Anti-TPO
Immunoassay Analyzer
REF C89004G 2×50 Tests
INTENDED USE
The iFlash-Anti-TPO assay is a paramagnetic particle
chemiluminescent immunoassay (CLIA) for the
quantitative determination of antibody to thyroid
peroxidase (TPO) in human serum and plasma using the
iFlash Immunoassay Analyzer.
SUMMARY AND EXPLANATION
TPO is a membrane-bound glycoprotein enzyme with an
approximate mass of 107kD. The in vivo function is the
iodination of tyrosine in the synthesis of T3 and T4.
Elevated serum titers of antibodies to TPO are found in
several forms of thyroiditis caused by autoimmunity.
Autoimmune reactivity to TPO is believed to be polyclonal
and heterogeneous in nature with a minimum of six
antigenic determinants being recognized, comprising both
conformational and linear epitopes.
Anti-TPO antibodies are found often in conjunction with
anti-Tg in the majority of cases of Hashimoto’s thyroiditis,
Primary Myxedema, and Graves’ disease. High anti-TPO
titers are found in up to 90% of patients with chronic
Hashimoto’s thyroiditis. In Graves’ disease, 70% of the
patients have an elevated titer. Anti-TPO antibodies are
also found in some other autoimmune disorders such as
type 1 diabetes mellitus or systemic lupus erythematosus.
ASSAY PRINCIPLE
The iFlash-Anti- TPO assay is a sandwich immunoassay.
 Incubation: Total Anti- TPO in the sample, TPO coated
paramagnetic microparticles and Anti-TPO
acridinium-ester-labeled conjugate react to form a
sandwich complex.
 Wash: The unbound materials are washed away from
the solid phase in a magnetic field.
 Trigger of signal: The Pre-Trigger and Trigger
Solutions are added to the reaction mixture. The
resulting chemiluminescent reaction is measured as
relative light units (RLUs).
 A direct relationship exists between the amount of total
Anti-TPO in the sample and the RLUs detected by the
iFlash optical system.
 Results are determined via a calibration curve, which
is instrument-specifically generated by 3-point
calibration and a master curve provided via the
reagent QR code.
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iFlash
REAGENTS
Reagent kit, 100 tests, 2 packs, 50 tests/pack
TPO antigen-coated paramagnetic
R1 microparticles, 3.5 mL/pack, 0.05%
ProClin 300.
Acridinium-ester-labeled anti-TPO
R2 conjugate; 6.5 mL/pack; 0.05% ProClin
300.
Sample diluent, phosphate buffer, 10
R3
mL/pack; 0.05% ProClin 300.
Calibrator 1, 1 bottle, 1.0 mL, phosphate
CAL1 buffer with protein stabilizers, 0.05%
ProClin 300.
Calibrator 2, 1 bottle, 1.0 mL, Anti-TPO in
CAL2 phosphate buffer with protein stabilizers,
0.05% ProClin 300.
Calibrator 3, 1 bottle, 1.0 mL, Anti-TPO in
CAL3 phosphate buffer with protein stabilizers,
0.05% ProClin 300.
MATERIALS REQUIRED (BUT NOT PROVIDED)
REF C89999/C89959/C89949, iFlash Pre-Trigger
Solution: hydrogen peroxide solution.
REF C89998/ C89958/ C89948, iFlash Trigger Solution:
sodium hydroxide solution.
REF C89997, iFlash Wash Buffer: phosphate buffered
saline solution with 0.05% ProClin 300.
REF C80001, iFlash Wash Buffer (10×): phosphate
buffered saline solution with 0.05% ProClin 300.
REF C89996, reaction vessels.
Controls: Commercial controls could be used.
WARNINGS AND PRECAUTIONS
IVD For in vitro diagnostic use
 The calibrators (CAL2 and CAL3) have been prepared
exclusively from the blood of donors tested individually
and shown to be free from HBsAg, Anti-HCV, and
anti-HIV-1/2 by approved methods.
 However, as no known test method can offer the
complete assurance that products derived from human
sources will not transmit infection. Therefore, all
humanized materials should be considered potentially
infectious.
 Exercise the normal precautions required for handling
all laboratory reagents.
 Disposal of all waste material should be in accordance
with local guidelines.
 Wear gloves when handling specimens or reagents.
 Clean and disinfect all spills of specimens or reagents
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Anti-TPO
using a suitable disinfectant.
 iFlash Trigger solution contains sodium hydroxide
(NaOH) and should be avoided contact with eyes.
REAGENT HANDLING
 The reagents may not be used after the stated
expiration date.
 Avoid the formation of foam with all reagents.
 The reagents in the pack and calibrators are ready for
use
 Close the bottles of calibrator right after calibration and
store at 2–8°C.
 Do not pool reagents within a reagent kit or between
reagent kits.
 Prior to loading the iFlash-Anti-TPO reagent pack on
the system for the first time, suspend the
microparticles by inverting the reagent pack slightly.
 For further information on reagent handling
precautions during system operation, refer to the
iFlash system operating instruction.
STORAGE AND STABILITY
Storage:
 Store at 2–8°C in an upright position.
 The kit may be used immediately after removal from
2-8°C storage.
Stability:
 Unopened at 2–8°C: up to the stated expiration date.
 Opened at 2–8°C: 28 days.
 Store on-board: 28 days.
SPECIMEN COLLECTION AND PREPARATION
 Serum or plasma (lithium heparin, sodium heparin
potassium EDTA, and sodium citrate) are the
recommended samples. Other anticoagulants have
not been validated for use with the iFlash-Anti-TPO
assay.
 Ensure that serum specimens to form complete clot
prior to centrifugation.
 Centrifuge the specimens.
 Store specimens at room temperature (20 to 25°C) for
no longer than 8 hours.
 If the testing will not be completed within 8 hours,
refrigerate the samples at 2 to 8°C.
 If the testing will not be completed within 14 days, or
for shipment of samples, freeze at -20°C or colder.
 Frozen specimens must be mixed thoroughly after
thawing.
 The samples may be frozen for maximum 1 time.
 Centrifuge specimens with a lipid layer on the top, and
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iFlash
Immunoassay Analyzer
transfer only the clarified specimen without the lipemic
material.
 Ensure that residual fibrin and cellular matter have
been removed prior to analysis.
 Use with caution in handling patient specimens to
prevent cross-contamination.
 Do not use heat-inactivated samples.
 Ensure that the patient samples, calibrators and
controls are at ambient temperature (20–25°C) before
measurement.
 Due to the possible evaporation, specimens and
calibrators on the analyzers should be measured
within 2 hours.
ASSAY PROCEDURE
 Refer to the system operating instruction or the online
help system for detailed information on preparing the
system.
 The test-specific parameters stored in barcode on the
reagent pack are read in. In case the barcode cannot
be read, enter the sequence numbers.
 Carry out calibration, if necessary.
 Place the calibrators CAL1, CAL2 and CAL3 in the
calibrator rack in the sample zone. Only keep
calibrators open during calibration.
 Test application.
 Load samples (Use 20 μL of sample for each
determination in addition to the sample container and
system dead volumes).
 Click RUN, the iFlash System performs all the
functions automatically and calculates the results.
CALIBRATION
 Traceability:This assay has been standardized against
Anti-TPO standard substance of China's food and drug
verification research institute.
 Every iFlash-Anti-TPO reagent kit has a QR code label
containing the specific information for calibration of the
particular reagent lot.
 To perform an iFlash-Anti-TPO calibration, test CAL1,
CAL2 and CAL3 in duplicate, and the predefined
master curve is adapted to the analyzer.
 Once an iFlash-Anti-TPO calibration is accepted and
stored, all subsequent samples may be tested without
further calibration unless:
 After 28 days when using the same reagent lot.
 A reagent kit with a new lot number is used.
 Controls are out of range.
 Required by pertinent regulations.
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Anti-TPO iFlash
Immunoassay Analyzer

MEASURING RANGE interchangeably.

 1 - 1,000 IU/mL  Samples containing an apparent Anti-TPO level as
high as 124,561 IU/mL did not exhibit a hook effect in
QUALITY CONTROL the iFlash-Anti-TPO assay.
Quality control materials should be run as single  The assay is unaffected by icterus (bilirubin < 10
determinations at least once every 24 hours when the test mg/dL), hemolysis (Hb < 500 mg/dL), lipemia
is in use, once per reagent kit and after every calibration. (Intralipid < 1,800 mg/dL) and total serum protein (< 10
Include commercially available quality control materials g/dL).
that cover at least two levels of analyte. Follow
 No interference was observed from rheumatoid factors
manufacturer’s instructions for reconstitution and storage.
up to a concentration of 2,000 IU/mL.
Each laboratory should establish mean values and
acceptable ranges to assure proper performance. Quality  No interference was observed from anti-nuclear
control results that do not fall within acceptable ranges antibodies up to a concentration of 500 AU/mL.
may indicate invalid test results.  No interference was observed from HAMA up to a
concentration of 600 ng/mL.
RESULT
PERFORMANCE CHARACTERISTICS
Calculation:
Below are the representative performance data, and the
The iFlash system automatically calculates the analyte results obtained in individual laboratories may differ.
concentration of each sample. The results are given in
IU/mL Precision
The precision of iFlash-Anti-TPO was determined using
Interpretation of Results
Anti-TPO reagents, samples and controls. Two serum
A study of with iFlash-Anti-TPO assay on samples from samples, consisting low and median concentration of
357 apparently healthy males of various age groups Anti-TPO were assayed.
yielded the following result:
th
The within run precision was determined by testing each
< 35 IU/mL (95 percentile) sample in replicates of 10 (n = 10), and calculating percent
It is recommended that each laboratory establish its own coefficient of variation (%CV). The results of the study are
expected reference range for the specific population. shown below:
Sample Mean (IU/mL) SD %CV
LIMITATIONS
 The iFlash-Anti-TPO assay is limited to the 1 40.24 2.15 5.34%
determination of Anti-TPO in human serum or plasma 2 397.12 18.86 4.75%
(lithium heparin, sodium heparin, potassium EDTA,
The between run precision was determined by testing
and sodium citrate). It has not been validated for use
each sample in duplicate, two separate runs daily for 20
with other types of plasma.
days (n = 80), and calculating percent coefficient of
 The use of serum separator (gel) blood collection variation (%CV). The results of the study are shown
tubes has been validated for use with this assay. below:
However, it is not possible to survey all manufacturers
Sample Mean (IU/mL) SD %CV
or tube types.
1 1.68
 The upper limit of the measuring range of this assay is 40.63 4.13%
1,000 IU/mL. Over-range samples may be diluted with 2 399.8 18.98 4.75%
wash buffer (1:20 recommended) and re-tested to
obtain an estimate of the actual concentration. Analytical Sensitivity
 If the results are inconsistent with clinical evidence, The detection limit representing the lowest measurable
additional testing is suggested to confirm the result. analyte level is 1 IU/mL, which can be distinguished from
zero. It is calculated as the value lying two standard
 For diagnostic purposes, the results should be
deviations above that of the lowest standard of the master
interpreted in light of the total clinical presentation of
curve (standard 1 + 2 SD, n = 20).
the patient, including symptoms, clinical history results.
 Specimens from heparinized patients may be partially Method Comparison
coagulated and erroneous results could occur due to A comparison of the iFlash-Anti-TPO assay (y) with a
the presence of fibrin. commercially available Anti-TPO assay (x) using clinical
 The results from an alternative assays (i.e. EIA or RIA) samples was performed, and the curve is fitted with Linear
may not be equivalent and cannot be used regression)
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Anti-TPO iFlash
Immunoassay Analyzer

y = 1.007x + 42.35 ANNEX A:

r = 0.9945 Explanation of abbreviation
Sample concentration: 1 – 1,000 IU/mL Abbreviation Explanation
Number of samples measured: 88

REFERENCES Product No.

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2. DeGroot LJ, Niepomniszcze H. Biosynthesis of thyroid Reagent
hormone: basic and clinical aspects. Metabolism 1977;
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Number of tests
3. Mariotti S, Caturegli P, Piccolo P, Barbesino G and
Pinchera A. Antithyroid peroxidase autoantibodies in
thyroid diseases. J Clin Endocrinol Metab 1990; 71: Manufactured by
661-9.
4. Toda S, Koike N, Sugihara H. Cellular integration of EU Representative
thyrocytes and thyroid folliculogenesis: a perspective
for thyroid tissue regeneration and engineering.
EC Declaration of Conformity
Endocrine Journal 2001; 48 (4): 407-425.
5. Feldt-Rasmussen, U. Analytical and clinical
performance goals for testing autoantibodies to Caution
thyroperoxidase, thyroglobulin and thyrotropin
receptor. Clinical Chemistry1996; 42:1, 160-3. Instructions for use
6. Feldt-Rasmussen U, Høier-Madsen M, Bech K,
In vitro diagnostic medical
Blichert-Toft M, Anti-thyroid peroxidase antibodies in
thyroid disorders and non-thyroid autoimmune device
diseases. Autoimmunity 1991; 9: 245–253.
Lot No.

SHENZHEN YHLO BIOTECH CO., LTD. Date of manufacture

1st-4th Floor, No.5 Building, Lishan Industrial
Area, Xinghai Road, Nanshan District, Expiry date
Shenzhen 518054, P.R. China

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Wellkang Ltd (www.CE-marking.eu)

Suite B, 29 Harley St., London W1G 9QR, UK Pictograms for Caution

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aquatic environment

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