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Procedure: N-Acetylprocainamide OSR4N229 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 Copies # of Distributed to © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. Copies CLSIOSR4N229.02 Page 1 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 2 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 3 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 4 of 14 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, AU5400 and AU5800 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. March 2012 All printed copies are considered to be copies of the electronic original. (Cat No. AU1063). CLSIOSR4N229.02 Page 5 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 6 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 7 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 8 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 9 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 10 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 11 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 12 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 13 of 14 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. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4N229.02 Page 14 of 14