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Procedure:
Valproic Acid
OSR4G229
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
Signature
# of
Distributed to
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All printed copies are considered to be copies of the electronic original.
Copies
CLSIOSR4G229.02
Page 1 of 14
Procedure:
Valproic Acid
OSR4G229
PRINCIPLE:
Valproic Acid (also known as Depakene or Depakote) is an anti-convulsant or
anti-seizure medication chemically related to most other anti-convulsants.
Monitoring valproic acid concentrations in serum helps to individualize drug
therapy for safe and effective control of absence seizures, other generalized
seizures, and partial seizures. Serum valproic acid monitoring is useful for
assessing patient compliance, or to explain changes in seizure control or drug
toxicity.1
Achieving and maintaining therapeutic concentrations of serum valproic acid
concentrations is difficult due to marked inter- and intra-patient variability
in pharmacokinetics. The pharmacokinetics of valproic acid may be altered
by age, pregnancy, renal failure, liver dysfunction, other drugs, low albumin,
and other factors.1
Valproic acid has pharmacokinetic parameters that make it susceptible to
drug interactions. Valproic acid is extensively metabolized by the liver.
Other co-administered drugs, including other anti-epileptics, may induce of
inhibit the drug metabolizing enzymes of the liver. When these drugs are
added or removed from the therapeutic regimen of a patient, the clearance
and concentration of valproic acid may be altered, requiring dosage
adjustment.1
Adverse reactions associated with high concentrations of valproic acid are
central nervous system depression, tremor, thrombocytopenia, and increases
in liver function tests. These reactions may be minimized by dosage titration.
Very high concentrations of valproic acid may also increase the risk of
developing fatal hepatotoxicity, stupor, coma, or cerebral edema.1
The method historically used to monitor valproic acid are non-isotopic
immunoassay and gas chromatography.2
INTENDED USE:
The Emit 2000 Valproic Acid Assay is intended for in vitro diagnostic use in
the quantitative analysis of valproic acid in human serum or plasma. The
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 2 of 14
Procedure:
Valproic Acid
OSR4G229
Emit 2000 assays are designed for use on multiple Beckman Coulter AU
analyzers.
METHODOLOGY:
The Emit 2000 Valproic Acid assay is a homogeneous enzyme immunoassay
technique used for the quantitative analysis of valproic acid (free and proteinbound) in human serum or plasma. The assay is based on competition
between drug in the sample and drug labeled with the enzyme glucose-6phosphate dehydrogenase (G6PDH) for antibody binding sites. Enzyme
activity decreases upon binding to the antibody, so the drug concentration in
the sample can be measured in terms of enzyme activity. Active enzyme
converts oxidized nicotinamide adenine dinucleotide (NAD) to NADH,
resulting in an absorbance change that can be 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
SAMPLE COLLECTION TIME:
To obtain a serum valproic acid concentration that best represents the
peak tissue level, draw the sample 1 to 3 hours after a dose is given.
Collect a trough sample just before the next scheduled dose.1
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 3 of 14
Procedure:
Valproic Acid
OSR4G229
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/fluoride, oxalate, and EDTA
have been tested and may be used with this assay.
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 may be stored frozen
(-20°C) for up to one year.2
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 sample in a 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. It is recommended that human
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 4 of 14
Procedure:
Valproic Acid
OSR4G229
specimens be handled in accordance with the OSHA Standard on
Bloodborne Pathogens or other appropriate local practices.3,4
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 Valproic Acid Assay
Antibody/Substrate Reagent 1 — mouse monoclonal antibodies
reactive to valproic acid, glucose-6-phosphate, nicotinamide adenine
dinucleotide, preservatives and stabilizers.
Enzyme Reagent 2 — valproic acid 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:
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 5 of 14
Procedure:
Valproic Acid
OSR4G229
Emit 2000 Valproic Acid Calibrators
(Cat No. 4G109)
The Emit 2000 Valproic Acid Calibrators contain the following stated
valproic acid concentrations: 0 g/mL, 10 g/mL, 25 g/mL, 50 g/mL,
100 g/mL, 150 g/mL. The calibrator kit is sold separately.
Storage location of the calibrator in this laboratory:
Preparation
The Emit 2000 Valproic Acid 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 Valproic Acid reagents and calibrators are for in
vitro diagnostic use.
2. Reagent 1 contains non-sterile mouse monoclonal antibodies.
Reagent kit contains non-sterile bovine serum albumin.
3. Do not use the reagents or calibrators after the expiration date.
4. Assay components contain sodium azide that 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 in accordance
with local regulations.
5. Reagents and calibrators contain materials that may cause
sensitivity on contact with skin.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 6 of 14
Procedure:
Valproic Acid
OSR4G229
Storage Requirements:
Any reagents not loaded in the reagent refrigerator on the analyzer or
any calibrators not in use should be store 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 reagent 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. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 7 of 14
Procedure:
Valproic Acid
OSR4G229
PRECISION
Within run precision was calculated by running two replicates of each
level of a tri-level control twice a day for twenty days (N=80). Total
precision was calculated according to Clinical and Laboratory Standards
Institute (CLSI EP5-A) from these data as well.
Within-Run Precision
Total Precision
Level 1
Level 2
Level 3
Level 1
Level 2
Level 3
Mean
(µg/mL)
26.2
79.0
130.2
26.2
79.0
130.2
CV %
2.8
2.5
3.2
4.3
3.1
3.8
COMPARISON
Samples from patients were analyzed using the Emit® 2000 Valproic Acid
Assay on the SYVA -30R Biochemical System and the AU600 analyzer.
The comparative analysis is shown below.
Slope
1.01
Intercept (g/mL)
-0.83
Mean (g/mL)
SYVA-30R
AU600
Correlation Coefficient
Number of Samples
70.9
70.9
0.98
50
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 8 of 14
Procedure:
Valproic Acid
OSR4G229
CALIBRATION:
Perform a multi-point calibration (5AB) using a water blank (blue rack) and
the Emit® 2000 Valproic Acid Calibrators: 10, 25, 50, 100, 150. Calibration
parameters are set to prepare the calibration curve. Refer to analyzer User’s
Guide or Analyzer Specific Protocol sheets for specific 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, recalibration is not required. Validate the system by assaying controls.
QUALITY CONTROL:
During operation of the Beckman Coulter AU analyzer at least one level of
control material should be tested every 8 hours. Alternate the control levels
tested and ensure that a minimum of 2 controls is assayed in every 24 hour
period. Controls should be performed after calibration, with each new lot or
set of reagents, 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. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 9 of 14
Procedure:
Valproic Acid
OSR4G229
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 valproic acid, multiply by 6.93.
REPORTING RESULTS:
REFERENCE RANGES:
In most patients, valproic acid serum concentrations of 50 – 100 g/mL
(347 – 693 mol/L) effectively control generalized and partial seizures.
Seizure control may improve at levels greater than 100 g/mL (693 
mol/L), but toxicity may occur at levels of 100 - 150 g/mL (693 – 1040 
mol/L).5
For effective treatment, some patients may require serum levels outside
this range. Therefore, the expected ranges are 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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 10 of 14
Procedure:
Valproic Acid
OSR4G229
Abnormal results are flagged by the listed analyzers according to the
normal values entered by the user into the analyzer parameters.
REPORTING FORMAT:
Results are automatically printed out for each sample in g/mL at 37C.
Interpretation of Results
The factors that can influence the relationship between valproic acid
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 valproic acid in serum or plasma depends on the
time of the last drug dose; time of sample collection; disease states that
affect drug clearance; age; concomitant drug therapy; and individual
variations in absorption, distribution, and elimination. These
parameters must be considered when interpreting results.1
An increase of the biologically active free fraction of the drug, caused
by saturation of the protein binding sites or disease states that alter
protein binding, can influence the relationship between serum or
plasma valproic acid concentration and clinical response. Although the
total drug concentration is within the therapeutic range, the patient
may exhibit toxic symptoms.1
Additional reporting information as designated by this laboratory:
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 11 of 14
Procedure:
Valproic Acid
OSR4G229
LIMITATIONS:
The Emit® 2000 Valproic Acid Assay accurately quantitates valproic acid
concentrations in human serum or plasma containing 10 - 150 g/mL (69 1040 mol/L) valproic acid.
To estimate valproic acid concentrations above the assay range, patient
samples containing more than 150 g/mL (1040 mol/L) valproic acid may be
diluted with one or two parts distilled or deionized water or Emit® 2000
Valproic Acid Calibrator 0. After diluting the sample, repeat the entire assay
sequence and multiply the results by the dilution factor.
Adulteration of reagents, use of analyzers without appropriate capabilities, or
other failure to follow instructions as set forth in this labeling can affect
performance characteristics and stated or implied labeling claims.
INTERFERING SUBSTANCES
No clinically significant interference has been found in sample 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 Valproic Acid Assay is 3.98 g/mL.
This level represents the lowest measurable concentration of valproic acid
that can be distinguished from 0 g/mL with a confidence level of 95%.
SPECIFICITY
The Emit Valproic Acid Assay measures the total (protein-bound plus
unbound) valproic acid concentration in serum or plasma. Compounds
whose chemical structure or concurrent therapeutic use would suggest
possible cross-reactivity have been tested.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 12 of 14
Procedure:
Valproic Acid
OSR4G229
The compounds listed in the following table do not interfere with the
Emit 2000 Valproic Acid Assay when tested in the presence of 50 g/mL
valproic acid. Levels tested were at or above maximum physiological or
pharmacological concentrations.
Compound
Concentration Tested
(g/mL)
Carbamazepine
1000
Clonazepam
100
Diazepam
100
Ethosuximide
1000
2-N-Propyl-3-hydroxy-pentanoic acid
100
2-N-Propyl-4-hydroxy-pentanoic acid
100
2-N-Propyl-5-hydroxy-pentanoic acid
50
2-N-Propyl-3-oxo-pentanoic acid
100
Phenobarbital
750
Phenytoin
1000
Primidone
1000
2-Propyl glutaric acid
400
2-Propyl-2-pentenoic acid
20
2-Propyl-4-pentenoic acid
10
2-Propyl succinic acid
500
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 13 of 14
Procedure:
Valproic Acid
OSR4G229
REFERENCES:
1. Garnett WR. Antiepileptics. In: Schumacher GE, ed. Therapeutic Drug
Monitoring. Norwalk, CT: Appleton & Lange; 1995:345-362.
2. Tietz, NW. Clinical Guide to Laboratory Tests Ed 3. Philadelphia, PA:
Saunders Co; 1995:884-885.
3. Occupational exposure to bloodborne pathogens (29 CFR 1910.1030)
Federal Register. December 06, 1991; 56:64004; amended April 13, 1992;
57:12717; July 01, 1992; 57:29206; February 13, 1996; 61:5507.
4. World Health Organization. Laboratory Biosafety Manual, Ed 2. Geneva:
World Health Organization; 1993.
5. Jacobs DS, DeMott WR, Grady HJ, Horvat RT, Huestis DW, Kasten BL
Jr. Laboratory Test Handbook, Ed 4. Hudson, OH: Lexi-Comp Inc;
1996:577-578.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
CLSIOSR4G229.02
Page 14 of 14