Download Prepared By - Beckman Coulter

Procedure:
N-Acetylprocainamide
OSR4N229
This procedure is valid for the following chemistry analyzers:

AU400/AU400e

AU640/AU640e

AU480

AU680

AU600

AU2700/AU5400
Prepared By
Date Adopted
Supersedes Procedure #
Review Date
Revision Date
Signature
# of
Distributed to
Copies
# of
Distributed to
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Copies
Page 1 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
PRINCIPLE:
N-Acetylprocainamide (NAPA) is the major metabolite of the antiarrhythmic
drug, procainamide. Measurements of procainamide in serum may not
accurately reflect the drug’s complete pharmacologic activity in the body.
Monitoring NAPA levels along with the procainamide is recommended during
procainamide therapy. NAPA is considered comparable in activity to its
parent compound; however, NAPA levels will vary widely. Procainamide is
metabolized to NAPA by N-acetyltransferase, an enzyme that is genetically
determined. Serum levels of N-acetylprocainamide increase in patients on
chronic procainamide therapy, particularly in those with renal impairment.
The average serum concentration ratio of N-acetylprocainamide to
procainamide is 0.8 to 1.2, depending on a genetically determined tendency to
acetylate procainamide rapidly or slowly. Because the ratio varies from
patient to patient, measuring serum NAPA and procainamide together helps
to achieve an optimum antiarrhythmic effect and reduce the risk of toxicity.
Methods historically used to monitor serum N-acetylprocainamide and
procainamide concentrations include chromatographic assay and
immunoassay.1-3
INTENDED USE:
The Emit 2000® N-Acetylprocainamide Assay is intended for use in the
quantitative analysis of N-acetylprocainamide in human serum or plasma.
The Emit® 2000 assays are designed for use on multiple Beckman Coulter AU
analyzers.
METHODOLOGY:
The Emit® 2000 N-Acetylprocainamide 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
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 2 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
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 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.1,2
SAMPLE COLLECTION TIME:
Collect the steady-state serum concentration representing the trough level
just before the next scheduled dose.
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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 3 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
Some sample dilution may occur when samples are collected in tubes
containing citrate anticoagulant. The amount of dilution and the possible
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 7 days or stored frozen (-20°C) for up to
one month.
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. 2010
All printed copies are considered to be copies of the electronic original.
Page 4 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
Additional handling conditions as designated by this laboratory:
EQUIPMENT AND MATERIALS:
EQUIPMENT:
Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e,
AU680, AU2700, and AU5400 analyzers.
MATERIALS:
Emit 2000 N-Acetylprocainamide Assay
Antibody/Substrate Reagent 1 — mouse monoclonal antibodies
reactive to N-acetylprocainamide, glucose-6-phosphate, nicotinamide
adenine dinucleotide, preservatives, and stabilizers
Enzyme Reagent 2 — N-acetylprocainamide labeled with glucose-6phosphate dehydrogenase, Tris buffer, preservatives, and stabilizers
Reagent storage location in this laboratory:
Test tubes 12 -16 mm in diameter or sample cups
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
(Cat No. AU1063).
Page 5 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
Storage location of test tubes or sample cups in this laboratory:
Emit® 2000 N-Acetylprocainamide Calibrators
(Cat No. 4N109)
The Emit 2000 N-Acetylprocainamide Calibrators contain the
following stated N-acetylprocainamide concentrations: 0 g/mL, 1.0
g/mL, 2.0 g/mL, 4.0 g/mL, 8.0 g/mL, 16 g/mL. The calibrator kit
is sold separately.
Preparation
The Emit 2000 N-Acetylprocainamide 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 N-Acetylprocainamide Assay and Calibrators are
for in vitro diagnostic use.
2. Reagent 1 contains non-sterile mouse monoclonal antibodies.
3. Do not use the reagents or calibrators after the expiration date.
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,
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 6 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
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.
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
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 7 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
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)
2.37
5.65
10.60
2.12
5.13
9.85
CV %
2.8
1.9
2.7
4.3
4.2
4.5
COMPARISON
Samples from patients were analyzed using the Emit® 2000 NAcetylprocainamide Assay on the Roche Diagnostics (RD)/Hitachi 704
analyzer and the AU600. The comparative analysis is given in the
following table.
Slope
0.91
Intercept (g/mL)
0.06
Mean (g/mL)
RD/Hitachi 704
AU600
Correlation Coefficient
Number of Samples
5.79
5.34
0.99
56
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 8 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
CALIBRATION:
Perform a multi-point calibration (5AB) using a water blank (blue rack) and
the Emit® 2000 N-Acetylprocainamide Calibrators: 1.0, 2.0, 4.0, 8.0, 16.0.
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 multi-level controls
should be tested in every run or 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. 2010
All printed copies are considered to be copies of the electronic original.
Page 9 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
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 N-acetylprocainamide, multiply by 3.61.
REPORTING RESULTS:
REFERENCE RANGES:
Since N-acetylprocainamide is a metabolite of procainamide, no
therapeutic range has been established exclusively for it. However, most
patients achieve a satisfactory therapeutic response when the sum of
procainamide and N-acetylprocainamide concentrations in serum is 10-30
g/mL.1,2
Because of patient-to-patient differences in metabolic activity, renal
function, and type and severity of cardiac arrhythmia, 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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 10 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
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 for each sample in g/mL at 37°C.
Interpretation of Results
The factors that can influence the relationship between the Nacetylprocainamide serum or plasma concentrations and clinical
response include the renal and circulatory function, rate of acetylation,
and the severity and type of cardiac arrhythmia, general state of
health, and use of other drugs.
The concentration of N-acetylprocainamide 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 N-Acetylprocainamide Assay accurately quantitates Nacetylprocainamide concentrations in human serum or plasma containing
1.0-16 g/mL (3.6-58 mol/L) N-acetylprocainamide.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 11 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
To estimate N-acetylprocainamide concentrations above the assay range,
patient samples containing more than 16 g/mL (58 mol/L) Nacetylprocainamide may be diluted with one or two parts distilled or
deionized water or Emit® 2000 N-Acetylprocainamide Calibrator 0. After
diluting the sample, repeat the entire assay sequence and multiply the
results by the dilution factor.
Adulteration of reagents, use of analyzer without appropriate capabilities, or
other failure to follow instructions as set forth in this protocol can affect
performance characteristics and stated or implied claims.
INTERFERING SUBSTANCES
No clinically significant interference has been found in sample to which
800 mg/dL hemoglobin, 1000 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 N-Acetylprocainamide Assay is
0.25 g/mL. This level represents the lowest measurable concentration of
N-acetylprocainamide that can be distinguished from 0 g/mL with a
confidence level of 95%.
SPECIFICITY
The Emit 2000 N-Acetylprocainamide Assay measures the total (proteinbound plus unbound) N-acetylprocainamide concentration in serum or
plasma. Compounds whose chemical structure or concurrent therapeutic
use would suggest possible cross-reactivity have been tested.
The compounds listed below do not interfere with the Emit 2000
N-Acetylprocainamide Assay when tested in the presence of 4.0 g/mL
N-acetylprocainamide. Levels tested were at or above maximum
physiological or pharmacological concentrations.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 12 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
Compound
Acetaminophen
Desethyl-Nacetylprocainamide
(DENAPA)
Digoxin
Disopyramide
Ephedrine
Furosemide
Glycinexylidide (GX)
Hydrochlorothiazide
Isoproterenol
Lidocaine
Monoethylglycinexylidide
(MEGX)
N-(2-diethylaminoethyl)
isonicotinamide
p-Acetamidobenzoic acid
p-Aminobenzoic acid (PABA)
Phenytoin
Procainamide
Procaine
Propranolol
Quinidine
Tocainide
Concentration
Tested
(g/mL)
100
100
0.1
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
REFERENCES:
1. Lima JJ, Lewis RP. Procainamide: Therapeutic Use and Serum
Concentration Monitoring, in Taylor WJ, Finn AL (eds.) Individualizing
Drug Therapy: Practical Applications of Drug Monitoring. New York:
Gross, Townsend, Frank, Inc. 1981, vol 3, pp 69-88.
2. Coyle JD, Lima JJ. Procainamide, in Evans WE, Schentag JJ, Jusko WJ
(eds.) Applied Pharmacokinetics: Principles of Therapeutic Drug
Monitoring. Washington: Applied Therapeutics, Inc. 1986, pp 682-711.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 13 of 14
Rev #1, Dec 31, 10
Procedure:
N-Acetylprocainamide
OSR4N229
3. Bauer LA, Black D, Gensler A et al. Influence of age, renal function, and
heart failure on procainamide clearance and N-acetylprocainamide serum
concentrations. Int J Clin Pharmacol Ther Tox. 1989; 27(5):213-216.
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. Mulberg E, Dalton P, Hefner A. Syva Emit 2000 N-Acetylprocainamide
Assay. Clin Chem. 1992; 38(6):336. Abstract.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 14 of 14
Rev #1, Dec 31, 10