This Procedure Is Valid for the Following Chemistry Analyzers s4

Procedure: tox Salicylic Acid OSR7S229 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 # of Distributed to Copies Distributed to Copies

Acetylsalicylic acid (aspirin) and other salicylates are rapidly metabolized to salicylic acid after ingestion.1 Aspirin is widely used for its analgesic, antipyretic, and anti- inflammatory properties. It is found in a number of over-the-counter and prescription medications, and as such can be inadvertently overdosed by adults or can cause accidental poisoning in children.

Chronic salicylate poisoning (salicylism) occurs most commonly in seniors who regularly take large doses of aspirin, often for osteoarthritis and then gradually increase their doses or develop renal insufficiency.2 Signs and symptoms of salicylism include fever, vomiting, and tachypnea.3

Acute overdosage can cause nausea, vomiting, dehydration, hyperpnea, oliguria, and tinnitus.3 Severe poisoning can cause coma, convulsions, severe hyperpnea, and metabolic acidosis.3 In suspected acute overdose cases, determination of salicylic acid concentrations in serum helps determine the severity of toxicity and the steps toward detoxification.1,4

In children, accidental poisoning or serious intoxication occurs frequently and is sometimes fatal1. Pediatric patients who are dehydrated are especially susceptible to salicylate intoxication5 Additionally, in children with varicella infections or influenza- like illnesses, testing for serum salicylates may either implicate or rule out Reyes syndrome.5

For patients on chronic aspirin therapy, monitoring salicylic acid concentrations in serum, along with careful clinical assessment, is the most effective means of ensuring adequate therapy. Therapeutic ranges for salicylic acid are frequently very close to the levels associated with toxic manifestations6. Salicylate concentrations in the blood generally correlate with both adverse and therapeutic effects. 6

The methods historically used to monitor serum salicylic acid concentrations include immunoassays and colorimetric, ultraviolet, fluorescent, chromatographic, and enzymatic assays.6

The Emit tox™ Salicylic Acid Assay is intended for use in the quantitative analysis of salicylic acid in human serum or plasma. This Emit Assay is packaged specifically for use on multiple Beckman Coulter AU analyzers.

METHODOLOGY

The Emitâ tox™ Salicylic Acid Assay is a homogeneous enzyme immunoassay technique used for the quantitative analysis of salicylic acid in human serum or plasma.7 This assay is based on competition 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 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 may influence the sample collection time.

Pharmacokinetic factors influence the relationship between the observed drug level and the time elapsed since drug ingestion. These factors included dosage form, mode of administration, concomitant drug therapy and biological variations affecting drug disposition.6

SAMPLE COLLECTION TIME:

Serum levels drawn less than 6 hours after a toxic dose can be used to confirm overdose. To use the Done nomogram to predict the severity of the toxic reaction, sample must be drawn at least 6 hours after ingestion of the toxic dose8. Repeat testing within 2 - 3 hours is recommended to ensure that absorption is complete and to determine the effectiveness of the therapeutic intervention.4

© Beckman Coulter, Inc. 2010 Page 3 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 After therapeutic doses of salicylates, peak levels are reached at 2 hours.8

Additional instructions for patient preparation as designated by this laboratory:

TYPE:

Serum or plasma is the recommended specimen. Whole blood cannot be used. The anticoagulants EDTA, sodium heparin, citrate, and oxalate/fluoride have been tested and may be used with this assay.

Some 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:

Preferably, blood specimens should be separated and tested immediately after collection.

According to the CAP Patient Preparation & Specimen Handling Fascicle IV: Therapeutic Drug Monitoring/Toxicology, samples are generally considered stable for 7 days when stored refrigerated at 2 - 8°C and stable for 6 month when stored frozen at < -20°C.

© Beckman Coulter, Inc. 2010 Page 4 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 Samples that contain particulate matter, fibrous material, 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 specimens be handled in accordance with the OSHA Standard on Bloodborne Pathogens or other appropriate local practices.9,10

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 tox™ Salicylic Acid Assay

© Beckman Coulter, Inc. 2010 Page 5 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 Antibody/Substrate Reagent 1 - mouse monoclonal antibodies reactive to salicylic acid, G6P, NAD, bovine serum albumin, Tris buffer, stabilizers, and preservatives.

Enzyme Reagent 2 – salicylic acid labeled with bacterial G6PDH, Tris buffer, bovine serum albumin, stabilizers, and preservatives.

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:

Emit tox™ Salicylic Acid Calibrators (Cat No 7S409)

The Emit tox™ Salicylic Acid Calibrators contain the following stated acetaminophen concentrations: 0 mg/dL, 5 mg/dL, 10 mg/dL, 20 mg/dL, 40 mg/dL, 80 mg/dL. The calibrators also contain phosphate buffer, and, preservatives. Source material from which the calibrators were derived is not biohazardous.

Storage location of the calibrator in this laboratory:

Preparation The Emit tox™ Salicylic Acid Calibrators are provided ready to use and may be used directly from the refrigerator. No reconstitution is necessary.

© Beckman Coulter, Inc. 2010 Page 6 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 Emit tox™ Salicylic Acid Reagents are provided ready to use; no preparation is necessary. 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® tox™ Salicylic Acid Assay and Calibrators are for in vitro diagnostic use.

2. Reagents contain non-sterile mouse antibodies. Reagent and calibrators contain non-sterile bovine serum albumin.

3. 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. Dispose of properly in accordance with local regulations.

4. Do not use the reagent kit or calibrators after the expiration date.

5. Reagents and calibrators contain materials that may cause sensitivity on contact with skin.

6. No known test method can offer complete assurance that products derived from human blood are pathogen-free. Handle all materials of human origin as though they were potentially infectious. If exposed to solution containing materials of human origin, the user should follow recommendations of the U.S. Occupational Safety and Health Administration.

Storage and Stability: Unopened Emit® tox™ Salicylic Acid reagents are stable until the expiration date printed on the label if stored upright and at 2 - 8°C. Refer to Assay Methodology Sheets for additional on-board stability information. When not in use, store reagents at 2 - 8°C, upright, and with the screw caps tightly closed.

The Emit® tox™ Salicylic Acid calibrators are light sensitive. Store calibrators capped in their original brown vials. Always store at 2 - 8°C when not in use. Store upright. When stored as directed the calibrators are stable until the expiration date printed on the vial labels.

© Beckman Coulter, Inc. 2010 Page 7 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 Do not freeze the reagents or calibrators or expose them to temperatures above 32°C.

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 calibrators 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

Precision was determined by assaying two replicates each of in-house tri-level controls for 20 days with 2 runs per day. Precision was calculated according to Clinical and Laboratory Standards Institute (CLSI EP5-A).

Within-Run Precision Total Precision

Level 1 Level 2 Level 3 Level 1 Level 2 Level 3

Mean (mg/dL) 3.3 19.3 49.2 3.3 19.3 49.2

SD 0.19 0.74 1.23 0.30 0.95 2.29

© Beckman Coulter, Inc. 2010 Page 8 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 CV % 5.8 3.8 2.5 9.0 5.0 4.7

COMPARISON

Samples from patients were analyzed on the Syva® -30R Biochemical System and the AU600. Results are shown in the following table.

Slope 1.049

Intercept (mg/dL) 0.175

Mean (mg/dL)

Syva® -30R 26.1 AU600 27.5

Correlation Coefficient 0.998

Number of Samples 62

CALIBRATION:

Perform a multi-point calibration (5AB) using a water blank (blue rack) and the Emit® tox™ Salicylic Acid Calibrators: 5, 10, 20, 40, 80. 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 calibration stability to be at least 14 days. Recalibrate as indicated by control results or whenever a new lot of reagents is used.

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.

During operation of the Beckman Coulter AU analyzer at least one level of control material should be tested every 8 hours, alternating the control levels tested. Ensure that a minimum of 2 controls is assayed in every 24 hour period. Controls should be performed after calibration, with each new set of reagent with the same lot number, 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.

CALCULATIONS:

Results are calculated automatically by the analyzer. No additional manipulation of data is required.

To convert from mg/dL to mmol/L salicylic acid, multiply by 72.4.

REPORTING RESULTS:

REFERENCE RANGES:

The therapeutic range for analgesic and antipyretic effects for salicylic acid is below 6 mg/dL (434 mol/L).1,4 The therapeutic range for anti-inflammatory effects is 15 - 30 mg/dL (1086 - 2172 mol/L) salicylic acid.1,6

Levels of 30 - 40 mg/dL (2172 - 2896 mol/L) salicylic acid are frequently maintained to manage acute rheumatic fever. These levels are within the toxic range for salicylates, which start at 30 mg/dL (2172 mol/L).6 Hemodialysis is indicated when serum salicylic acid concentrations exceed 100 mg/dL (7240 mol/L).8

© Beckman Coulter, Inc. 2010 Page 10 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 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 should be interpreted in light of other clinical signs and symptoms.6

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.

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 mg/dL at 37°C.

Interpretation of Results The concentration of Salicylic Acid in serum or plasma depends on the time of drug ingestion, 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.6

In acute aspirin overdose in pediatric patients, a single serum or plasma level determination plotted on the Done nomogram provides a good indication of the severity of intoxication.3,4,11

© Beckman Coulter, Inc. 2010 Page 11 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 Additional reporting information as designated by this laboratory:

LIMITATIONS:

The Emit® tox™ Salicylic Acid Assay accurately quantitates salicylic acid concentrations up to 80 mg/dL (5792 mmol/L) in human serum or plasma.

To estimate salicylic acid concentrations above the assay range, patient samples containing more than 80 mg/dL (5792 mmol/L) salicylic acid may be diluted with one or two parts distilled or deionized water or Emit® 2000 Salicylic 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 protocol or the package insert can affect performance characteristics and stated or implied labeling claims.

INTERFERING SUBSTANCES

No clinically significant interference has been found in samples 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® tox™ Salicylic Acid Assay is 0.2 mg/dL. This level represents the lowest concentration of salicylic acid that can be distinguished from 0 mg/dL with a confidence level of 95%.

SPECIFICITY

The Emitâ tox™ Salicylic Acid Assay measures the total (protein-bound plus unbound) salicylic 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 Page 12 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 The compounds listed in the following table do not interfere with the Emitâ tox™ Salicylic Acid Assay when tested in the presence 30 mg/dL salicylic acid. Levels tested were at or above maximum physiological or pharmacological concentrations.

Compound Concentration Compound Concentration Tested (μg/mL) Tested (μg/mL)

Acetaminophen 1000 Gentistic Acid 10

Acetylsalicylic Acid 1000 Ibuprofen 1000

p-Aminosalicylic Acid 400 Indomethacin 100

Benzoic Acid 1000 Methyl Salicylate 500

Caffeine 1000 Naproxen 150

Codeine 100 Salicylamide 500

Diflunisal 600 Salicylsalicylic Acid 1000

2,3 Dihydroxybenzoic Acid 500 Salicyluric Acid 1000

Fenoprofen 1000

REFERENCES:

1. Insel P: Analgesic-antipyretic and anti-inflammatory agents, In Hardman JG, Limbird LE (eds): Goodman and Gillman’s The Pharmacologic Basis of Therapeutics. 9th ed. New York, NY: McGraw-Hill; 1996:625-31.

2. Olson KR (ed): Management of poisoning and overdose, In Dale DC, Federman DD (eds), Scientific American Medicine. 1996; 8(I):20.

3. Jacobs DS, DeMott WR, Grady HJ, et al. (eds) Laboratory Test Handbook. 4th ed. Cleveland, OH: Lexi-Comp Inc.; 1996:573-4.

4. Porter WH: Clinical toxicology, in Burtis CA, Ashwood ER, (eds): Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia, Pennsylvania: WB Saunders. 1999:929-32.

© Beckman Coulter, Inc. 2010 Page 13 of 14 All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10 Procedure: tox Salicylic Acid OSR7S229 5. Central nervous system agents, in McEvoy GK, Litvak K, Welsh OH (eds): AHFS Drug Information. Bethesda, MD: American Society of Health-System Pharmacists. 1997:1466.

6. Dromgoole SH, Furst DE: Salicylates, in Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring. 3rd ed. Vancouver, WA: Applied Therapeutics. 1996; (32):1-34.

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.

8. Wallach J: Interpretation of Diagnostic Tests. 6th ed. Boston, MA: Little, Brown, and Company. 1996: 848-9.

9. 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.

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.