3 METHODOLOGY The Emit® tox™ Salicylic Acid Assay is a homogeneous enzyme immunoassay technique used for the quantitative analysis of salicylic acid in human serum or plasma.7 In the performance of the Emit® tox™ Salicylic Acid Assay, serum or plasma is mixed with Reagent 1, which contains antibodies to salicylic acid and the coenzyme nicotinamide adenine dinucleotide (NAD). Subsequently, Reagent 2, which contains salicylic acid ™ labeled with the enzyme glucose‑6‑phosphate dehydrogenase (G6PDH), is added. Salicylic acid in Emit tox Salicylic Acid Assay the sample and salicylic acid‑labeled G6PDH compete for antibody binding sites. Enzyme activity ® decreases upon binding to the antibody, so the salicylic acid concentration in the sample can be measured in terms of enzyme activity. Active enzyme converts oxidized NAD to NADH, resulting May 2012 7S052.2D_C in an absorbance change that is measured spectrophotometrically. Endogenous G6PDH does not interfere because the coenzyme functions only with the bacterial (Leuconostoc mesenteroides) enzyme employed in the assay. See shaded sections: Updated information from August 2010 version. 4 REAGENTS Reagents contain the following substances: Mouse monoclonal antibodies reactive to salicylic acid (60.4 µg/mL),* salicylic acid labeled with bacterial G6PDH (0.34 U/mL), glucose-6-phosphate (22 mM), nicotinamide adenine dinucleotide (18 mM), Tris buffer, bovine serum albumin, preservatives, and stabilizers. Catalog Quantity/ *The antibody titer and enzyme conjugate activity may vary from lot to lot. Number Product Description Volume For in vitro diagnostic use. OSR7S229 Emit® tox™ Salicylic Acid Assay Precautions OSR7S518 R1 (Antibody/Substrate Reagent 1) 2 x 32 mL • Reagents contains nonsterile mouse monoclonal antibodies. Reagents contain nonsterile OSR7S548 R2 (Enzyme Reagent 2) 2 x 16 mL bovine serum albumin. 7S109UL Emit® tox™ Salicylic Acid Calibrators* 1 x 5 mL,† • Reagent 2 contains sodium azide, which may react with lead or copper plumbing to form 5 x 2 mL highly explosive metal azides. If waste is discarded down the drain, flush it with a large volume of water to prevent azide buildup. *Required for calibrating the Emit ® tox™ Salicylic Acid Assay. Sold separately. • Reagents and calibrators contain materials that may cause sensitivity on contact with skin. †Additional negative calibrator is provided. • Do not use kit after the expiration date. Note: Reagents and calibrators are shipped ready to use in liquid form. No reconstitution is necessary. • Turbid or yellow reagents may indicate contamination or degradation and must be discarded. Note: Reagents 1 and 2 are provided as a matched set. They should not be interchanged with Safety data sheets (MSDS/SDS) available on www.siemens.com/diagnostics components of kits with different lot numbers. Preparation of Reagents The Emit® tox™ Salicylic Acid Calibrators contain the following salicylic acid concentrations: The Emit® tox™ Salicylic Acid Assay reagents are provided ready to use; no preparation is necessary. Calibrator 0 5 10 20 40 80 Storage of Assay Components Salicylic acid (mg/dL) 0 5 10 20 40 80 • Improper storage of reagents can affect assay performance. Salicylic acid (µmol/L) 0 362 724 1448 2896 5792 • When not in use, store reagents at 2–8°C (36–46°F), upright, and with the screw caps tightly closed. • Unopened reagents are stable until the expiration date printed on the label if stored upright at 1 INTENDED USE 2–8°C. The Emit® tox™ Salicylic Acid Assay is a homogeneous enzyme immunoassay intended for • Do not freeze reagents or expose them to temperatures above 32°C. use in the quantitative analysis of salicylic acid in human serum or plasma. These reagents are packaged specifically for use on a variety of AU® Clinical Chemistry Systems. 5 SPECIMEN COLLECTION AND PREPARATION 2 SUMMARY • Each assay requires serum or plasma. Whole blood cannot be used. The anticoagulants EDTA, sodium heparin, citrate, and oxalate/fluoride have been tested and may be used with Acetylsalicylic acid (aspirin) and other salicylates are rapidly metabolized to salicylic acid after this assay. Some sample dilution may occur when samples are collected in tubes containing ingestion.1 Aspirin is widely used for its antipyretic, analgesic, and anti-inflammatory properties. citrate anticoagulant. The amount of dilution and the possible need to correct for it should be It appears in many over-the-counter and prescription medications, and as such, can be considered when interpreting assay results for these samples. inadvertently overdosed by adults or can cause accidental poisoning in children. • Sample volume is instrument-dependent. Refer to the appropriate application sheet. Chronic salicylate poisoning (salicylism) occurs most commonly in seniors who regularly take • Pharmacokinetic factors influence the relationship between the observed drug level and the large doses of aspirin, often for osteoarthritis, and then gradually increase their doses or develop 2 3 time elapsed since drug ingestion. These factors include dosage form, mode of administration, renal insufficiency. Signs and symptoms of salicylism include fever, vomiting, and tachypnea. concomitant drug therapy, and biological variations affecting drug disposition.6 Acute overdosage can cause nausea, vomiting, dehydration, hyperpnea, oliguria, and tinnitus.3 3 • Serum levels drawn less than 6 hours after a toxic dose can be used to confirm overdose, Severe poisoning can cause coma, convulsions, severe hyperpnea, and metabolic acidosis. In but to use the Done nomogram to predict the severity of the toxic reaction, samples must be suspected acute overdose cases, determination of salicylic acid concentrations in serum helps 8 1,4 drawn at least 6 hours after ingestion of the toxic dose. Repeat testing within 2–3 hours is determine the severity of toxicity and the steps toward detoxification. recommended to ensure that absorption is complete and to determine the effectiveness of the In children, accidental poisoning or serious intoxication occurs frequently and is sometimes therapeutic intervention.4 1 5 fatal. Pediatric patients who are dehydrated are especially susceptible to salicylate intoxication. • After therapeutic doses of salicylates, peak levels are reached at 2 hours.8 Additionally, in children with varicella infections or influenza-like illnesses, testing for serum salicylates may either implicate or rule out Reyes syndrome.5 • Store and transport the serum or plasma refrigerated at 2–8°C. For patients on chronic aspirin therapy, monitoring salicylic acid concentrations in serum, along • Human serum or plasma samples should be handled and disposed of as if they were with careful clinical assessment, is the most effective means of ensuring adequate therapy. potentially infectious. It is recommended that human specimens be handled in accordance Therapeutic ranges for salicylic acid are frequently very close to the levels associated with toxic with the OSHA Standard on Bloodborne Pathogens or other appropriate local practices.9,10 manifestations.6 Salicylate concentrations in the blood generally correlate with both adverse and therapeutic effects.6 Methods historically used to monitor serum salicylic acid concentrations are immunoassays and colorimetric, ultraviolet, fluorescent, chromatographic, and enzymatic assays.6 6 PROCEDURE 9 SPECIFIC PERFORMANCE CHARACTERISTICS Materials Provided The information presented in this section is based on Emit® tox™ Salicylic Acid Assay studies Emit® tox™ Salicylic Acid Assay performed on the AU600® Clinical Chemistry System. Refer to the Application Sheet for other AU Clinical Chemistry Systems and for additional information. Results may vary due to analyzer- Reagent 1 to-analyzer differences. The following performance characteristics represent total system Reagent 2 performance and should not be interpreted to pertain only to reagents. Materials Required But Not Provided Emit® tox™ Salicylic Acid Calibrators Endogenous Substances Multilevel commercial controls No significant interference has been found in samples to which 800 mg/dL hemoglobin, 30 mg/dL bilirubin, or 750 mg/dL triglycerides were added to simulate hemolytic, icteric, or lipemic samples. Calibration Recalibrate whenever a new lot of reagents is used or as indicated by control results (see Quality Precision Control, below). If a new set of reagents with the same lot number is used, validate the system Precision was determined by assaying two replicates each of in-house tri-level controls on by assaying controls. 20 days with 2 runs per day. Precision was calculated according to National Committee for Clinical Laboratory Standards (NCCLS) Guideline EP5-A (February 1999). Table 1 summarizes Quality Control the results. • Validate the calibration by assaying multi-level controls. Commercial controls are available for this purpose. Ensure that control results fall within acceptable limits as defined by your own Table 1 — Precision laboratory. Once the calibration is validated, run samples. Mean Standard Coefficient of • Follow government regulations or accreditation requirements for quality control frequency. Control (mg/dL) Deviation (mg/dL) Variation (%) At least once each day of use, analyze two levels of a Quality Control (QC) material with known salicylic acid concentrations. Follow your laboratory internal QC procedures if the Within-Run results obtained are outside acceptable limits. 1 3.3 0.19 5.8 • Refer to the instrument operator’s manual for appropriate instrument checks. 2 19.3 0.74 3.8 3 49.2 1.23 2.5 Diluting High Concentration Samples To estimate salicylic acid concentrations above the assay range, patient samples containing Total more than 80 mg/dL (5792 µmol/L) salicylic acid may be diluted with one or two parts of distilled 1 3.3 0.30 9.0 or deionized water or Emit® tox™ Salicylic Acid Calibrator 0. After dilution, test the sample and multiply the results by the dilution factor. See the analyzer User’s Guide or appropriate 2 19.3 0.95 5.0 Application Sheet for instructions. 3 49.2 2.29 4.7
Evaluation and Interpretation of Results • Results are calculated automatically by the analyzers. No additional manipulation of data is Comparative Analysis required unless samples have been manually diluted. In this study, samples from patients taking salicylic acid and samples spiked with salicylic acid were analyzed on the SYVA®-30R Biochemical System and the AU600 Clinical Chemistry • Consult the appropriate instrument operating manual and Application Sheet for complete System, and the results were compared. Table 2 summarizes the results. instructions. • The concentration of salicylic acid in serum or plasma depends on the time of drug ingestion; Table 2 — Comparative Analysis Results concomitant drug therapy; sample condition; time of sample collection; and individual Slope 1.049 variations in absorption, distribution, biotransformation, and excretion. These parameters must be considered when interpreting results.6 Intercept (mg/dL) 0.175 • In acute aspirin overdose in pediatric patients, a single serum or plasma level plotted on the Mean (mg/dL) 3,4,11 Done nomogram provides a good indication of the severity of intoxication. SYVA®-30R 26.1 • Results of this test should always be interpreted in conjunction with the patient’s medical AU600 27.5 history, clinical presentation and other findings. Correlation Coefficient 0.998 Number of Samples 62 7 LIMITATIONS OF THE PROCEDURE When diluting patient samples containing high salicylic acid concentrations, the following factors Specificity can affect the accuracy of the result: the use of the correct diluent (Emit® tox™ Salicylic Acid The Emit® tox™ Salicylic Acid Assay measures the total (protein-bound plus unbound) salicylic Calibrator 0 or distilled or deionized water) and the accuracy of the dilution. acid 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 Table 1 do not interfere with the Emit® tox™ Salicylic Acid Assay 8 EXPECTED VALUES when tested in the presence of 30 mg/dL salicylic acid. Levels tested were at or above maximum physiological or pharmacological concentrations. The Emit® tox™ Salicylic Acid Assay accurately quantitates salicylic acid concentrations in human serum or plasma containing up to 80 mg/dL (5792 µmol/L) salicylic acid. Table 3 — Compounds That Do Not Interfere The therapeutic range for analgesic and antipyretic effects is below 6 mg/dL (434 µmol/L) salicylic acid.1,4 The therapeutic range for anti-inflammatory effects is 15–30 mg/dL Compound Concentration Tested (µg/mL) 1,6 (1086–2172 µmol/L) salicylic acid. Levels of 30–40 mg/dL (2172–2896 µmol/L) salicylic Acetaminophen 1000 acid are frequently maintained to manage acute rheumatic fever; these levels are within the toxic range for salicylates, which starts at 30 mg/dL (2172 µmol/L).6 Hemodialysis is indicated Acetylsalicylic Acid 1000 when serum salicylic acid concentrations exceed 100 mg/dL (7240 µmol/L).8 See Section 6, p-Aminosalicylic Acid 400 Procedure, Evaluation and Interpretation of Results, for a discussion of overdose cases. Benzoic Acid 1000 Note: To convert from mg/dL to µmol/L salicylic acid, multiply by 72.4. Caffeine 1000 For effective treatment, some patients may require serum or plasma levels outside these ranges. Therefore, the expected ranges are provided only as guidelines, and individual patient results Codeine 100 should be interpreted in light of other clinical signs and symptoms.6 Diflunisal 600 2,3 Dihydroxybenzoic Acid 500 Fenoprofen 1000 Gentistic Acid 10 Ibuprofen 1000 Indomethacin 100 Methyl Salicylate 500 Naproxen 150 Salicylamide 500 Salicylsalicylic Acid 1000 Salicyluric Acid 1000
2 7S052.2D_C Sensitivity The sensitivity level of the Emit® tox™ Salicylic Acid Assay is 0.2 mg/dL salicylic acid. This level Symbols Key represents the lowest concentration of salicylic acid that can be distinguished from 0 mg/dL with a confidence level of 95%. Do not reuse Calibration Stability Use By Studies have shown calibration stability of at least 14 days. Calibration stability may vary from laboratory to laboratory depending on handling of reagents, maintenance of instruments, Batch Code adherence to operating procedures, establishment of control limits, and verification of calibration. Catalogue Number
Caution, consult accompanying 10 REFERENCES documents
1. Insel P: Analgesic-antipyretic and anti-inflammatory agents, in Hardman JG, Limbird LE Manufacturer (eds): Goodman and Gillman’s The Pharmacologic Basis of Therapeutics. 9th ed. New York, NY: McGraw-Hill; 1996:625–31. Authorized Representative in the European Community 2. Olson KR (ed): Management of poisoning and overdose, in Dale DC, Federman DD (eds): Scientific American Medicine. 1996;8(I):20. Contains sufficient for
4. Porter WH: Clinical toxicology, in Burtis CA, Ashwood ER (eds): Tietz Textbook of Clinical Temperature Limitation Chemistry. 3rd ed. Philadelphia, Pennsylvania: WB Saunders. 1999:929–32. 5. Central nervous system agents, in McEvoy GK, Litvak K, Welsh OH (eds): AHFS Drug Consult Instructions for Use Information. Bethesda, MD: American Society of Health-System Pharmacists. 1997:1466. 6. Dromgoole SH, Furst DE: Salicylates, in Applied Pharmacokinetics: Principles of Therapeutic Non-sterile Drug Monitoring. 3rd ed. Vancouver, WA: Applied Therapeutics. 1996;(32):1–34. CE Mark 7. Hendeles L, Edwards C: Clinical assessment of an enzyme immunoassay (EMIT) for measurement of serum salicylate. J Clin Pharmacol Ther. 1988;(13):131–8. Contents 8. Wallach J: Interpretation of Diagnostic Tests. 6th ed. Boston, MA; Little, Brown, and Company. 1996:848–9. Reconstitution Volume
9. Occupational exposure to bloodborne pathogens (29 CFR 1910.1030). Federal Register. Level December 06, 1991;56:64004; amended April 13, 1992;57:12717; July 01, 1992;57:29206; 2010-07_BC February 13, 1996;61:5507. 10. World Health Organization. Laboratory Biosafety Manual. 2nd ed. Geneva: World Health Organization; 1993. 11. Done AK: Aspirin overdosage: incidence, diagnosis, and management. Pediatrics. 1978;62(suppl 5):890–7.
For technical assistance: Beckman Coulter customers contact their technical assistance center. 1-800-223-0130
Siemens Healthcare Diagnostics customers contact their technical assistance center. 1-800-227-8994 in the USA 1-800-264-0083 in Canada
The Beckman Coulter logo and AU® are trademarks of Beckman Coulter, Inc.
The Syva logo, Syva ®, Emit ® and tox™ are trademarks of Siemens Healthcare Diagnostics.
Made in USA for Beckman Coulter, Inc. 250 S. Kraemer Blvd. Brea, CA 92821
® Revised May 2012 Printed in USA 7S052.2D_C