DRI® Salicylate Serum Tox Assay
For In Vitro Diagnostic Use
0977 (1 x 25 mL) Reagent Kit
0980 (1 x 5 mL, 1 x 2 mL ) Calibrator Set
Intended Use
The DRI® Salicylate Serum Tox Assay is intended for the quantitative determination of salicylate
in human serum or plasma.
Summary and Explanation of the Test
Salicylates are a group of non-addictive analgesics which also have antipyretic, anti-inflammatory,
and antirheumatic effects in humans. Accidental ingestion of salicylates remains the single
most common cause of drug toxicity in preschool children.1,2 Toxicity has also been observed
with patients on chronic salicylate therapy. Chronic salicylate dosing generates a therapeutic
concentration of approximately 60 µg/mL. In rheumatoid arthritis therapy, large doses of salicylate
are administered to obtain a therapeutic salicylate concentration of 150-300 µg/mL. Serum
salicylate concentration in excess of 300 µg/mL is often associated with toxicity. 3 Severe
toxic effects occur when the serum concentration reaches 500 µg/mL.4 Monitoring salicylate
concentration can help a physician evaluate the extent of poisoning and determine the steps
for detoxification.
Many techniques are available for the determination of salicylate concentrations in serum.
These methods include gas chromatography (GC), enzyme immunoassay, spectrophotometric
method and fluorescence immunoassay.
The DRI Salicylate Serum Tox Assay is a spectrophotometric method using ready-touse liquid reagents. The assay measures the absorbance intensity at 540 nm, which is a
characteristic of an iron complex formed by salicylate with ferric nitrate in an acidic medium.5
The intensity measured at 540 nm is directly proportional to the concentration of salicylate in
the serum sample.
Reagents
Salicylate Assay Reagent. Contains 25 mL of ferric nitrate in an aqueous nitric acid solution.
Additional Materials Required (sold separately):
Salicylate Calibrator Kit: Contains 1 x 5 mL of Negative Calibrator (Tris buffer) and 1 x 2 mL of
800 µg/mL salicylate in Tris buffer.
Precautions and Warning
The reagent components are harmful if swallowed.
Quality Control and Calibration
Good laboratory practice suggests the use of control specimens to ensure proper assay
performance. Various commercial controls are available for the quality control of this
assay. Ensure the control results are within established ranges, as determined by your
laboratory. Establish a two-point linear calibration curve by using the negative (0 µg/mL) and
800 µg/mL calibrators. Recalibrate the instrument when using a different reagent lot or
when control results are outside established ranges. The sample salicylate concentration is
determined by interpolation of the reaction rate from the two point linear calibration curve. All
quality control requirements should be performed in conformance with local, state and/or federal
regulations or accreditation requirements.
Results and Expected Values
Clinical chemistry analyzers automatically determine the concentration of salicylate in serum or
plasma. No additional data manipulation is required.
Persons not under salicylate therapy should have no salicylate in their serum. Chronic salicylate
dosing normally generates a therapeutic level of 60 µg/mL. When salicylates are used in
rheumatoid arthritis therapy, an optimal therapeutic level of 150-300 µg/mL is desirable.6
Severity of salicylate toxicity correlates well with serum salicylate levels. Based on the quantity of
salicylate ingested, the Done nomogram can help the physician evaluate the extent of poisoning
and determine steps for detoxification.7
Limitations
1. Sodium azide interferes with the assay and should not be used for sample collection.
2. Hemolyzed samples can cause interference in the assay resulting in falsely elevated
concentrations for Salicylate.
3. The test is designed for use with human serum or plasma only but not for whole blood.
Typical Performance Characteristics
The following typical data was generated with a Hitachi 704 clinical chemistry analyzer.
Precision
The within-run and run-to-run precision (evaluated over a three-week period) was evaluated
using three serum samples with the following results:
Within-run (n=20)
Sodium azide interferes with assay and should not be used as a preservative.
Do not use the reagents beyond their expiration dates.
The reagent contains dilute nitric acid. Nitric acid is corrosive. Wear gloves when handling
the reagent. If reagent comes in contact with skin or eyes, flush the contact area for at least
15 minutes with water. If redness persists, consult a physician immediately.
Reagent Preparation and Storage
The reagents are ready-to-use. No reagent preparation is required. The assay components should
be stored at room temperature. The reagent is good until the expiration date stated on the label.
Specimen Collection and Handling
Either serum or plasma can be used with the assay. Anticoagulants such as heparin,
citrates, oxalates and EDTA will not interfere with the assay. Plasma samples collected
with these anticoagulants may be used, although a fresh serum sample is preferred.
Sodium azide should not be used for sample preservation or collection. Store the sample
refrigerated. An effort should be made to keep pipetted samples free of gross debris. Handle all
serum specimens as if they were potentially infectious.
Assay Procedure
Analyzers capable of maintaining a constant temperature, pipetting samples, mixing reagents,
and measuring absorbance at 540 nm and timing the reaction accurately can be used to perform
this homogenous enzyme immunoassay.
Before performing the assay, refer to the analyzer-specific protocol sheet, which contains
parameters and/or additional instructions for use.
Mean ± SD
(μg/mL)
% CV
01
49 ± 1.7
02
194 ± 2.8
03
785 ± 7.7
Sample
Run-to-run (n=12)
Mean ± SD
(μg/mL)
% CV
3.5
53 ± 3.5
6.6
1.4
202 ± 7.6
3.7
1.0
798 ± 29.0
3.7
Sensitivity
Sensitivity, defined as the lowest concentration that can be differentiated from the 0 µg/mL with
95% confidence, is 4.4 mg/dL (44 µg/mL).
Linearity
A serial dilution of the 800 µg/mL calibrator was made with the negative calibrator to prepare
400, 200, 100, and 50 µg/mL solutions. These diluted solutions were tested for salicylate with the
assay. Regression analysis with a correlation coefficient of 0.999 was obtained between the
expected and observed salicylate concentrations.
Specificity
The percent (%) cross-reactivity of compounds with similar chemical structure to salicylate was
determined at a concentration of 1000 µg/mL with the following results:
Compound
Concentration Tested (μg/mL)
% Cross Reactivity
Acetylsalicylic Acid
1000
91.8 %
Benzoic Acid
1000
0.0 %
Gentisic Acid*
1000
19.1 %
Methyl Salicylate
1000
1.0 %
Salicyluric Acid*
1000
38.9 %
Salicylamide
1000
68.2 %
*Metabolites of salicylate.
The following compounds were tested at a concentration of up to 1000 µg/mL and showed crossreactivity results less than the detection limit of the assay.
Acetaminophen
Caffeine
Chlorpromazine
Dextromethorphan
Dihydroxycodeine
Ephedrine
Guaiacol Glyceryl Ether
Homatropine
Ibuprofen
Indomethacin
Phenobarbital
Phenylbutazone
Propoxyphene
Pyrilamine
No interference with lipemic and icteric samples was found with cholesterol and bilirubin
concentrations as high as 400 mg/dL and 30 mg/dL, respectively.
Accuracy and Correlation
Forty-six clinical serum samples were assayed with DRI Salicylate Serum Tox Assay (y) and a
commercially available Salicylate Assay (x). A correlation with a linear regression equation of y
= 0.802 (x) + 11.8 and a correlation coefficient (r) of 0.999 was obtained. Mean sample salicylate
concentration for the DRI assay was 90.2 µg/mL with a range of 0 µg/mL to 890 µg/mL. Mean sample
salicylate concentration for the commercial Salicylate Assay was 97.6 µg/mL with a range of
0 µg/mL to 1100 µg/mL.
Bibliography
1.
2.
3.
4.
Andrews HB: Salicylate Poisoning. Am Fam Physician, 8:102 (1973).
Pierce AW: Salicylate Poisoning. Pediatrics, 54:342 (1974).
Proudfoot AT: Toxicity of Salicylates. Am J Med, 14:99 (1983).
Irey NS: Blood and Tissue Concentrations of Drugs Associated with Fatalities. Med Clin
North Am, 58:1093 (1974).
5. Natelson S: Microtechniques of Clinical Chemistry for the Routine Laboratory, 2nd ed.,
Springfield, Ill, Charles C. Thomas, Publisher, 1961.
6. Mongan E, Kelly P, Nies K, Porter WW and HE Pamlus: Tinnitus as An Indication of
Therapeutic Serum Salicy late Levels. JAMA, 226:142 (1973).
7. Dugandizc RM, Tierney MG, Dickinson GE, Dolan MC and McKnight DR. Evaluation of
the Validity of the Done Nomogram in the Management of Acute Salicylate Intoxication.
Ann Emerg Med 18: 1186 (1989).
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