This package insert follows the Pharmaceuticals, Medical devices and Other Therapeutic Products Act of Japan.
In Vitro Diagnostics
Marketing Notification No. 13A2X00197218081**Revised: January 2017 (10th edition)*Revised: April 2008 (9th edition)

This package insert must be read carefully prior to use.
Heparin assay kit(Classification No.: 30564000)

plasma, p-nitroaniline corresponding to theresidual activity of Factor Xa releases. Because
the residual activity Factor Xa is related to theheparin concentration in the test plasma, theheparin concentration can be determined throughmeasurement of the released p-nitroaniline bycolorimetry at 405 nm.

Testzym HeparinS
General Precautions
1. This product is for in vitro diagnostic use, andmust not be used for any other purposes.
2. Clinicians should make a comprehensive clinicaldecision based on assay results in conjunction withclinical symptoms and other examination results.
3. This product should be used only as directed in thispackage insert. Reliability of results cannot be
guaranteed if there are any deviations from the
instructions in this package insert.
4. If the reagent accidentally comes in contact witheyes and/or mouth, rinse immediately with ample
water as first aid, and consult the doctor if
required.
5. Perform a quality control test prior to assay toensure accuracy.

Antithrombin
III

Heparin

Heparin

Antithrombin
III

Heparin(Complex)AA

AA

Factor Xa

Factor Xa(Residual activity)

Factor Xa

AA

Antithrombin
III(Inactive)
AA

pNA

AA

Substrate (S-2222)

AA

AA

AA

pNA
Colorimetry(405 nm)2. Features
1) Heparin can be measured without any
influence of other coagulation factors.
2) Handling is easy, and results can be obtained
rapidly.
3) This product shows good quantification and
excellent reproducibility.

Description (Kit Components) ＊＊Component
Ingredients(1) Substrate: N-Benzoyl-L-isoleucyl-L-glutamyl(γ-OR)-glycyl-L-arginyl-p-nitroan
ilide hydrochloride (S-2222)(2) Antithrombin III:
Antithrombin III (human origin)(3) Factor Xa: Factor Xa (bovine origin)(4) Buffer Solution:
2-Amino-2-hydroxymethyl-1,3-pr
opanediol buffer(5) Normal Plasma :Normal human plasma＊＊

Procedural Precautions ＊1. Properties of Samples and Sampling Methods
1) Samples(1)Plasma (Citrated plasma) may be used.
(2)Heparin plasma or EDTA plasma should not be
used.
2) Storage of samples(1) If the isolated plasma sample cannot be testedon the same day, specimens should be stored asfollows:
2–10C: for tests within 1 week
≤ -20C : for tests within 1 month
Bring samples to room temperature (15–30C)
before use.
(2) Avoid repeated freezing and thawing, or errors
in the assay results may occur.
(3) Use plastic containers and test tubes fordilution of plasma.
2. Interfering substances
Assay results are not affected by bilirubin (up to
20 mg/dL) or hemoglobin (up to 500 mg/dL).
3. Others
1) Use JP heparin as the calibration material.
Caution must be exercised, because results
will not necessarily be the same due tovariation between manufacturers and lots. In
addition, take care to prepare the heparinstandard solution accurately, because thedilution ratio of the heparin standard solution
is high.
2) Precautions for assay range
If the heparin level in the test plasma is high(≥ 0.8 IU/mL), dilute the sample with Normal
Plasma and perform re-measurement.

Intended Use
Measurement of heparin in plasma
It is known that heparin binds to antithrombin III in
the blood to form a complex, which in turn binds toactivated coagulation factors (such as thrombin andFactor Xa) and inhibits their activity.
Because of its action, heparin is widely used inclinical practice for prevention and treatment ofdisseminated intravascular coagulation (DIC) andthrombosis, as well as for anticoagulation during
extracorporeal circulation. Measurement of heparin is
useful for monitoring the effect of heparin therapy.
Assay Principle
1. Assay Principle
Add antithrombin III to the test plasma containing
heparin to form heparin/antithrombin III
complexes. When a fixed excess of Factor Xa is
subsequently added to the sample, theheparin/antithrombin III complexes bind to Factor
Xa,
forminginactive
complexes(heparin/antithrombin III/Factor Xa).
When the Substrate (S-2222) is added to the test1

Dosage/Administration (Assay Procedure) ＊＊1. Equipment
Centrifuge (for separation of plasma)
Thermostat
Spectrophotometer
Stopwatch
Mixer
Semi-microcuvette or 10  10 mm cuvette
Various micropipettes
Various measuring pipettes
Measuring cylinder
Plastic test tube
Tissue paper without waste textiles (such as Kim
Wipe).
2. Methods of preparing the reagents andsamples1) Substrate Solution: Before use, dissolve 1 vialof the Substrate in 20 mL of purified water.
After preparation, the solution is stable whenstored for 3 months at 2–10 °C.
2) Antithrombin III Solution: Before use,
dissolve 1 vial of the Antithrombin III in 10
mL of purified water. After preparation, thesolution is stable when stored for 1 month at
2–10 °C.
3) Factor Xa Solution: Before use, dissolve 1 vialof the Factor Xa in 10 mL of purified water.
After preparation, the solution is stable whenstored for 1 month at 2–10 °C.
4) Buffer Solution: Buffer Solution is supplied asa ready-to-use reagent. After opening, store the
Buffer Solution at 2–10 °C, and use it as soon
as possible.
5) Normal Plasma: Before use, dissolve 1 vial of
the Normal Plasma in 1.0 mL of purified water.
After preparation, the plasma is stable whenstored for 1 week at 2–10°C or for 1 month at–20°C.
6) Stop Solution (prepared in-house): Before use,
add purified water to 20 g (20 mL) of glacialacetic acid to make a volume of 40 mL.
7) Heparin standard solution (tertiary diluted
heparin solution) (prepared in-house):(1) Primary diluted heparin solution (10 IU/mL)
Add physiological saline to 1 mL of 1000
IU/mL heparin sodium solution to make avolume of 100 mL, and use this solution as
the primary diluted heparin solution.
(2) Secondary diluted heparin solution (0.2
IU/mL)
Add exactly 4.9 mL of the Buffer Solution
to 100 μL of the primary diluted heparinsolution (10 IU/mL), and use this solution as
the secondary diluted heparin solution.
(3) Heparin standard solution (tertiary diluted
heparin solution)
Dilute the secondary diluted heparinsolution as directed in the following table
and mix to prepare serial dilutions of theheparin standard solution.

Standard solution Buffer
AntiNormal SecondarySolu- thrombin Plasma
dilutedNo.
HeparinIII(µL)
heparinconcentration tion(μL) Solution
solution(IU/mL(µL)(µL)
plasma)
1
0
800
100
100
0
2
0.2
700
100
100
100
3
0.4
600
100
100
200
4
0.6
500
100
100
300
5
0.8
400
100
100
400

8) Sample(1) Collect approximately 5 mL of citrated
blood (whole blood: 0.1 mol/L sodium
citrate = 9 volumes: 1 volume) from thepatient by the dual syringe method.
(2) Centrifuge the collected blood at 2000g for
20 minutes at 4°C, and immediately transfer
the supernatant to a plastic tube for use as
the test plasma.
(3) Add 100 µL of the Antithrombin III Solution
and 800 µL of the Buffer Solution to 100 µLof the test plasma, mix without bubbling,
and use this solution as the sample. (Start thenext operation as soon as possible.)
3. Assay Procedure
1) Endpoint method(1) Measure exactly 200 µL each of the sample
for measurement and the blank sample into
separate test tubes, and warm for 2–6
minutes at 37°C.
(2) Add 100 µL of the Factor Xa Solution to thetest tube for measurement, mix thoroughly,
and warm for exactly 30 seconds at 37°C.
(3) Add 200 μL of the Substrate Solution(previously warmed at 37°C) to the test tube
for measurement, mix thoroughly, and warm
for exactly 180 seconds at 37°C.
(4) Add 300 μL of the Stop Solution to each test
tube, and mix immediately.
(5) Add 300 µL of purified water to the blanktest tube, and mix.
(6) Determine the absorbance of this solution at
405 nm using the blank as the control.
(7) Proceed with each heparin standard solution
as described for the sample for measurement,
and determine the absorbance.
StandardBlanksolution(Heparin(Sample or(Sample)standard
heparin standard
Specimen
200 μL
solution)solution)
200 μL200 μL
Warm for 2–6 minutes at 37°C.
Factor Xa
100 μL100 μL—
SolutionMix, and warm for exactly 30 seconds at 37°C.
(Previously
warmed to
37°C)
200 μL200 μL—
Substrate
SolutionMix, and warm for exactly 180 seconds at 37°C.
Stop
300 μL
300 μL
300 μL
Solution
Mix immediately.
Purified——
300 μLwater
Determine the absorbance at 405 nm after mixing
Sample2(2) Avoid vigorous mixing when preparing theAntithrombin III Solution, Factor Xa
Solution, and Normal Plasma, because it
may lead to deterioration of enzymes anddegeneration of proteins.
(3) Warm the Factor Xa Solution (previously
stored in a refrigerator) to room temperature
before use.
(4) The components of the Substrate Solution
may separate during storage in a refrigerator.
If separation occurs, warm the solution at
50°C and use it after dissolution.
(5) If a standard 10 x 10 mm cuvette is used inplace of the semi-microcuvette for theendpoint method, 1900 µL of 10% acetic
acid may be added instead of 300 μL of theStop Solution to stop the reaction. In thiscase, plot the calibration curve by adding1900 μL of 10% acetic acid.
(6) After the reaction is stopped, the color
remains stable for 4 hours.

2) Method of measuring the initial rate(1) Add 200 μL of the sample to the test tube,
and warm for 2–6 minutes at 37°C.
(2) Add 100 µL of the Factor Xa Solution to thetest tube, mix, and warm for exactly 30
seconds at 37°C.
(3) Add 200 μL of the Substrate Solution(previously warmed to 37°C) to the test tube,
mix, and immediately transfer to thesemi-microcuvetteequipped
withathermostat (37°C).
(4) Determine the change of absorbance at 405
nm per minute (ΔA/min) from 10 to 70
seconds after the start of the reaction.
(5) Proceed with each heparin standard solution
as described for the sample, and determine
the change of absorbance (ΔA/min).
Standard
solution(Heparin(Sample)standard
Specimen
200 μL
solution)
200 μL
Warm for 2–6 minutes at 37°C.
Factor Xa Solution
100μL
100μL
Mix, and warm for exactly 30 seconds at 37°C.
(Previously warmed
to 37°C)
200μL
200μL
Substrate Solution
Immediately after mixing, transfer the mixture to asemi-microcuvette equipped with a thermostat (37°C),
and determine the change of absorbance at 405 nm per
minute (ΔA/min) from 10 to 70 seconds after the start of
the reaction.
SampleAssessment of Assay Results
1. Reference treatment concentration2)
0.2–1.2 IU/mL
2. There may be reactions or interfering reactions
with non-target substances. If assay results appear
to be unreliable, repeat the measurement (ifnecessary, after dilution) or try another analyticalmethods.
Performance
1. Sensitivity
1) Slope of the calibration curve: -0.78 to -0.42
2) Intercept of the calibration curve: 0.58 to 1.00
2. Accuracy: 90–110 % of the expected assay value
3. Within-run Reproducibility:
Coefficient of variation ≤ 5 %(Test methods used for 1.–3. are in-housemethods.)
4. Measurement Range3): (Manual method)
0.15–0.8 IU /mL
5. Correlation3)
Plasma N=50
r=0.996y=0.986x+0.00
Control method: Synthetic chromogenic substrate
method
6. Standard Material
Heparin reference standard (NIBSC)

3) Method of calculation
On a graph with the absorbance (or change ofabsorbance) on the y-axis and the heparinconcentration on the x-axis, plot theabsorbance (or change of absorbance)corresponding to the heparin concentrations inthe serial dilutions of the heparin standard
solution, and draw a line passing through eachpoint to create the calibration curve. Use thiscalibration curve to determine the heparinconcentration from the absorbance (or changeof absorbance) of the sample.
Example ofcalibration
curves＊＊Precautions for Use or Handling ＊1. Precautions for Handling (to Ensure Safety)
1) All samples used in the test should be handled
as a material possibly infected with HIV, HBV,HCV, or other viruses. To prevent infection, use
disposable gloves and avoid mouth pipetting
during the test.
2) Antithrombin III and Normal Plasma containhuman-derived components that have beenshown to be negative for HBs antigens, HIVantibodies, and HCV antibodies. However,these reagents (as well as the samples) should
be considered potentially infectious and handledwith great care.
2. Precautions for use
1) This product should be stored as directed,
without freezing. Freezing can deteriorate the

Heparin concentration(IU/mL)4) Precautions for testing(1) Cool the collected blood on ice and prepare
the test plasma as soon as possible,
preferably within 90 minutes of bloodcollection. Measurement may be interfered if
platelets are destroyed or there are too many
contaminating platelets.

3reagents, which can produce inaccurate results.
Therefore, avoid using the reagents which havebeen previously frozen.
2) Do not use expired reagents. Use of suchreagents cannot guarantee the reliability ofmeasurement values.
3) Do not replenish the reagents.
4) Do not perform the assay under direct sunlight
3. Precautions for Disposal
1) Before disposal, used samples and theircontainers must be immersed in sodium
hypochlorite solution at a concentration ofgreater than 0.1% for longer than 1 hour or
autoclaved at 121ºC for 20 minutes.
2) To prevent infections from spilled samples orsolutions containing samples, wipe the spilledarea thoroughly with disinfectants such as
sodium hypochlorite solution at a concentrationof greater than 0.1%.
3) The reagents and treated samples should bediscarded as medical waste or industrial waste
according to the waste disposal regulations.
4) The reagents should be disposed of inaccordance with the Water Pollution Control act
or related regulations.
4. Other precautions
Do not use the containers for other purposes.
Storage and Shelf Life ＊＊1. Storage temperature: 2–10C
2. Shelf life: 2 years months from the date of
manufacture(The expiration date is printed on the outer
package.)
Packaging
Name
Substrate
Antithrombin III
Testzym
Factor Xa
HeparinS
Buffer Solution
Normal Plasma

Package
1 × for 20 mL
1 × for 10 mL
1 × for 10 mL
1 × 100mL
4 × for 1 mL

References ＊1) Teien A.N. et al.: Thromb Res, 11, 107, 1977.
2) Hasegawa H. et al.: Jap Heart J, 21, 367, 1980.
3) In house data, SEKISUI MEDICAL CO., LTD.
Contact ＊SEKISUI MEDICAL CO., LTD.
international@sekisui.com＊＊＊

Manufacturer
SEKISUI MEDICAL CO., LTD.
1-3, Nihonbashi 2-chome, Chuo-ku, Tokyo, Japan

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