4 Reagents
Reagents contain the following substances: Mouse monoclonal antibodies reactive to quinidine (1.35 µg/mL), glucose‑6‑phosphate (22 mM), nicotinamide adenine dinucleotide (18 mM), quinidine labeled with glucose‑6‑phosphate dehydrogenase (0.41 U/mL), 0.1% sodium azide, Tris buffer, Quinidine Assay preservatives, and stabilizers Precautions • For in vitro diagnostic use. August 2010 4Q052.2D_B • Contains nonsterile mouse monoclonal antibodies. • 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. • Do not use the kit after the expiration date. • This kit contains streptomycin sulfate. Please dispose of appropriately. • Turbid or yellow reagents may indicate contamination or degradation and must be discarded.
Preparation of Reagents Catalog Quantity/ The Emit® 2000 Quinidine Assay reagents are provided ready to use; no preparation is Number Product Description Volume necessary.
OSR4Q229 Emit® 2000 Quinidine Assay Storage of Assay Components OSR4Q518 R1 (Antibody/Substrate Reagent 1) 2 x 23 mL • Improper storage of reagents can affect assay performance. OSR4Q548 R2 (Enzyme Reagent 2) 2 x 13 mL • When not in use, store reagents upright at 2–8°C and with screw caps tightly closed. • Unopened reagents are stable until the expiration date printed on the label if stored upright at 4Q109UL Emit® 2000 Quinidine Calibrators* 1 x 5 mL†, 2–8°C. 5 x 2 mL • Do not freeze reagents or expose them to temperatures above 32°C. *Required for calibrating the Emit ® 2000 Quinidine Assay. Sold separately. †Additional negative calibrator is provided. 5 Specimen Collection and Preparation Note: Reagents and calibrators are shipped ready to use in liquid form. Note: Reagents 1 and 2 are provided as a matched set. They should not be interchanged with • Each assay requires serum or plasma. Whole blood cannot be used. The anticoagulants components of kits with different lot numbers. 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 The Emit® 2000 Quinidine Calibrators contain the following stated quinidine concentrations: anticoagulant. The amount of dilution and the possible need to correct for it should be considered when interpreting assay results for these samples. Calibrator 0 0.5 1 2 4 8 • Sample volume is instrument-dependent. Refer to the appropriate Application Sheet for Quinidine (µg/mL) 0 0.5 1.0 2.0 4.0 8.0 specific volumes. Quinidine (µmol/L) 0 1.5 3.1 6.2 12 25 • 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 1 month. 1 Intended Use • 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, The Emit® 2000 Quinidine Assay is a homogeneous enzyme immunoassay intended for use in and biological variations affecting drug disposition.1–3 the quantitative analysis of quinidine in human serum or plasma. These reagents are packaged • Measure the steady-state serum concentration representing the trough level just before the specifically for use on a variety of AU® Clinical Chemistry Systems. next scheduled dose. • Human serum or plasma samples should be handled and disposed of as if they were 2 Summary potentially infectious.
Monitoring serum quinidine concentrations, along with careful clinical assessment, is the most 6 Procedure effective means of achieving an optimum antiarrhythmic effect and reducing the risk of toxicity for the following reasons: Materials Provided • Serum quinidine concentrations correlate better with pharmacologic activity than does Emit® 2000 Quinidine Assay dosage.1,2 Reagent 1 • The quinidine dosage required to control cardiac arrhythmias varies substantially in different Reagent 2 patients and in the same patient at different times. These variations are due to differences Materials Required But Not Provided inabsorption and metabolism and to the influences of disease states and coadministered drugs.1–3 Emit® 2000 Quinidine Calibrators Multi-level commercial controls • Quinidine is safe and effective only in a narrow range of serum concentrations.1–3 • The symptoms of serious quinidine toxicity can mimic those of an ineffectively controlled Calibration cardiac disorder.1 Recalibrate whenever a new lot of reagents is used or as indicated by control results (see Quality Methods historically used to monitor serum quinidine concentrations include spectroscopic Control, below). If a new set of reagents with the same lot number is used, validate the system 1, 2, 4 assay, chromatographic assay, and immunoassay. by assaying controls.
Quality Control 3 Methodology • Validate the calibration by assaying multi-level (eg, low, medium, and high) controls in every run. Commercial controls are available for this purpose. Ensure that control results fall within The Emit® 2000 Quinidine Assay is a homogeneous enzyme immunoassay technique used for acceptable limits as defined by your own laboratory. Once the calibration is validated, run the analysis of specific compounds in biological fluids.4,5 The assay is based on competition samples. 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 • Refer to the instrument User’s Guide for appropriate instrument checks and maintenance to the antibody, so the drug concentration in the sample can be measured in terms of enzyme instructions. 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. Diluting High Concentration Samples Comparative Analysis To estimate quinidine concentrations above the assay range, patient samples containing more In this study, samples from patients were analyzed on the TDx analyzer and on the AU600 Clinical than 8.0 µg/mL (25 µmol/L) quinidine may be diluted with one or two parts distilled or deionized Chemistry System. A summary of the results is as follows: water or Emit® 2000 Quinidine Calibrator 0. After diluting the sample, repeat the entire assay sequence and multiply the results by the dilution factor. Some analyzers dilute and retest high Table 3 — Comparative Analysis Results concentration samples automatically. See the analyzer User’s Guide or appropriate Application Sheet for instructions. Slope 1.02 Intercept -0.06 Evaluation and Interpretation of Results Mean TDx 2.85 • This assay uses Math Model No. 1. AU600 2.86 • The factors that can influence the relationship between quinidine serum or plasma concentrations and clinical response include the type and severity of arrhythmia, liver Correlation Coefficient 0.98 1–3 function, kidney function, general state of health, and use of other drugs. Number 53 • The concentration of quinidine 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 of absorption, distribution, biotransformation, and Specificity excretion. These parameters must be considered when interpreting results.1–3 The Emit® 2000 Quinidine Assay measures the total (protein-bound plus unbound) quinidine concentration in serum or plasma. In addition, dihydroquinidine (DHQ) makes a small, but measurable, contribution to the assayed value (see Section 8, Expected Values). 7 Limitations of the procedure Compounds whose chemical structure or concurrent therapeutic use would suggest possible cross-reactivity have been tested. o-Desmethylquinidine (DMQ), a minor metabolite of quinidine, o-Desmethylquinidine (DMQ), a minor metabolite of quinidine, cross-reacts with this assay (see cross-reacts with this assay. Section 9, Specific Performance Characteristics, Specificity). The compounds listed in Table 4 do not interfere with the Emit® 2000 Quinidine Assay when tested in the presence of 2.0 µg/mL quinidine. Levels tested were at or above maximum 8 Expected Values physiological or pharmacological concentrations. Table 4 — Compounds That Do Not Interfere The Emit® 2000 Quinidine Assay accurately quantitates quinidine concentrations in human serum or plasma containing 0.5–8.0 µg/mL (1.5–25 µmol/L) quinidine. Dihydroquinidine Concentration Tested Concentration Tested (DHQ), a constituent of pharmaceutical quinidine preparations,6,7 has antiarrhythmic activity and Compound (µg/mL) Compound (µg/mL) 7 pharmacokinetic properties similar to those of quinidine. DHQ makes a small, but measurable, Structurally Related Compounds Structurally Unrelated Compounds contribution to the assayed value. Levels of DHQ in serum are reported to be approximately 5% to 10% of the total quinidine level.6 3-Hydroxyquinidine 5 Clonidine 100 Note: To convert from µg/mL to µmol/L quinidine, multiply by 3.08. 2’-Oxoquinidinone 2 Digoxin 0.1 Clinicians should be aware of the differences among the various analytical methods used to Quinine 20 Disopyramide 100 measure quinidine and how these differences may affect the range of quinidine concentrations Furosemide 100 that are defined as therapeutic. Regardless of the method used to measure quinidine concentrations, the therapeutic range is variable between patients. Plasma quinidine levels Hydrochlorothiazide 100 between approximately 2 and 5 µg/mL have commonly been considered effective.1–4,8 In all Lidocaine 100 cases, the clinician should carefully consider patient response and evidence of toxicity along with blood levels in determining optimal quinidine therapy. Methyldopa 100 N-Acetylprocainamide 100 9 Specific Performance Characteristics Procainamide 100 Propranolol 100 The information presented in this section is based on Emit® 2000 Quinidine Assay studies Tocainide 100 on the AU400®/AU600® Clinical Chemistry System. Refer to the Application Sheets for other AU Clinical Chemistry Systems and for additional information. Results may vary due Sensitivity to analyzer‑to-analyzer differences. The following performance characteristics represent total system performance and should not be interpreted to pertain only to reagents. The sensitivity level of the Emit® 2000 Quinidine Assay is 0.25 µg/mL. This level represents the lowest measurable concentration of quinidine that can be distinguished from 0 µg/mL with a Endogenous Substances confidence level of 95%. No clinically significant interference has been found in samples to which 800 mg/dL hemoglobin, Calibration Stability 1000 mg/dL triglycerides, or 30 mg/dL bilirubin were added to simulate hemolytic, lipemic, or icteric samples. Studies have shown calibration stability of more than two weeks. When proper reagent handling, instrument maintenance, and operating procedures are followed, the calibration should remain Precision stable for at least two weeks. Within-run precision was determined by assaying 20 replicates of each level of a tri-level control. Table 1 summarizes the data.
Table 1 — Within-Run Precision Level 1 Level 2 Level 3 Mean (µg/mL) 1.45 3.28 4.67 %CV 2.9 3.4 3.0
Total precision was calculated according to NCCLS guideline EP5-T2 using data collected from controls run in duplicate twice daily over twenty (20) days. Table 2 summarizes the data.
Table 2 — Total Precision Level 1 Level 2 Level 3 Mean (µg/mL) 1.39 3.34 4.68 %CV 8.5 6.7 9.3
2 4Q052.2D_B 10 References Symbols Key
1. Covinsky JO, Conn RD: Quinidine: Therapeutic use and serum concentration monitoring, in Taylor WJ, Finn AL (eds): Individualizing Drug Therapy: Practical Applications of Drug Do not reuse Monitoring. New York, Gross, Townsend, Frank, Inc, 1981, vol 3, pp 111–132. Use By 2. Ueda CT: Quinidine, in Evans WE, Schentag JJ, Jusko WJ (eds): Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring. Washington, Applied Therapeutics, Inc, 1986, Batch Code pp. 712–734. 3. Bigger JT Jr, Hoffman BF: Antiarrhythmic drugs, in Gilman AG, Rall TW, Nies AS, et al (eds): Catalogue Number Goodman and Gilman’s the Pharmacological Basis of Therapeutics, ed 8. New York, Caution, consult accompanying Pergamon Press Inc, 1990, pp 840–873. documents 4. Pincus, MR, Abraham NZ Jr: Toxicology and therapeutic drug monitoring, in Henry JB (ed): Manufacturer Clinical Diagnosis and Management by Laboratory Methods, ed 18, Philadelphia, WB Saunders Co, 1991, pp. 349–384. Authorized Representative in the European Community 5. Pope E, Huster M: Syva® Emit® 2000 Quinidine Assay. Clin Chem 1992;38(6):338. Abstract. 6. Guentert TW, et al: Determination of quinidine and its major metabolites by high- Contains sufficient for
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