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Acetaminophen
(Paracetamol)/
Salicylate Test
A rapid visual test for the qualitative detection
of Acetaminophen (Paracetamol)
and Salicylate in human serum, plasma or venipuncture whole blood.
This test is NOT AVAILABLE FOR RETAIL SALE.
FOR HEALTHCARE PROFESSIONAL PURCHASES ONLY.
INTENDED USE
The Acetaminophen (Paracetamol)/Salicylate Test is an in vitro
diagnostic test for the rapid detection of Acetaminophen (Paracetamol)
and/or Salicylate in human serum, plasma and venipuncture whole
blood at a cut-off level of 25 µg/mL for Acetaminophen (Paracetamol)
and 100 µg/mL for Salicylate. This test is intended as an
aid for the determination of acetaminophen (paracetamol) and/or
salicylate overdose. The test is used to obtain a visual, qualitative
result and is intended for professional and laboratory use only.
Positive results should be confirmed with quantitative acetaminophen
(paracetamol) and salicylate tests.
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SUMMARY
Salicylate and acetaminophen (paracetamol) are two of the most
frequently used analgesic/antipyretic drugs. Recently, the number
of self-poisonings and suicides with acetaminophen (paracetamol)
or salicylate has grown alarmingly. Over 111,000 acetaminophen (paracetamol)
overdoses are reported to poison control centers with 40,000 associated
ED cases each year in the United States.
Acetaminophen (Paracetamol)
After therapeutic doses of acetaminophen (paracetamol), more than
90% of the drug is metabolized through conjugation to glucuronate
or sulphate in the liver, a non-toxic route. The remaining 10% is
oxidized by P450 within the liver to a highly toxic free-radical
form, N-acetyl-p-benzoquinone imine. At normal doses, this hazardous
metabolite is immediately inactivated by liver glutathione to a
harmless water-soluble product and eliminated in urine or bile.
In the case of overdose, the liver is unable to produce sufficient
glutathione to inactivate the N-acetyl-p-benzoquinone imine. Thus,
the toxic metabolite accumulates in liver cells and eventually causes
hepatic cell death. Acute acetaminophen (paracetamol) toxicity shows
few early signs; liver damage is not clinically manifested for three
days. N-acetylcysteine (NAC) is a viable antidote for acetaminophen
overdose provided therapy begins as quickly as possible. The therapeutic
concentration of acetaminophen (paracetamol) is 10-20µg/mL.
Serum acetaminophen (paracetamol) levels greater than 200µg/mL
at 4 hours post dose or 50µg/mL at 12 hours post dose are
associated with serious liver damage.
Salicylate
Aspirin (acetylsalicylic acid) is a common drug used in many formulations
due to its analgesic and anti-inflammatory properties. Aspirin is
quickly metabolized into salicylate after ingestion and is eliminated
by conjugation with glycine and glucuronic acid.
Salicylate has a direct effect on the respiratory center of the
brain, with overdoses causing hyperventilation. The resulting increased
elimination of CO 2 causes the blood to become abnormally alkaline.This
respiratory alkalosis is complicated by a metabolic acidosis, which
results from an accumulation of salicylic acid and other metabolic
acids in the blood. The therapeutic concentration of salicylate
is 50-350µg/mL. Symptoms of severe salicylate toxicity generally
occur at blood levels in excess of 700µg/mL although some
mild toxicity can occur with patients on long-term, high-dose aspirin
therapies. Determination of Acetaminophen (Paracetamol) and Salicylate
overdose is essential for doctors to properly treat patients in
the emergency department. While quantitative methods are widely
used to measure acetaminophen and salicylate concentrations, these
techniques require instrumentation. Lateral flow immunoassays may
reduce the time for acetaminophen (paracetamol) and salicylate determinations
and serve as a screening method for emergency clinical treatment.
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TEST PRINCIPLE
The Acetaminophen (Paracetamol)/Salicylate Test is a rapid immunoassay,
in which acetaminophen/salicylate-protein conjugates compete with
acetaminophen/salicylate that may be present in the specimen for
limited antibody binding sites. The test device contains a membrane
strip that has been pre-coated with acetaminophen-protein and salicylate-protein
conjugates on the test band regions. Colored anti-acetaminophen
/salicylate antibody-colloidal gold conjugates are placed on a pad
at the end of the membrane. In the absence of drug in test specimen,
the colored antibody gold particles move along with the sample solution
by capillary action across the membrane to the test band regions
forming visible lines as the antibodies and drug conjugates complex.
Therefore, formation of a visible precipitant in the specific test
line occurs when the test specimen is negative for that particular
drug. When a drug is present in test specimen, it competes with
the drug conjugate on the test band region for the limited antibody
binding sites. When an adequate amount of drug is present, it will
fill the limited antibody binding sites and prevent the formation
of red complex on the test line. Therefore, absence of the color
band on the test region indicates a positive result for that particular
drug.
A control or reference band with a different antigen/antibody reaction
is also added to the immunochromatographic membrane strip to indicate
that 1) a sufficient volume of specimen has been added and 2) that
proper flow was obtained. This control line should always appear
regardless of the presence of acetaminophen (paracetamol)/salicylate.
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STORAGE AND STABILITY
The test kit should be stored refrigerated 2-8ºC or at room
temperature (15-30ºC). Each device should remain in its sealed
pouch prior to use.
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PRECAUTIONS
- For in vitro diagnostic use only.
- For professional or laboratory use only.
- Specimens may be potentially infectious. Proper handling and
disposal methods should be established.
- Avoid cross-contamination of samples by using a new specimen
collection container and specimen pipette for each specimen.
- The Dilution buffer contains 0.1% sodium azide. Sodium azide
may react with lead or copper plumbing to form metal azides that
may be explosive. Large quantities of water should be used to
flush the buffer down the sink.
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REAGENTS AND MATERIALS SUPPLIED
- 25 individually wrapped test devices with disposable transfer
pipette. Each test cassette contains a membrane and a colloidal
gold conjugate pad. The membrane is coated with acetaminophen/salicylate-BSA
derivatives in the test region and goat anti-mouse IgG in the
control region. The colloidal gold conjugate pad contains acetaminophen/salicylate
antibody-colloidal gold conjugate and mouse IgG-colloidal gold
conjugate.
- Dilution buffer (8mL): 50mM Tris-HC1 buffer with 0.1% sodium
azide.
- One instruction sheet.
MATERIAL REQUIRED BUT NOT PROVIDED
- Specimen collection container.
- Timer.
- External controls.
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SPECIMEN COLLECTION AND HANDLING
Whole blood
1. A phlebotomist should collect blood into a purple, blue or green
top collection tube (containing EDTA, citrate or heparin, respectively)
by venipuncture.
2. The whole blood may be used for testing immediately or may be
stored at 2-8ºC up to three days.
Plasma
1. Blood should be collected by a phlebotomist into a purple, blue
or green top collection tube (containing EDTA, citrate or heparin,
respectively) by venipuncture.
2. Separate the plasma by centrifugation.
3. Carefully withdraw the plasma for testing or label and store
at 2-8ºC for up to three days.
Serum
1. A phlebotomist should collect blood into a red top collection
tube (containing no anticoagulants) by venipuncture. 2. Allow the
blood to clot and separate serum by centifugation. 3. Carefully
withdraw the serum for testing, or label and store at 2-8ºC
for up to three days.
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TEST PROCEDURE
Review " Specimen Collection" instructions. If stored
refrigerated, the test device, patient samples, and controls should
be brought to room temperature (20-30ºC) prior to testing.
Do not open pouches until ready to perform the test.
- Remove the test device from its protective pouch. Label the
device with patient or control identification.
- Add the specimen to the sample well.
Serum or plasma sample in collection tube:
Hold the provided transfer pipette in vertical position.
Draw the serum sample to the pipette.
Add 2 drops (about 40µL) into the sample well.
Venipuncture whole blood sample in collection tube:
Mix sample before using.
Hold the provided transfer pipette in vertical position.
Draw the serum sample to the pipette.
Add 4 drops (about 80µL) of whole blood into the sample
well.
- After the sample in the sample well has been completely
absorbed, add 3 drops of dilution buffer.
- Read result between 5 to 8 minutes after the addition of dilution
buffer. Do not read result after 8 minutes.
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INTERPRETATION OF RESULTS
Negative:
A colored line appears in the control region. A sample is negative
for the drug if a colored line appears in the Test Region adjacent
to its name.
Positive:
A colored line appears in the control region. A sample is positive
for the drug if no colored line appears in the Test Region adjacent
to its name.
Invalid:
No line appears in the control region. Under no circumstances should
a positive sample be identified until the control line forms in
the viewing area. If the control line does not form, the test result
is inconclusive and the assay should be repeated.
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QUALITY CONTROL
Good laboratory practice recommends the use of control materials.
Each laboratory should follow the appropriate federal, state and
local guidelines concerning external control requirements.
Quality control specimens are available from commercial sources.
The controls should be challenging to the cutoff concentration.
When testing the positive and negative controls, use the same assay
procedure as with a serum, plasma or venipuncture whole blood specimens.
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LIMITATIONS
1. There is the possibility that other substances and/or factors
may interfere with the test and cause erroneous results (e.g. technical
or procedural error). Refer to the
Specificity section in this insert for a list of substances that
will produce positive results and substances that do not interfere
with test performance.
2. A positive result with this test only indicates the presence
of acetaminophen (paracetamol) and/or salicylate. It does not reflect
the degree of toxicity.
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EXPECTED RESULTS
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PERFORMANCE CHARACTERISTICS
Accuracy Study
The accuracy study was performed at three point-of-care sites. Acetaminophen
(Paracetamol) and Salicylate were spiked into the individual serum
samples at various concentrations (from 0 µg/mL to over 300
µg/mL) including ten near-cutoff concentrations (five within
25% below and five within 25% above). The serum samples were tested
with the Acetaminophen (Paracetamol)/Salicylate Test. The acetaminophen
(paracetamol) and salicylate concentrations were confirmed with
the Sigma Diagnostics Acetaminophen (Paracetamol) and Salicylate
tests. The summarized results are shown in Tables I and II.
B. Specificity
The following structurally related compounds at levels equal to
or greater than the concentrations listed below produced positive
results when tested with Acetaminophen (Paracetamol)/Salicylate
Test device:
Acetaminophen (Paracetamol), Acetaminophen
(Paracetamol) 25µg/mL, Bezafibrate 5µg/mL, Salicylate,
Salicylate 100µg/mL, Aspirin 50µg/mL, p-Aminosalicylic
Acid 12.5µg/mL
The following structurally related compounds were found not to
cross- react when tested at concentrations of 10 µg/mL and
100 µg/mL:
Acetaminophen (Paracetamol), Aniline, Acetophenetidin,
Hippuric Acid, o-Hydroxyhippuric Acid, p-Benzoquinone, 4-Aminophenol,
Salicylate, 3-Methyl salicylate, Gentisic Acid, Sulfasalazine, 3-Aminosalicylic
Acid, 5-Aminosalicylic Acid, Diflunisal, Salsalate
The following structurally unrelated compounds, which are likely
to be present in human body, were tested with Acetaminophen (Paracetamol)/Salicylate
Test. They were found not to be positive when tested at concentrations
of 10 µg/mL and 100 µg/mL:
Albumin, Amitriptyline, D-Amphetamine, L-Amphetamine,
Amobarbital, Amoxapine, Ampicillin, Aspartame, Atropine, Baclofen,
Benzocaine, Benzoylecgonine, (+)-Brompheniramine, Bupivacaine, Buspirone,
Butabarbital, Caffeine, Carbamazepine, Carisoprodol, Chlordiazepoxide,
Chloroquine, (+)-Chlorpheniramine, (+/-)-Chlorpheniramine, Chlorpromazine,
Chlorprothixene, Chlorthalidone, Clobazam, Clofibrate, Cocaine,
Codeine, Creatine, Creatinine, Cyclobenzaprine, g-Cyclodextrin,
Cyproheptadine, Dantrolene, Delorazepam, ( _ )-Deoxyephedrine, Dexamethasone,
Dextromethorphan, Diazepam, Dicyclomine, 4-Dimethylaminoantipyrine,
Diphenhydramine, 5,5-Diphenylhydrantoin, Dopamine, Doxepin, Doxylamine,
Ecgonine, Ecgonine methyl ester
EDDP, ( _ )-Ephedrine, (+/-)-Ephedrine, (+/-)-Epinephrine, Erythromycin,
Famprofazone, Fenofibrate, Fluoxetine, Furosemide, Gemfibrozil,
Glucose, Guaiacol Glyceryl Ether, Hemoglobin, d,l-Hematropine, Hydrochlorothiazide,
Hydrocodone, Hydromorphone, Ibuprofen, Imipramine, (+/-)-Isoproterenol,
Ketamine, Lidocaine, MDA (methylenedioxyamphetamine), (+/-)-3,4-MDMA
(Methylenedioxy- methamphetamine), Mebeverine, Mephentermine, Maprotiline,
Meperidine, MeS, Methadol, Methadone, Methamphetamine, Methapyrilene,
Methaqualone, (1R,2S)-( _ )-N-Methyl-Ephedrine, Methylphenidate,
Metoclopramide, Morphine, Morphine-3-ß-D-glucuronide, Naloxone,
Naltrexone, ß-Naphthaleneacetic acid, (+)-Naproxen, ( _ )-Nicotine,
Nicotinic acid, (+/-)-Norephedrine, Nortriptyline, Noscapine hydrochloride,
Ofloxacin, Orphenadrine, Oxalic Acid, Oxazepam, Oxycodone, Penicillin-G,
Pentobarbital, Perphenazine, Phencyclidine, Phenelzine, Pheniramine,
Phenobarbital, Phenothiazine, Phentermine, L-Phenylethylamine, ß-Phenylethylamine,
Primidone,Procaine, Promazine, Promethazine, d-Propoxyphene, Protriptyline,
Pseudoephedrine, Quinidine, Quinine, Riboflavin, Ritodrine, Ranitidine,
Secobarbital, Sodium Chloride, Sulindac, Thioridazine, Trehalose,
Trifluoperazine, Tyramine, Vitamin C
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