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Procedure:
Phenobarbital
OSR4D229
This procedure is valid for the following chemistry analyzers:

AU400/AU400e

AU640/AU640e

AU480

AU680

AU600

AU2700

AU5400

AU5800
Prepared By
Date Adopted
Supersedes Procedure #
Review Date
Revision Date
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CLSIOSR4D229.02
Page 1 of 14
Procedure:
Phenobarbital
OSR4D229
PRINCIPLE:
Phenobarbital is an anticonvulsant medication used in the treatment of all
types of seizures except absence seizures (petit mal).
Monitoring serum phenobarbital concentrations, along with careful clinical
assessment, is the most effective means of improving seizure control,
reducing the risk of toxicity, and minimizing the need for additional
anticonvulsant medication for the following reasons:1-3

Serum phenobarbital concentrations correlate better with concentration in
the brain than does dosage once steady state is reached.

Patients taking the same dosage of phenobarbital show considerable
variation in serum phenobarbital concentrations because of individual
differences in absorption, metabolism, disease states, and compliance.

Serum level monitoring helps physicians individualize dosage regimens.
Methods historically used to monitor serum phenobarbital concentrations
include gas-liquid chromatography, high-performance liquid chromatography
(HPLC), radioimmunoassay, and immunoassay.1,2
INTENDED USE:
The Emit® 2000 Phenobarbital Assay is intended for use in the quantitative
analysis of phenobarbital in human serum or plasma. The Emit® 2000 Assay
is designed for use on multiple Beckman Coulter AU analyzers.
METHODOLOGY:
The Emit® 2000 Phenobarbital Assay is a homogeneous enzyme
immunoassay technique used for the analysis of specific compounds in
biological fluids.4,5 The assay is based on competition between drug in the
sample and drug labeled with the enzyme glucose-6-phosphate
dehydrogenase (G6PDH) for antibody binding sites. Enzyme activity
decreases upon binding to the antibody, so the drug concentration in the
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CLSIOSR4D229.02
Page 2 of 14
Procedure:
Phenobarbital
OSR4D229
sample can be measured in terms of enzyme activity. Active enzyme converts
nicotinamide adenine dinucleotide (NAD) to NADH, resulting in an
absorbance change that is measured spectrophotometrically. Endogenous
serum G6PDH does not interfere because the coenzyme functions only with
the bacterial (Leuconostoc mesenteroides) enzyme employed in the assay.
SPECIMEN:
PATIENT / SAMPLE PREPARATION:
No special preparation for the patient is required. The patient’s clinical
condition and dosage regimen may influence the sample collection time.
Pharmacokinetic factors influence the correct time of sample collection
after the last drug dose. These factors include dosage form, mode of
administration, concomitant drug therapy, and biological variations
affecting drug disposition.1-3
SAMPLE COLLECTION TIME:
Measure the steady-state serum concentration representing the trough
level just before the next scheduled dose.1
Additional instructions for preparation as designated by this laboratory:
TYPE:
Serum or plasma is the recommended specimen. Whole blood cannot be
used. The anticoagulants heparin, citrate, oxalate, and EDTA have been
tested and may be used with this assay.
Some sample dilution may occur when samples are collected in tubes
containing citrate anticoagulant. The amount of dilution and the possible
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All printed copies are considered to be copies of the electronic original.
CLSIOSR4D229.02
Page 3 of 14
Procedure:
Phenobarbital
OSR4D229
need to correct for it should be considered when interpreting assay results
for these samples.
Additional type conditions as designated by this laboratory:
HANDLING CONDITIONS:
Store the serum or plasma refrigerated at 2-8°C. For transporting,
maintain the sample temperature at 2-8°C. Samples can be stored
refrigerated at 2-8°C for up to one month or stored frozen (-20°C) for up
to 3 months.6
Samples that contain particulate matter, fibrous material, or gel-like
masses; appear unusual; or are frozen require preparation. Use the
following instructions to prepare such samples:
1. If sample is frozen, thaw at a room temperature of 15-25°C.
2. Vigorously mix all samples: Vortex for at least 30 seconds.
3. Centrifuge sample at > 2000 rpm for 15 minutes.
4. Collect a specimen from the middle portion of the sample. Avoid
collecting lipids from the top portion or particulate matter from the
bottom portion.
Human serum or plasma samples should be handled and disposed of as if
they were potentially infectious.
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4D229.02
Page 4 of 14
Procedure:
Phenobarbital
OSR4D229
Additional handling conditions as designated by this laboratory:
EQUIPMENT AND MATERIALS:
EQUIPMENT:
Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e,
AU680, AU2700, AU5400 and AU5800 analyzers.
MATERIALS:
Emit 2000 Phenobarbital Assay
Antibody/Substrate Reagent 1 — mouse monoclonal antibodies
reactive to phenobarbital, glucose-6-phosphate, nicotinamide adenine
dinucleotide, preservatives, including 0.1% sodium azide and
stabilizers.
Enzyme Reagent 2 — phenobarbital labeled with glucose-6-phosphate
dehydrogenase, Tris buffer, preservatives, including 0.1% sodium azide
and stabilizers.
Reagent storage location in this laboratory:
Test tubes 12 -16 mm in diameter or sample cups
(Cat No. AU1063).
Storage location of test tubes or sample cups in this laboratory:
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CLSIOSR4D229.02
Page 5 of 14
Procedure:
Phenobarbital
OSR4D229
Emit® 2000 Phenobarbital Calibrators
(Cat No. 4D109)
The Emit 2000 Phenobarbital Calibrators contain the following stated
phenobarbital concentrations: 0 g/mL, 5.0 g/mL, 10 g/mL, 20
g/mL, 40 g/mL, 80 g/mL. The calibrator kit is sold separately.
Storage location of the calibrator in this laboratory:
Preparation
The Emit 2000 Phenobarbital Assay reagents and calibrators are
packaged in a ready to use liquid form and may be used directly from
the refrigerator. Close the calibrator vials when not in use. Caps must
always be replaced on the original containers.
Note: Reagents 1 and 2 are provided as a matched set. They should
not be interchanged with components of kits with different lot
numbers.
Precautions:
1. The Emit® 2000 Phenobarbital Assay and Calibrators are for in
vitro diagnostic use.
2. Reagent 1 contains non-sterile mouse monoclonal antibodies.
3. Do not use the reagent kit or calibrators after the expiration date.
4. Assay components contain sodium azide, which may react with lead
and copper plumbing to form highly explosive metal azides. If
waste is discarded down the drain, flush it with a large volume of
water to prevent azide buildup. Dispose of properly according to
local regulations.
5. The reagent kit and calibrators contain streptomycin sulfate.
Please dispose of appropriately.
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4D229.02
Page 6 of 14
Procedure:
Phenobarbital
OSR4D229
Storage Requirements:
Any reagents not loaded in the reagent refrigerator on the analyzer or
any calibrators not in use should be stored at 2-8°C (36-46°F), upright,
and with caps tightly closed. Do not freeze reagents or calibrators or
expose them to temperatures above 32°C.
Unopened reagents and calibrators are stable until the expiration date
printed on the label if stored as directed. Refer to Assay Methodology
Sheets for additional on-board stability information.
Improper storage of reagents or calibrators can affect assay
performance. Stability depends on handling reagents or calibrators as
directed.
Additional storage requirements as designated by this laboratory:
Indications of Deterioration:
Discoloration (especially yellowing) of the reagents or calibrators,
visible signs of microbial growth, turbidity, or precipitation in reagent
or calibrator may indicate degradation and warrant discontinuance of
use.
PERFORMANCE PARAMETERS:
The following performance characteristics represent total system
performance and should not be interpreted to refer only to reagents. Studies
were performed on the Beckman Coulter AU analyzer series. Results may
vary due to analyzer-to-analyzer differences.
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4D229.02
Page 7 of 14
Procedure:
Phenobarbital
OSR4D229
PRECISION
Within run precision was calculated by running twenty replicates of each
level of a tri-level control. Total precision was calculated according to
Clinical and Laboratory Standards Institute (CLSI EP5-A) using data
collected from controls run in duplicate twice daily over twenty days
(N=80).
Within-Run Precision
Total Precision
Level 1
Level 2
Level 3
Level 1
Level 2
Level 3
Mean
(µg/mL)
9.8
28.0
53.0
10.6
28.9
48.6
CV %
2.2
2.5
3.2
4.0
4.7
5.5
COMPARISON
Samples from patients were analyzed using the Emit® 2000 Phenobarbital
Assay on the Roche Diagnostics (RD)/Hitachi 704 analyzer and the
AU600. The comparative analysis is shown in the following table.
Slope
0.922
Intercept (g/mL)
0.324
Mean (g/mL)
RD/Hitachi 704
AU600
Correlation Coefficient
Number of Samples
33.23
30.98
0.992
82
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4D229.02
Page 8 of 14
Procedure:
Phenobarbital
OSR4D229
CALIBRATION:
Perform a multi-point calibration (5AB) using a water blank (blue rack) and
the Emit® 2000 Phenobarbital Calibrators: 5.0, 10, 20, 40, 80. Calibration
parameters are set to prepare the calibration curve. Refer to analyzer User’s
Guide or Analyzer Specific Protocol sheets for analyzer settings.
CALIBRATION STABILITY
Studies have shown the median calibration stability to be at least 14 days.
Recalibrate as indicated by control results or with a new lot of reagent.
Calibration stability may vary from laboratory to laboratory depending on
the following: handling of reagents, maintenance of analyzer, adherence to
operating procedures, establishment of control limits, and verification of
calibration.
Note: When using a new set of reagents with the same lot number,
validate the system by assaying controls.
QUALITY CONTROL:
During operation of the Beckman Coulter AU analyzer at least two levels of
control material should be tested a minimum of once a day. Controls should
be performed after calibration, with each new set or lot of reagent, and after
specific maintenance or troubleshooting steps described in the appropriate
User’s Guide. Quality control testing should be performed in accordance with
regulatory requirements and individual laboratory’s standard procedures. If
more frequent verification of test results is required by the operating
procedures within your laboratory, those requirements should be met.
PARAMETERS
A complete list of test parameters and operating procedures can be found in
the appropriate User’s Guide and at www.beckmancoulter.com.
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4D229.02
Page 9 of 14
Procedure:
Phenobarbital
OSR4D229
CALCULATIONS:
Results are calculated automatically by the analyzer. No additional
manipulation of data is required.
This assay uses Math Model No. 1.
To convert from g/mL to mol/L phenobarbital, multiply by 4.31.
REPORTING RESULTS:
REFERENCE RANGES:
Most patients achieve a satisfactory therapeutic response in the serum
concentration range of 10-40 g/mL (43-172 mol/L).7,8 Peak
concentrations above 60 g/mL (259 mol/L) are often associated with
toxicity.1,9
For effective treatment, some patients may require serum levels outside
this range. Therefore, the expected range is provided only as a guide, and
individual patient results should be interpreted in light of other clinical
signs and symptoms.
Expected reference ranges in this laboratory:
PROCEDURES FOR ABNORMAL RESULTS
The laboratory must define procedures to be used in reporting high
concentration (toxic) results to the patient’s physician.
Abnormal results are flagged by the listed analyzers according to the
normal values entered by the user into the analyzer parameters.
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4D229.02
Page 10 of 14
Procedure:
Phenobarbital
OSR4D229
REPORTING FORMAT:
Results are automatically printed for each sample in g/mL at 37°C.
Interpretation of Results
The factors that can influence the relationship between the
phenobarbital serum or plasma concentrations and clinical response
include the type and severity of seizures, age, general state of health,
and use of other drugs.
The concentration of phenobarbital in serum or plasma depends on the
time of the last drug dose; mode of administration; concomitant drug
therapy; sample condition; time of sample collection; and individual
variations in absorption, distribution, biotransformation, and
excretion. These parameters must be considered when interpreting
results.1,2
Additional reporting information as designated by this laboratory:
LIMITATIONS:
The Emit® 2000 Phenobarbital Assay accurately quantitates phenobarbital
concentrations in human serum or plasma containing 5.0-80 g/mL (22-345 
mol/L) phenobarbital.
To estimate phenobarbital concentrations above the assay range, patient
samples containing more than 80 g/mL (345 mol/L) phenobarbital may be
diluted with one or two parts distilled or deionized water or Emit® 2000
Phenobarbital Calibrator 0. After diluting the sample, repeat the entire
assay sequence and multiply the results by the dilution factor.
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4D229.02
Page 11 of 14
Procedure:
Phenobarbital
OSR4D229
Adulteration of reagents, use of analyzer without appropriate capabilities, or
other failures to follow instructions as set forth in this protocol or the package
insert can affect performance characteristics and stated or implied claims.
INTERFERING SUBSTANCES
No clinically significant interference has been found in samples to which
800 mg/dL hemoglobin, 750 mg/dL triglycerides, or 30 mg/dL bilirubin
were added to simulate hemolytic, lipemic or icteric samples.
SENSITIVITY
The sensitivity level of the Emit® 2000 Phenobarbital Assay is 0.6 g/mL.
This level represents the lowest measurable concentration of
phenobarbital that can be distinguished from 0 g/mL with a confidence
level of 95%.
SPECIFICITY
The Emit® 2000 Phenobarbital Assay measures the total (protein-bound
plus unbound) phenobarbital concentration in serum or plasma.
Compounds whose chemical structure or concurrent therapeutic use
would suggest possible cross-reactivity have been tested.
The compounds listed in the following table do not interfere with the
Emit® 2000 Phenobarbital Assay when tested in the presence of 20 g/mL
phenobarbital. Levels tested were at or above maximum physiological or
pharmacological concentrations.
© Beckman Coulter, Inc. March 2012
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CLSIOSR4D229.02
Page 12 of 14
Procedure:
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Compound
Concentration
Tested
(g/mL)
Compound
Concentration
Tested
(g/mL)
Amitriptyline
25
5-(p-Hydroxyphenyl)-5phenylhydantoin
Amobarbital
30
Imipramine
Butabarbital
100
Mephenytoin
200
Carbamazepine
500
Mephobarbital
100
Carbamazepine-10,11epoxide
500
Methsuximide
150
Chlordiazepoxide
60
Nortriptyline
10
Chlorpromazine
8
Pentobarbital
100
Chlorazepate
500
Phensuximide
500
Diazepam
60
2-Phenyl-2-ethylmalondiamide (PEMA)
500
Ethosuximide
500
Phenytoin
200
Ethotoin
200
Primidone
200
5-Ethyl-5-phenyhydantoin
200
Promethazine
10
Glutethimide
200
Secobarbital
25
Heptabarbital
200
Valproic Acid
1000
100
5
REFERENCES:
1. Saunders GH and Penry JK. Phenobarbital/Primidone: Therapeutic Use
and Serum Concentration Monitoring. In Taylor WJ and Finn AL eds.
Individualizing Drug Therapy: Practical Applications of Drug Monitoring.
New York: Gross, Townsend, Frank, Inc. 1981, vol 2, pp 49-62.
2. Levy RH, Wilensky AJ, Friel PN: Other Antiepileptic Drugs. In Evans
WE, Schentag JJ, Jusko WJ eds. Applied Pharmacokinetics: Principles of
© Beckman Coulter, Inc. March 2012
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CLSIOSR4D229.02
Page 13 of 14
Procedure:
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Therapeutic Drug Monitoring. Washington: Applied Therapeutics, Inc.
1987, pp 540-569.
3. Morrow JI, Richens A: Disposition of anticonvulsants in childhood. Clin
Pharmacokinet 1989; 17 (Suppl 1):89-104.
4. Pincus MR, Abraham NZ Jr: Toxicology and Therapeutic Drug
Monitoring. In Henry JB, ed. Clinical Diagnosis and Management by
Laboratory Methods, Ed 18. Philadelphia: WB Saunders Co. 1991, pp 349–
384.
5. Dalton P: Syva Emit 2000 Phenobarbital Assay, San Jose, CA, Syva
Co.
6. Kumps AH: Therapeutic drug monitoring: A comprehensive and critical
review of analytical methods for anticonvulsive drugs. J Neurol 1982;
228:1-16.
7. Larkin JG, Herrick AL, McGuire GM, et al: Antiepileptic drug monitoring
at the epilepsy clinic: A prospective evaluation, Epilepsia 1991;32(1):8995,
8. Brodie MJ, Feely J. Practical clinical pharmacology: Therapeutic drug
monitoring and clinical trials. BMJ. 1988; 269(1):1110-1114.
9. Rall TW, Schleifer LS. Drugs Effective in the Therapy of the Epilepsies. In
Gilman AG, Rall TW, Nies AS, et al. eds. Goodman and Gilman's The
Pharmacological Basis of Therapeutics Ed 8. New York: Pergamon Press,
1990, pp 436-462.
© Beckman Coulter, Inc. March 2012
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CLSIOSR4D229.02
Page 14 of 14