This package insert follows the Pharmaceuticals, Medical devices and Other Therapeutic Products Act of Japan.
In Vitro Diagnostics
Marketing Notification No. 13A2X00197218104**Revised: January 2020 (6th edition)*Revised: January 2017 (5th edition)

This package insert must be read carefully prior to use.
Phenobarbital assay kit(Classification No.: 30401000)

without further metabolism or as a glucuronic acidconjugate.１）
Toxicities of phenobarbital can be dose-dependent or
dose-independent. Most of the dose-dependent
symptoms of toxicity are neurological symptoms, suchas sedation, nystagmus, ataxia, and coma. Reflex
excitation; coagulopathy; abnormal hepatic function;skin rash; osteomalacia; and neck, shoulder and arm
syndrome are symptoms of toxicity that are considered
to be dose-independent. Farewell et al. recently
reported that intellectual disability is observed inchildren on long-term phenobarbital therapy.2)
Monitoring the blood level of phenobarbital is usefulfor adjusting the dosage so that the optimum clinicalresponse can be obtained without symptoms oftoxicity.3),4)

Nanopia TDM Phenobarbital
General Precautions
1. 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.
7. PB Antibody Solution 1 contains human-derived
components that have been shown to be negative for
HBs antigens, HIV antibodies (AIDS virus
antibodies), and HCV antibodies. However, thesereagents (as well as the samples) should beconsidered potentially infectious and handled withgreat care.

Assay Principle
1. Assay Principle
When a certain amount of anti-phenobarbital
antibody is added and reacted with a sample,
consumption of the antibody depends on its content
in the sample. When phenobarbital-coated latex isadded, residual anti-phenobarbital antibody reacts
with the latex and forms aggregates.
Since the extent of aggregation depends on thephenobarbital concentration in the sample, thephenobarbital concentration can be determined bymeasuring aggregation as the change ofabsorbance.
Sample (phenobarbital) + Anti-phenobarbital
antibody ─────▶ Antigen-antibody reaction

Description (Kit Components)
Component: Ingredients
PB Antibody Solution 1:
Anti-phenobarbital mousemonoclonal antibody
PB Latex Reagent 2:
Phenobarbital-coated latex

Unreacted
anti-phenobarbital
antibody+
Phenobarbital-coated latex───▶ Aggregation by antigen-antibody reaction
2. Features
1) Because a highly specific monoclonal antibody
is used, this product shows excellent sensitivity
and accuracy.
2) Liquid reagents, ready-to-use.
3) Applicable to various automated analyzers.

Intended Use ＊＊Measurement of phenobarbital in serum or plasma

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 samples
If serum or plasma samples cannot be measured
on the day of separation, store them as follows.
Avoid repetition of freezing and thawing.
2–8C: for tests within 7 days
≤ -20C: for tests within 3 months
Bring samples to room temperature (15–30C) before use.
3) Caution must be exercised, because aseparating agent, etc. in the blood collection
tube may affect assay values.5)

Since Hauptmann introduced phenobarbital into
clinical practice in 1912, this drug has been widelyused as an anticonvulsant for the treatment of epilepsy,
especially focal seizures, sensory seizures, and grandmal epilepsy. The maximum blood level ofphenobarbital is attained at 12−18 hours after oral
administration at a dose of 2−3 mg/kg. The proteinbinding rate of phenobarbital is relatively low, andapproximately 40−50% of this drug is bound to plasma
protein in the bloodstream. Free phenobarbital notbound to plasma protein exhibits a pharmacologicaleffect. Phenobarbital is mostly absorbed from thestomach and partially absorbed from the intestine, andis metabolized in the liver. With is hydroxylation of thebenzene ring, it is metabolized to phydroxyphenobarbital and then excreted in the urine

14) Sampling should be performed after removing
insoluble matter from the sample.
2. Interfering substances
1) Assay results are not affected by free bilirubin(up to 20 mg/dL), conjugated bilirubin (up to 20mg/dL), hemoglobin (up to 500 mg/dL),ascorbic acid (up to 50 mg/dL), formazine
turbidity (up to 2500 FTU), or rheumatoid
factors (up to 450 U/mL).
2) Because mouse antibody is used in the assay,
artifactual elevation of results may occur if thesample contains human anti-mouse antibody. In
this case, perform re-measurement by anothermethod.
3) Cross-reactivity
The following table summarizes drugs related
to phenobarbital and cross-reactivity between
phenobarbital and other drugs.

Substance
1,3-Dimethylbarbituricacid
2-Phenyl-2ethylmalonamide5-(p-Hydroxyphenyl)5-phenylhydantoin
Amitriptyline
Aprobarbital
Barbital
Butabarbital
Carbamazepine
Carbamazepine-10,11epoxide
Clorazepate
Chlorpromazine
Diazepam
Ethosuximide
Ethotoin
Glutethimide
Imipramine
Mephenytoin
Methsucimide
Phenytoin
p-Hydroxyphenobarbital
Primidone
Promethazine
Secobarbital
Valproic acid

Concentration
tested(µg/mL)
1000

Crossreactivity (%)

1000

0.24

1000

0.17

1000
1000
2000
1000
1000
1000

0.09
0.11
0.04
0.21
0.28
0.26

2000
2500
1000
1000
1000
1000
4000
3000
5000
400
2000
1000
1500
2000
7000

0.08
0.10
0.13
0.16
0.17
0.14
0.07
0.100.05
0.55
0.02
0.33
0.12
0.15
0.00

use.
Reagent (2): PB Latex Reagent 2 is ready to use.
Before using this product, gently invert the PB
Latex Reagent 2 bottle to mix it thoroughly, andcheck that there are no bubbles.
2. Assay Procedure
This product is compatible with various types ofautomated analyzer. An example of the assay
procedure is indicated below.
37C Reagent 37CSample Reagent
Measurement
+2 μL
(2)
(1)
270
53 (Absorbance I※)
180 μL sec 50 μL sec
37CMeasurement
265 (Absorbance II※)sec
Calculation ofconcentration※Absorbance I and II: Absorbance at 700 nm

Calibration material: TDM Calibrator for Nanopia(manufacture's assigned value)

0.19

＊＊

Assessment of Assay Results ＊1. Reference standard range
The effective concentration of phenobarbital has
been reported to be 10−40 µg/mL.6) However,there is individual variation of drug metabolism, so
higher blood levels beyond this range may berequired in some patients. Therefore, interpretationof assay results should also be based on thepatient's clinical findings and other examination
results.
It is recommended that the blood concentration ofphenobarbital should be measured in a sufficientnumber of samples for statistical analysis and thatits clinical effective concentration should bedetermined by each medical institution.
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) The change of absorbance per minute with thestandard solution (0 µg/mL) is 0.110−0.200.
2) The ratio between the change of absorbance perminute with the standard solution (0 µg/mL)
and that with another standard solution (5µg/mL) is 60−80%.
2. Accuracy: 80–120 % of the expected assay value
3. Within-run Reproducibility:
Coefficient of variation ≤ 10 %(Test methods used for 1.–3. are in-house methods.)
4. Measurement Range7): (On Hitachi 7170S
automated analyzer)
0.8–80 μg/mL
5. Correlation7)
1) Serum
N=60
r=0.992
y=0.95x+0.21
Control method: Approved in vitro diagnostic(enzyme immunoassay)
2) Plasma
N=103 r=0.992
y=0.96x+0.88
Control method: Approved in vitro diagnostic

3. Others
1) Always use TDM Calibrator for Nanopiafor calibration.
2) Precautions for assay range
If the concentration of a target substance in thesample exceeds the measurement range, dilutethe sample with a separately sold diluent(manufactured by SEKISUI MEDICAL CO.,LTD.), and perform re-measurement.
Dosage/Administration (Assay Procedure) ＊1. Preparation of reagents
Reagent (1): PB Antibody Solution 1 is ready to2

(enzyme immunoassay)
6. Standard Material
Phenobarbital (U.S. Pharmacopoeia)

Name
Package
PB Antibody
1 × 36 mL
Solution 1
Nanopia TDM
Phenobarbital
PB Latex
1 × 10 mL
Reagent 2
Constituent reagents are available in otherconfigurations. For further details please contact
SEKISUI MEDICAL CO., LTD.

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) Sodium azide is added as an antiseptic agent in
the PB Antibody Solution 1 and PB Latex
Reagent 2. Therefore, if the reagent comes in
accidentally contact with eyes, mouth or skin,rinse immediately with ample water as first aid,
and consult the doctor if required.
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 mix materials from different kit lotnumbers.
5) 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 PB Antibody Solution 1 and PB
Latex Reagent 2. It can react with lead or copper
pipes to produce the highly explosive metal azide.
Therefore, the reagent should be flushed withlarge amounts of water during disposal.
4. Other precautions
Do not use the containers for other purposes.

＊＊References ＊1) Buchthal F., Svensmark O.：Serum concentrationsof diphenylhydantoin （ phenytoin ） andphenobarbital and their relation to therapeutic andtoxic effects, Psychiat. Neurol. Neurochir., 74 ：117−135, 1971.
2) Farwell J. R., Lee Y. J., Hirtz D. G., Sulzbacher S.
I., Ellenberg J. H., Nelson K. B.：Phenobarbital for
febril seizures−effects on intelligence and onseizure recurrence, N. Engl. J. Med., 322：363−369,1990.
3) Kutt He., Penry K.：Usefulness of blood levels ofantiepileptic drugs, Arch. Neurol., 3 ： 283−288,1974.
4) Waddell W. J., Butler T. C.：The distribution andexcretion of phenobarbital, J. Clin. Invest., 36 ：1217−1226, 1957.
5) Sawada T. et al.: J Med Pharm Sci, 51(1), 131−141,2004.
6) The Japanese Society of Therapeutic Drug
Monitoring. [Guideline for therapeutic drug
monitoring (TDM) for antiepileptic drug 2018].
Tokyo: Kanehara Shuppan; 2018. pp. 10-11, pp.
40-48.
7) 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

Storage and Shelf Life ＊1. Storage temperature: 2–8C
2. Shelf life: 2 years from the date of manufacture(The expiration date is printed on the outerpackage.)

Packaging3