This package insert follows the Pharmaceuticals, Medical devices and Other Therapeutic Products Act of Japan.
In Vitro Diagnostics
Marketing Notification No. 13A2X00197218046**Revised: January 2017 (8th edition)*Revised: September 2016 (7th edition)

This package insert must be read carefully prior to use.
Cholesterol assay kit(Classification No.: 30159000)

Total cholesterol is determined by measuring theabsorbance of the red-purple dye. The influence ofascorbic acid is blocked by ascorbate oxidase.

Cholestest CHO

Esterified cholesterol
Fatty acidCODFree cholesterol

General Precautions1. This product is for in vitro diagnostic use, and mustnot 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 beguaranteed if there are any deviations from theinstructions 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. Carefully read the operating instructions for eachtype of automated analyzers prior to using thisproduct. Parameters for each type of analyzers areavailable, and can be requested from SEKISUIMEDICAL CO., LTD. if required.
6. Perform a quality control test prior to assay toensure accuracy.

CEFree cholesterol +Δ4-cholestenone + H2O2

POD
H2O2 + ESBmT + 4-Aminoantipyrine Redpurple color
2. Features
1) Liquid reagents, ready-to-use.
2) With this product, assay results are hardly
affected even if cross-contamination occurs.
3) Bilirubin, hemolysis, ascorbic acid and chylehave minimal effects on results.
4) Applicable to various automated analyzers.
Procedural Precautions ＊1. Properties of Samples and Sampling Methods
1) Samples
Serum and plasma (heparin plasma, EDTA
plasma and citrated plasma) may be used.
2) Storage of samples1)
If the isolated serum or plasma sample cannot
be tested on the same day, specimens should be
stored as follows:
2–10C: for tests within 1 week≤ -20C: for tests after more than 1 week
Bring samples to room temperature (15–30C)
before use.
2. Interfering substances
Assay results are not affected by free bilirubin (upto 50 mg/dL), conjugated bilirubin (up to 50mg/dL), hemoglobin (up to 500 mg/dL), ascorbicacid (up to 50 mg/dL), or Intralipos (up to 5%).
3. Others
1) Always use Seronorm Lipid, Cholestest N
Calibrator, or QUALIGENT N Calibrator for
Labospect 008 for calibration.
2) Precautions for assay range
If the concentration of sample exceeds assay
range, dilute the sample with saline and repeat
the measurement.

Description (Kit Components) ＊Component: Ingredients
Enzyme Solution 1:
4-Aminoantipyrine
Cholesterol esterase
Peroxidase
Enzyme Solution 2:
Cholesterol oxidase (microbialorigin)
N-ethyl-N-sulfobutyl-m-toluidine
sodiumIntended Use
Measurement of total cholesterol in serum orplasma
Cholesterol in serum or plasma is derived from dietaryintake and is also synthesized in the liver.
Measurement of cholesterol is important as an index
of abnormal lipid metabolism and is also considered to
be useful for the diagnosis of liver disorders andmetabolic disease.

Dosage/Administration (Assay Procedure) ＊1. Preparation of reagents
Reagent (1): Enzyme Solution 1 is ready touse.
Reagent (2): Enzyme Solution 2 is ready touse.
2. Assay Procedure
This product is compatible with various types ofautomated analyzer. An example of the assay
procedure is indicated below.

Assay Principle
1. Assay Principle
Cholesterol exists in samples as both the esterified
and free types. Esterified cholesterol is converted
to free cholesterol by the action of cholesterolesterase (CE). Then free cholesterol is oxidized bycholesterol oxidase (COD) to form hydrogenperoxide. Hydrogen peroxide causes oxidativecondensation of 4-aminoantipyrine and N-ethyl-Nsulfobutyl-m-toluidine (ESBmT) in the presenceof peroxidase (POD) to form a red-purple dye.

137CSample + Reagent (1)4 μL300 μL
5 min
Reagent (2) 37C100 μL

5 min

the Enzyme Solution 2. Therefore, if the reagent
comes in accidentally contact with eyes, mouthor skin, rinse immediately with ample water as
first aid, and consult the doctor if required.
4) Proclin 300, which possesses skin-irritative
potential, is added as an antiseptic agent in theEnzyme Solution 1. Therefore, if the reagent
comes in contact with skin or clothes, rinse
immediately with ample water, and consult thedoctor if skin irritation develops.
2. Precautions for use
1) This product should be stored as directed,
without freezing. Freezing can deteriorate thereagents, 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.
5) Sodium azide has been added as an antiseptic
agent in the Enzyme Solution 2. It can react withlead or copper pipes to produce the highly
explosive metal azide. Therefore, the reagent
should be flushed with large amounts of water
during disposal.
4. Other precautions
Do not use the containers for other purposes.

Measurement(Absorbance I※)
Measurement(Absorbance II※)
Calculation ofConcentration※

Absorbance I and II: The difference in absorbance
between 800 nm and 600 nm.
Calibration material: Seronorm Lipid, Cholestest
N Calibrator (manufacturer’s assigned value), orQUALIGENT N Calibrator for Labospect 008(manufacture's assigned value)
Reagent blank: Purified water or saline

Assessment of Assay Results ＊＊1. Reference standard range2)
142–248 mg/dL
2. Diagnostic criterion3)
Hypercholesterolemia: ≥ 220 mg/dL
3. 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) Reagent blank: absorbance being equal to or
lower than 0.05
2) Sensitivity: The absorbance is 0.40–0.72 per
300 mg/dL of cholesterol.
2. Accuracy: 95–105 % of the expected assay value
3. Within-run Reproducibility:
Coefficient of variation: ≤ 3 %(Test methods used for 1.–3. are in-house methods.)
4. Measurement Range4): (On Hitachi 7150
automated analyzer)
5–1000 mg/dL
5. Correlation4)
1) Serum
N=50
r=0.999
y=0.99x+0.39
Control method: Approved in vitro diagnostic(enzymatic method)
2) Plasma
N=50
r=0.999
y=1.00x–1.26
Control method: Approved in vitro diagnostic(enzymatic method)
6. Standard Material
SRM 911b（NIST）

Storage and Shelf Life
1. Storage temperature: 2–10C
2. Shelf life: 18 months from the date of manufacture(The expiration date is printed on the outerpackage.)

Precautions for Use or Handling ＊1. Precautions for Handling (to Ensure Safety)
1) All samples used in the test should be handled asa material possibly infected with HIV, HBV,HCV, or other viruses. To prevent infection, use
disposable gloves and avoid mouth pipetting
during the test.
2) Cholestest N Calibrator and QUALIGENT N
Calibrator for Labospect 008 contains humanderived components determined as HBsAgnegative, HIV antibody (AIDS virus antibody)
negative, and HCV antibody negative. Whenusing, however, it should be handled very
carefully as with samples, considering the risk ofinfectious.
3) Sodium azide is added as an antiseptic agent inPackaging
Name
Package
Cholestest (1) Enzyme Solution 1 2 × 400mL
CHO(2) Enzyme Solution 2 2 × 200mL
Constituent reagents are available in otherconfigurations. For further details please contact
SEKISUI MEDICAL CO., LTD.
References ＊＊1) Sasaki M. et al.: Sampling of constituents of thehuman body, 246, Kodansha, 1972.
2) Kanai M. (supervising editor): Kanai’s manual of2

clinical laboratory medicine. 34th ed. 512, Kanehara
Shuppan, 2015.
3) Japan Atherosclerosis Society, ed. Japan
Atherosclerosis Society Guidelines for Diagnosis
and Treatment of Atherosclerotic CardiovascularDiseases, 2002, 5.
4) 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, Japan3