA Well carriers can be stored on-board for 28 days at 2-8°C or 24 hours at room
temperature.
The calibration curve is obtained with EliA IgG Calibrators which are run in duplicate. The
curve is stored and subsequent tests are evaluated against the stored curve using only
the EliA IgG Curve Control (run in duplicate).
The IgG Calibrators are traceable via an unbroken chain of calibrations to the International
Reference Preparation (IRP) 67/86 of Human Serum Immunoglobulins A, G and M from
World Health Organization (WHO).
• EliA Calibrator Strips, EliA Curve Control Strips
Can be stored on-board for 28 days.
• EliA Sample Diluent
Can be stored on-board for 7 days at room temperature. Re-cap bottles every night.
A new calibration curve must be run when:
• the last calibration was made more than one month ago or
• a new lot of EliA IgG Conjugate is introduced or
• when the EliA IgG Curve Control is outside the specified limits (defined in Phadia 250
Instrument Software).
• EliA Conjugate
Single use reagent, open vials must not be stored.
• Development Solution
Can be stored on-board for a total of 40h at room temperature. Can be used 5 times
during shelf life and be stored at room temperature for 8 hours on each occasion. Recap bottles every night. During weekends or longer interval between instrument usage
it is recommended to store bottles at 2-8°C.
• Stop Solution
Can be stored on-board for 7 days at room temperature. Re-cap bottles every night.
EliA anti-TPO is calibrated against the NIBSC research standard for anti-thyroid
microsome serum code 66/387. Results are given in International Units (IU/ml).
QUALITY CONTROL
Control Specimens
Good laboratory practice requires that quality control specimens should be included in every
run. Any material used should be assayed repeatedly to establish mean values and acceptance ranges. EliA Controls are available for the quality control of the measurements.
• Washing Solution
Prepared solution can be stored on-board for 7 days at room temperature. Discard every
seventh day and perform weekly maintenance according to instrument user manual.
CALCULATION AND INTERPRETATION OF RESULTS
Volumes per determination
Reagent volumes per determination
Calibrator
90 μl
EliA IgG Conjugate
90 μl
Development Solution
90 μl
Stop Solution
200 μl
Presentation of Results
Phadia 250 measures specific IgG concentrations in μg/l. By using a conversion factor
given by the lot-specific code of the EliA anti-TPO Well, the results are automatically converted to IU/ml.
Interpretation of Test Results
The ranges (negative, equivocal, positive) recommended for the evaluation of the results
are given in the table below.
Sample volumes per determination
Manual dilution:
90 μl of diluted sample
Instrument dilution (1:200):
10 μl of non diluted sample
For tube-specific dead volumes see Phadia 250 User's Guide/Reference Manual.
negative
equivocal
positive
IU/ml
< 25
25 – 35
> 35
LIMITATIONS
* The residual volume depends on the number of samples and dilution method used.
A definitive clinical diagnosis should not be based on the results of a single diagnostic
method, but should only be made by the physician after all clinical and laboratory findings
have been evaluated.
Procedural comments
• From one sample diluted by the instrument (1:200), up to 11 determinations can be
made.
• When using software default, samples are run in single determination.
• Washing Solution must be at room temperature when used.
• The first result is available after approx. 2 hours and further results at one minute intervals afterwards. Up to 5 x 10 samples can be loaded continuously and are processed
by random access.
• Incubations are automatically performed at 37 °C (98.6 °F).
Issued March 2016
Unit
EliA anti-TPO
Good laboratory practice requires that each laboratory establishes its own range of expected values.
In case of equivocal results, we recommend to retest the patient after 8 –12 weeks.
Reagent volumes per 200 determinations
Washing Solution
5-7 l*
Rinse Solution
5-6 l*
250-5641- 020 / US
Test
EXPECTED VALUES
Antibody prevalence in autoimmune patients varies widely depending on disease area.
A significant percentage (>10%) of healthy subjects was reported to have anti-thyroid
peroxidase antibodies.5
Expected values may vary depending on the population tested.
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anti-TPO
Results Obtained for Healthy Subjects
The frequency distribution for TPO antibodies was investigated in a group of apparently
healthy subjects equally distributed by age and gender, using sera from Caucasian,
African American, Hispanic and Asian population obtained from a blood bank. The results
are given in the table below.
Test
EliA anti-TPO
Unit
IU/ml
No. of
samples
Median
value
90th
percentile
95th
percentile
604
4.4
14.2
59.9
METHOD COMPARISON
For analysis of agreement with the predicate device 498 sera were measured.
n=498
297
167
331
498
Sample
Unit
Mean value
Diagnostic groups
Graves` Disease (= Thyroid-AID)
Autoimmune thyroiditis (= Thyroid-AID)
Non-AI Thyroid Disease
Thyroid cancer
Multi-nodular goiter
Connective Tissue Disease
Crohn`s disease
Ulcerative colitis
Celiac disease
Primary Biliary Cirrhosis
HIV infection
HCV infection
HBV infection
Other infection
Cancer6,7
Rheumatoid arthritis
Hypergammaglobulinemia
Systemic lupus erythematosus
Sjögren`s syndrome
Type I diabetes mellitus8
Type II diabetes mellitus
Pregnant women (all trimesters)9
Pre-eclampsia10
Miscarriage10
Myasthenia gravis
Pernicious anemia
1
IU/ml
2
Coefficients of variation (%)
Intra-Run
Inter-Run
15.7
5.7
2.3
IU/ml
27.4
4.6
1.5
3
IU/ml
39.4
3.8
0.5
4
IU/ml
66.7
3.9
1.0
5
IU/ml
218.0
4.6
1.3
6
IU/ml
480.1
4.7
1.0
7
IU/ml
814.9
3.9
0.4
8
IU/ml
1212.6
4.0
0.0
Issued March 2016
100% x 167/167
Agreement [%]
95% CI
100.0
97.8 – 100.0
100% x 297/331
89.7
85.9 – 92.8
100% x (167+297)/498
93.2
90.6 – 95.2
CLINICAL PERFORMANCE
Precision
To determine the precision of the assay, the variability was assessed in a study with 21
runs by examining the samples in 252 replicates on 3 instruments over 7 days with a calibration curve included into each run.
250-5641- 020 / US
201
297
Total Agreement
Interferences
Undiluted samples were analyzed for interference with EliA anti-TPO. No interference
could be observed for the following substances at the levels indicated: Bilirubin
C/F (201/192 mg/dl), Lipemic factor (ClinOleic) (1%), Hemoglobin (4960 mg/dl),
Rheumatoid factor (RF) (500 IU/ml), Thyroxine (2.5 ng/dl) and Iodide (3 mM).
EliA anti-TPO
34
0
Negative Percent Agreement
Specificity
The EliA anti-TPO test permits the determination of IgG antibodies directed against the
TPO antigen as described in section “Reagents”.
Test
167
EliA anti-TPO Negative (≤ 35 IU/ml)
Positive Percent Agreement
Measuring Range
The technical measuring range (Limit of Detection, upper limit of the calibration curve) for
EliA anti-TPO is from 4 to 1542 IU/ml. Results above the upper limit are generally reported
as “above”.
No hook effects could be observed for concentrations up to 20725 IU/ml.
Linearity was shown for samples in the range from 4 – 1542 IU/ml (linear range). Please
note that due to differing binding characteristics of the antibodies in patient samples, not
all sera can be diluted linearly within the technical measuring range.
Total
EliA anti-TPO Positive (> 35 IU/ml)
Total
PERFORMANCE CHARACTERISTICS
Predicate Device VarelisA TPO
Positive
Negative
(> 100 IU/ml)
(≤ 100 IU/ml)
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Published 2016-04-15
n
100
115
48
21
4
15
20
20
15
10
20
20
20
20
56
12
15
24
14
15
15
90
5
9
7
8
No (%) Positive
EliA anti-TG
81 (81%)
96 (83.5%)
5 (10.4%)
2 (9.5%)
0 (0%)
1 (6.7%)
0 (0%)
0 (0%)
2 (13.3%)
0 (0%)
1 (5%)
1 (5%)
0 (0%)
0 (0%)
11 (19.6%)
1 (8.3%)
0 (0%)
1 (4.2%)
1 (7.1%)
2 (13.3%)
2 (13.3%)
10 (11.1%)
4 (80%)
2 (22.2%)
1 (14.3%)
1 (12.5%)
No (%) Positive
VarelisA TG
68 (68%)
88 (76.5%)
5 (10.4%)
1 (4.8%)
0 (0%)
1 (6.7%)
0 (0%)
0 (0%)
2 (13.3%)
0 (0%)
0 (0%)
1 (5%)
0 (0%)
0 (0%)
5 (8.9%)
1 (8.3%)
0 (0%)
0 (0%)
1 (7.1%)
2 (13.3%)
2 (13.3%)
10 (11.1%)
3 (60%)
1 (11.1%)
0 (0%)
0 (0%)
anti-TPO
WARRANTY
The performance data presented here was obtained using the procedure indicated. Any
change or modification in the procedure not recommended by Phadia AB may affect the
results, in which event Phadia AB disclaims all warranties expressed, implied or statutory,
including the implied warranty of merchantability and fitness for use.
Phadia AB and its authorized distributors, in such event, shall not be liable for damages,
indirect or consequential.
REFERENCES
1. Trotter WR, Belyavin G, Wadhams A. Precipitating and complement-fixing antibodies in Hashimoto's disease.
Proc R Soc Med 1957; 50:961
2. Portmann L, Hamada N, Heinrich G, de Groot LJ. Anti-thyroid peroxidase antibody in patients with autoimmune
thyroid disease: Possible identity with antimicrosomal antibody. J Clin Endocrinol Metab 1985; 61:1001-1003
3. Mariotti S, Caturegli P, Piccolo P, et al. Antithyroid Perixodase autoantibodies in thyroid disease. J Clin
Endocrinol Metab 1990; 71:661-9
4. Berthold H, Steffens U, Northemann W. Human Thyroid Peroxidase: Autoantibody Recognition Depends on the
Natural Conformation. J Clin Lab Anal 1993; 7:401-4
5. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T4, and Thyroid Antibodies in the United States
Population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III) J Clin Endocrinol
Metabol 2002; 87: 489
6. Turken O, Narin Y, Demirbas S, et al. Breast cancer in association with thyroid disorders. Breast Cancer
Research Vol 5 2003; No 5
7. Jiskra J, Límanová Z, Barkmanová J, et al. Autoimmune Thyroid Diseases in Women with Breast Cancer and
Colorectal Cancer. Physiol R 2004; 53:693-702
8. Grzelka A, Araszkiewicz A, Uruska A, et al. Prevalence of Anti-Thyroid Peroxidase in Adults with Type 1
Diabetes Participating in Poznań Prospective Study. Adv Clin Med 2015; 24(1):79-84
9. Stagnaro-Green A, Abalovich M, Alexander E, et al. Guidelines of the American Thyroid Association for the
Diagnosis and Management of Thyroid Disease During Pregnancy and Postpartum. Thyroid 2011; 21(10):
1081-1125
10. Karakosta P, Alegakis D, Georgiou V, et al. Thyroid Dysfunction and Autoantibodies in Early Pregnancy Are
Associated with Increased Risk of Gestational Diabetes and Adverse Birth Outcomes. J Clin Endocrinol Metab
2012; 97(12):4464–4472
ßLOTß
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(
ßIVDß
Batch code
À
Contains x determinations
Biological Risk
§
Read Directions for Use
Store at 2-8°C/35-46°F
&
Manufactured by
Expiration date
"
Do not reuse in a second run
For in vitro diagnostic use
& Phadia AB
)
Rapsgatan 7P
P.O. Box 6460
751 37 Uppsala
Sweden
Tel: +46-18-16 50 60
Fax: +46-18-14 03 58
Autoimmunity@phadia.com
www.phadia.com
© Phadia GmbH, Freiburg, Germany
250-5641- 020 / US
Issued March 2016
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Published 2016-04-15
anti-TPO