Download Prepared By - Beckman Coulter

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
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All printed copies are considered to be copies of the electronic original.
Copies
Page 1 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
PRINCIPLE:
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
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All printed copies are considered to be copies of the electronic original.
Page 2 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
INTENDED USE:
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 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 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
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All printed copies are considered to be copies of the electronic original.
Page 3 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
ensure that absorption is complete and to determine the effectiveness of
the therapeutic intervention.4
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
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All printed copies are considered to be copies of the electronic original.
Page 4 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
considered stable for 7 days when stored refrigerated at 2 - 8C and stable
for 6 month when stored frozen at < -20C.
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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 5 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
MATERIALS:
Emit tox™ Salicylic Acid Assay
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:
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All printed copies are considered to be copies of the electronic original.
Page 6 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
Preparation
The Emit tox™ Salicylic Acid Calibrators are provided ready to use
and may be used directly from the refrigerator. No reconstitution is
necessary.
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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 7 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
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.
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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 8 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
PRECISION
Precision was determined by assaying two replicates each of in-house trilevel 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
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
AU600
Correlation Coefficient
Number of Samples
26.1
27.5
0.998
62
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 9 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
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.
QUALITY CONTROL:
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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 10 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
CALCULATIONS:
Results are calculated automatically by the analyzer. No additional
manipulation of data is required.
To convert from mg/dL to mol/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 antiinflammatory 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
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:
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All printed copies are considered to be copies of the electronic original.
Page 11 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
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
Additional reporting information as designated by this laboratory:
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 12 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
LIMITATIONS:
The Emit® tox™ Salicylic Acid Assay accurately quantitates salicylic acid
concentrations up to 80 mg/dL (5792 mol/L) in human serum or plasma.
To estimate salicylic acid concentrations above the assay range, patient
samples containing more than 80 mg/dL (5792 mol/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 Emit 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.
The compounds listed in the following table do not interfere with the
Emit 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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 13 of 15
Rev # 1, Dec 31, 10
Procedure:
tox Salicylic Acid
OSR7S229
Compound
Concentration
Tested (μg/mL)
Compound
Concentration
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
All printed copies are considered to be copies of the electronic original.
Page 14 of 15
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Procedure:
tox Salicylic Acid
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5. Central nervous system agents, in McEvoy GK, Litvak K, Welsh OH (eds):
AHFS Drug Information. Bethesda, MD: American Society of HealthSystem 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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 15 of 15
Rev # 1, Dec 31, 10