K.O. Troubleshooting Guide v.3 0
K.O. Troubleshooting Guide v.3 0
K.O. Troubleshooting Guide v.3 0
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TROUBLESHOOTING GUIDE
DRUGS OF ABUSE
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INTRODUCTION:
The purpose of this guide is to assist our partners in troubleshooting any foreseen or unforeseen events
that may occur while running Innovacon drugs of Abuse tests. All Innovacon drug of abuse panels are
rapid visual immunoassay for the qualitative detection of drug and drug metabolites in human urine. All
panels and all formats provide only a preliminary analytical test result. A more specific alternate
chemical method must be used in order to obtain a confirmed analytical result; Gas
Chromatography/mass spectrometry (GC/MS) is the preferred confirmatory method, but the use of
Liquid Chromatography/mass spectrometry (LC/MS) for confirmation testing is increasing. Clinical
consideration and professional judgment should be applied to any drug of abuse test result, particularly
when preliminary results are indicated.
In addition to the drugs of abuse assays, the One Step S.V.T. (Specimen Validity Test) is a semi-
quantitative color comparison screen for the detection of adulterants. Within one minute (1) of the pads
being activated by the urine sample, the color that appears on the pads can be compared with the
printed color chart on the canister or color chart enclosed in the kit box. The S.V.T. also provides a
preliminary screen only. A more specific alternate chemical method must be used in order to obtain a
confirmed analytical result. Abnormal results should be sent to a laboratory for confirmation.
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BACKGROUND:
Amphetamine(s)
Amphetamine is a stimulant drug that is used in treatment of ADHD, narcolepsy and obesity. Most
positive results originate from legitimate, prescription use of the drug. Illicitly, the drug is occasionally
diverted from legitimate prescriptions and sold on the streets.
Technical Notes
1. Common cross-reactants: Prescription appetite suppressants such as Phentermine, other
amphetamine-like prescription drugs; Ecstasy-like club drug called MDA (which is a metabolite
of MDMA). In addition, there are several therapeutic agents, such as Benzphetamine, that
metabolize to amphetamine (and methamphetamine) in the body and will produce positive drug
test results. It is also important to note that amphetamine is a metabolite of methamphetamine
and will appear in the urine of a person who has taken methamphetamine.
2. Current US SAMHSA “screening” cutoff is 1,000 ng/mL
3. Current GC/MS (industry standard) confirmation cutoff is 500 ng/mL.
4. Proposed changes in SAMHSA are:
a. To lower the screening cutoff to 500 ng/mL
b. Change target drug from D-amphetamine (historical) to D-methamphetamine
c. Change the GC/MS confirmation to 250 ng/mL
5. Outside the US, amphetamine screening cutoffs may be 300 ng/mL.
6. INNOVACON manufactures amphetamine assays with cutoff levels of 1,000 and 300 ng/mL.
Both assays are FDA cleared and target d-amphetamine as the primary antigen.
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Methamphetamine(s)
Methamphetamine is a stimulant drug that is very rarely prescribed, but there is a form
(stereoisomer) of methamphetamine that is used in the Over-the-Counter Vick’s Inhalers. Most
positives are the result of illegal use; the drug is made illegally from pseudoephedrine.
Technical Notes
1. About 4-7% of a methamphetamine dose is broken down to amphetamine by the body
2. Common cross-reactants: metabolites of Ephedrine, Ranitidine (Zantac), and MDMA. In
addition, there are several therapeutic agents, such as Benzphetamine, that metabolize to
methamphetamine (and amphetamine) in the body and will produce positive drug test results.
It is also important to note that amphetamine is a metabolite of methamphetamine and will
appear in the urine of a person who has taken methamphetamine.
3. Current US SAMHSA cutoff: grouped together with Amphetamine(s) is 1,000 ng/mL
4. Current GC/MS (SAMHSA) confirmation cutoff is 500 ng/mL; MUST ALSO HAVE AT LEAST
200 ng/mL of amphetamine in the urine to report a viable “methamphetamine” positive
5. Proposed changes in SAMHSA are:
z Change the screening cutoff to 500 ng/mL
z Change target drug from D-amphetamine (historical) to D-methamphetamine
z Change the GC/MS confirmation to 250 ng/mL; amphetamine must still be present in
the urine “around cutoff” to report a methamphetamine positive GC/MS result.
6. Innovacon manufactures methamphetamine assays with cutoff levels of 1,000, 500 and 300
ng/mL. The 500 and 1,000 ng/mL assays are FDA cleared; the 300 ng/mL assay is for outside
the US only. All three assays target d-methamphetamine as the primary antigen.
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Cocaine
Cocaine is a stimulant drug and local anesthetic drug that is derived from the coca plant; the drug is
rarely prescribed. Most drug test positives are from illegal use; “crack” is a “free-base” form of
cocaine that is more readily bio-available.
Technical Notes
1. Cocaine is extensively metabolized in the body to benzoylecgonine, ecognine methyl ester and
ecognine
2. Common cross-reactants: there are No KNOWN cross-reactive compounds with urine-based
cocaine immunoassays but the Innovacon Cocaine assay is considered very sensitive to low
levels of benzoylecgonine in urine.
3. Current US SAMHSA “screening” cutoff: 300 ng/mL
4. Current GC/MS (industry standard) confirmation cutoff: 150 ng/mL of benzoylecgonine
5. Proposed changes in SAMHSA are to lower the screening cutoff to 150 ng/mL
6. Proposed change to GC/MS confirmation level is to lower it to 100 ng/mL of benzoylecgonine
7. INNOVACON manufactures Cocaine assays with cutoff levels of 300 and 150 ng/mL. Both are
FDA cleared and target benzoylecgonine as the primary antigen.
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Opiates
Opiates are a large “class” of drugs used legally for pain-relief (morphine) and cough suppression
(codeine). Heroin (diacetylmorphine) is an illegal opiate made from the opium poppy. In addition to
the primary opiates, “synthetic opiates” are often used in pain relief. The original intent of creating
synthetic opiates was to increase pain relief tendencies and reduce likelihood of dependence;
synthetic opiates include hydrocodone (Vicodan), oxycodone (Oxycontin), hydromorphone
(Dilaudid). Most synthetic opiates do not cross react very well with morphine-based opiate tests.
Technical Notes
1. Common cross-reactants: Poppy seeds contain codeine/morphine and, as such, can cause a
“true positive” opiate test.
2. Current US SAMHSA “screening” cutoff: 2,000 ng/mL
3. Current GC/MS (industry standard) confirmation cutoff: 2,000 ng/mL for codeine and/or
morphine
4. Other cutoff: 300 ng/mL Opiate cutoff was the historical SAMHSA cutoff; still used on occasion
outside regulated drug testing (clinical, OUS)
5. Codeine and heroin metabolize to morphine in the body. Codeine is also eliminated
unchanged.
6. Innovacon manufactures Opiate assays with cutoff levels of 300 and 2,000 ng/mL. Both are
FDA cleared and target morphine as the primary antigen. In addition, INNOVACON has an
oxycodone-specific assay which is described in more detail later in this guide.
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PCP
Phencyclidine (PCP) is a hallucinogen drug originally made for anesthetic use in humans. It’s use
in humans was discontinued due to “negative side effects.” There is no current legal use of the
drug in humans. The use of PCP illegally is rarer now than before, but is sometimes seen in large,
metropolitan areas such as LA and Detroit.
Technical Notes
1. PCP is liquid; marijuana joints are sometimes dipped into PCP (“sherm”)
2. Common cross-reactants: Venlafaxine (Effexor), Lamotrigine (Lamictal)
3. Current SAMHSA “screening” cutoff: 25 ng/mL
4. Current GC/MS (industry standard) confirmation cutoff: 25 ng/mL
5. Innovacon manufactures a PCP assay with a cutoff level of 25 ng/mL.
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Marijuana (THC)
THC (tetrahydrocannabinol) is a hallucinogenic, sedative drug considered the primary active
compound of the 400+ chemicals in the cannabis sativa plant. While there is some legal use of
THC, such as the prescription Marinol (Dronabinol) used for wasting in cancer/HIV patients, most
THC drug test positives are the result of illicit use. Marijuana is the number 1 illegal drug in the US
Technical Notes
1. THC breaks down extensively in the body; most common metabolite is THC-COOH. This
metabolite is also referred to as “carboxy-THC” or “THCA.”
2. Common cross-reactants: the consumption of Sustiva (efavirenz), an HIV treatment drug, is
known to produce metabolites that cross-react on many THC assays. Also, the INNOVACON
test is considered “very sensitive” to low levels in urine
3. Current US SAMHSA “screening” cutoff: 50 ng/mL
4. Current GC/MS (industry standard) confirmation cutoff: 15 ng/mL of THC-COOH
5. No proposed changes to cutoffs in SAMHSA
6. Innovacon manufactures a THC assay with a cutoff level of 50 ng/mL for THC-COOH.
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Benzodiazepines
Benzodiazepines are a large “class” of drugs used primarily for sedation and anxiety relief. Examples
of Benzodiazepines include diazepam (Valium), chlordiazepoxide (Librium), alprazolam (Xanax), and
clonazepam (Klonopin). Most benzodiazepine use in the US is due to prescription use, but some
benzodiazepines are diverted for illegal consumption. Outside the US, particularly in Europe,
benzodiazepines are more widely abused.
Technical Notes
1. Common cross-reactants: Sertraline (Zoloft). Of note, given the large number of
benzodiazepine drugs (35+), be aware that GC/MS labs will not be able confirm for all of them.
2. NOT A SAMHSA-regulated drug, so cutoffs are usually country specific
3. Common US “screening” cutoff: 300 ng/mL
4. Some products/countries use 200 ng/mL screening cutoff
5. GC/MS confirmation cutoffs vary but usually range from 100-200 ng/mL
6. Again, GC/MS labs are limited by the number of benzodiazepines they identify on their GC/MS
analyses
7. Innovacon manufactures BZO assays with cutoff levels of 300 and 200 ng/mL; both target
oxazepam as the primary antigen.
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Barbiturates
Barbiturates are another large “class” of drugs. They are used primarily for controlling epilepsy; some
use for migraine relief (butalbital) and drug-induced comas for head trauma (pentobarbital). Examples
of Barbiturates include phenobarbital, pentobarbital (used for head trauma) and butalbital (Fiorinal).
Most barbiturate positive drug tests are due to prescription use; barbiturates are rarely diverted for
illegal consumption. Some countries have heroin “cut” with phenobarbital.
Technical Notes
1. Common cross-reactants: Possibly Phenytoin which is chemically-related to barbiturates and
used for epilepsy
2. NOT A SAMHSA-regulated drug
3. Common US “screening” cutoff: 300 ng/mL
4. GC/MS confirmation cutoffs vary but usually range from 100-200 ng/mL
5. GC/MS labs are limited by the number of barbiturates they identify on their GC/MS analyses
6. Innovacon manufactures a barbiturate assay with a cutoff level of 300 ng/mL; the target antigen
is secobarbital
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Methadone
Methadone is an analgesic compound that is most commonly used to treat heroin/opiate addiction. In
addition, the drug may be prescribed for pain relief. Patients prescribed methadone for opiate addiction
are considered to be in “methadone maintenance.” Patients in methadone maintenance are drug tested
commonly to ensure they are (1) taking their methadone and (2) not taking heroin/opiates or other
drugs.
Technical Notes
1. Methadone is eliminated in the urine as parent drug and as metabolites. The metabolites
EDDP and EDMP are VERY different in structure from parent methadone, so they do not react
with the drug test.
2. NOT A SAMHSA-regulated drug
3. Common US “screening” cutoff: 300 ng/mL
4. GC/MS confirmation cutoffs: 300 ng/mL
5. Most screening tests do not detect MTD metabolites (EDDP or EDMP). If a patient is an
“extensive metabolizer,” they may not have any parent MTD in the urine
6. Innovacon manufactures a methadone assay with a cutoff level of 300 ng/mL.
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Tricyclic Antidepressants
TCAs are a “class” of drugs used for treating depression. TCAs are the “older” antidepressants.
Examples of TCAs include amitriptyline, nortriptyline, desipramine and clomipramine. New
antidepressant drugs like Prozac, Paxil, Zoloft are NOT TCAs and, as such, will not cross react on the
TCA assay.
Technical Notes
1. TCAs have a very low “lethal dose.” Since patients taking TCAs have a higher likelihood of
suicidal tendencies, TCAs are often implicated in overdose situations.
2. Common cross-reactants: large doses of carbamazepine (Tegretol) metabolites, other
prescription drugs may also cross react
3. NOT A SAMHSA-regulated drug class
4. Common US “screening” cutoff: 1,000 ng/mL
5. Other “screening” cutoff: 300 ng/mL
6. TCAs cannot be analyzed by GC/MS very well, so they are often “confirmed” by HPLC or
another method
7. Innovacon manufactures a TCA assay with a cutoff level of 1,000 ng/mL
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Oxycodone
Oxycodone is a member of the “synthetic” opiate class. The drug is used for pain relief (most
commonly for chronic pain); oxycodone prescriptions are often diverted and sold on the street for illegal
use. Brand names of oxycodone include: Percodan, Percocet and Oxycontin. Oxycontin is a “time
released” formula of oxycodone. Overdoses are most often the result of people crushing the time-
released Oxycontin pills and consuming the crushed powder in one dose.
Technical Notes
1. The structure of oxycodone is not close enough to codeine or morphine to ensure the drug is
detected by regular Opiate immunoassays
2. Not part of the SAMHSA Opiate Class
3. Screening cutoff: 100 ng/mL
4. GC/MS Confirmation cutoff: 100 ng/mL
5. Most laboratories will not automatically confirm for oxycodone if a specimen is “opiate positive.”
Oxycodone-specific confirmation must be requested.
6. Innovacon manufactures an oxycodone-specific assay with a cutoff level of 100 ng/mL. The
OXY assay also shows some cross reactivity to hydrocodone (Vicodan, Lortab); 1,562 ng/mL of
hydrocodone produces a positive OXY test result.
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Propoxyphene
Propoxyphene is a narcotic analgesic structurally related to Methadone; the drug is used for the relief of
mild to moderate pain. Brand names of propoxyphene include: Darvon and Darvocet. Propoxyphene is
often dosed in combination with Aspirin and Acetaminophen.
Technical Notes
1. Although structurally related to methadone, ppx will not cross react on the INNOVACON
Opiates or Methadone assays.
2. PPX is not part of the SAMHSA-regulated drug testing menu
3. US Screening cutoff: 300 ng/mL
4. GC/MS Confirmation cutoff: 300 ng/mL for PPX or metabolite, Nor-PPX
5. Most laboratories will not automatically confirm for Propoxyphene. Propoxyphene-specific
confirmation must be requested.
6. Innovacon manufactures a propoxyphene-specific assay with a cutoff level of 300 ng/mL. The
assay has an equal affinity to the metabolite nor-propoxyphene.
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MDMA/Ecstasy
Ecstasy (MDMA) is a stimulant and hallucinogen structurally related to Methamphetamine; the drug was
once used for psychotherapy, but is now a Schedule 1 drug in the US (no approved medical use). The
chemical name for Ecstasy is 3,4-methylenedioxymethamphetamine, hence the “MDMA” abbreviation.
MDMA metabolizes to many compounds, but the most notable one is MDA
(methylenedioxyamphetamine) which is chemically related to amphetamine.
Technical Notes
1. The structure of MDMA is similar enough to cross-react on the MAMP assays, but the
sensitivity (cutoff) isn’t ideal. As a result, the MDMA-specific assay was developed. The
MDMA assay is reactive to Ecstasy/MDMA at level of 500 ng/mL. This assay is also sensitive
to MDA and MDEA (methylenedioxyethylamphetamine).
2. Not part of current SAMHSA guidelines
3. Screening cutoff: 500 ng/mL
4. Confirmation cutoff: varies, but 250-500 ng/mL is typical
5. Proposed changes in SAMHSA are to add MDMA as a component of the
“Amphetamine/Methamphetamine” drug class.
6. Innovacon manufactures an MDMA-specific assay with a cutoff level of 500 ng/mL. The
MDMA-specific assay is also sensitive to MDA and MDEA (methylenedioxyethylamphetamine).
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Definitions:
FALSE POSITIVES:
False positives are defined by a human urine specimen yielding positive results with an Innovacon drug
of abuse test, then testing negative on a LOD (Limit of Detection) gas chromatography/ mass
spectrometry test. A false positive result in any Immunoassay is most likely cause by cross-reactivity.
The collector needs to verify that the donor has given an accurate list of any medication, prescription
and/or over-the-counter. Please review with cross-reactivity table enclosed in this guide.
FALSE NEGATIVES:
False negatives are defined by a human urine specimen yielding negative results with an Innovacon
drug of abuse test, then testing positive on a gas chromatography/ mass spectrometry test at levels at
least 2 times the stated cut-off level.
INVALID RESULTS:
Invalid results are defined by an absence of a control line on an Innovacon drug of abuse test.
Insufficient specimen volume or incorrect procedural techniques are the most likely reasons for control
line failure.
SCREENING TESTS:
All Innovacon drug of abuse tests are screening tests that provide only a preliminary analytical test
result. The purpose of a screening test is to rule out negative specimens from specimens that may
contain drugs. As with most immunoassays the test is looking for class of drug, many different
chemicals and compounds fall into this category. Not all specimens that have screened positive will
confirm positive.
CONFIRMATION TESTING:
All preliminary positive immunoassay results should be confirmed by a more specific alternative
method. In drugs of abuse testing the gold standard of confirmation is Gas Chromatography/mass
spectrometry (GC/MS) and/or Liquid Chromatography/mass spectrometry (LC/MS). GC/MS and/or
LC/MS testing is performed on any specimen that screens positive it will confirm a specific analyte in
the class of drug such as Benzoylecgonine when a specimen screens positive for Cocaine. Liquid
Chromatography/mass spectrometry is a fairly new confirmation method in drugs of abuse testing, it is
believed that it will become prevalent in the field.
SAMHSA:
SAMHSA is an acronym for Substance Abuse Mental Health Services Administration which is the
governing agency that establishes the cutoff concentrations used in regulated drug testing in the United
States. SAMHSA was formally known as NIDA (National Institute for Drug Abuse) hence the term of
the NIDA 5 drug panel. Regulated drug testing in the United States is limited to testing for 5 drugs
(Cocaine, THC, PCP, Opiates, Amp/Methamp).
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CREATININE (DILUTION):
Dilution is the most common type of adulteration. Dilution can be either “in vivo” (consuming excessive
quantities of fluids in an attempt to dilute the urine) or “in vitro” (introducing liquid into a specimen that
has already been collected). The intention of dilution is to make the concentration of drug in the urine
lower then the detection limit (cutoff) of the test. Creatinine testing in conjunction with specific gravity
testing is a good indication of dilution of the urine sample. The absence of Creatinine (<5 mg/dl) is
indicative of a specimen not consistent with human urine.
SPECIFIC GRAVITY:
Specific gravity tests for sample dilution. Values outside the normal range may be the result of
specimen dilution or adulteration.
pH:
pH tests for the presence of acidic or alkaline adulterants in urine. Values outside the normal range
may indicate that the specimen has been altered or spiked with acidic or alkaline compounds.
NITRITE:
Nitrite is a compound that is introduced into a urine specimen after collection. Nitrite works by oxidizing
the major cannabinoid (THC-COOH) metabolite and making it undetectable. While this mechanism
does work, the time needed for the reaction to occur is usually several hours. This means that after
collection of the urine the rapid test maybe positive and when the sample is tested at the laboratory the
nitrate will have modified the THC metabolite making it undetectable. Some commonly used
commercial adulterants that contain nitrates are “Klear, Whizzes, Mary Jane 13”. Nitrites are
sometimes found in people with urinary tract or bacterial infections.
GLUTARALDEHYDE:
Glutaraldehyde is an older adulterant that is introduced into the urine specimen after collection. It is not
believed to affect the performance of lateral flow tests. Glutaraldehyde denatures the enzyme used in
EMIT-like autoanalyzer reagents. Adulterants such as UrinAid and Clear Choice contain
glutaraldehyde. Glutaraldehyde is not normally found in urine. However certain metabolic
abnormalities such as ketoacidosis (fasting, uncontrolled diabetes, high-protein diets) may interfere with
the test results.
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CROSS-REACTIVITY GUIDE:
Innovacon™ Drugs of Abuse Assays Cross-Reactivity Manual by Trade Name
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1. SPECIMEN
Q: How can specimens be stored prior to testing?
A: Urine specimens may be stored at 2-8°C for up to 48 hours prior to testing. For
prolonged storage, specimens may be frozen and stored below -20°C. Frozen
specimens should be thawed and mixed well before testing. For specimen undergoing
Specimen Validity testing, for best results test specimens immediately. Storage of
urine specimens should not exceed 2 hours at room temperature or 4 hours
refrigerated.
2. TEST PROCEDURE
Q: Q: How does the test work?
A: The test is a lateral flow chromatographic immunoassay for the qualitative detection of
an identified drug or the identified drug metabolites in human urine. The S.V.T. test is a
semi-quantitative color comparison screen for the detection of adulterants.
Q: If the test is used before the refrigerated specimen reaches room temperature, is
the result reliable?
A: No. If refrigerated, the test and the specimen must be at room temperature (15-30°C)
before the test is performed. Specimen at body temperature does not need to reach
room temperature before running the test.
Q: If the test strip was removed from the foil pouch and dropped on the floor prior
to using it, will it still work?
A: If the test is intact and the exposed membrane in the “reaction window” was not
damaged, it can still be used and expected to function properly.
3. INTERPRETATION
Q: Do I have to wait the full amount of time before reading my results?
A: Yes. It is important that you wait to see if a line in the test region appears before
reading your result. This might take the entire suggested number of minutes for the test
region line to appear. Within one minute of the urine specimen activating the pads on
the adulteration strips the colors can be compared.
Q: Can the test results be read after the suggested number of minutes?
A: Yes. The test card and panel results remain stable for up to 1 hour after test initiation.
For specimen validity testing test results cannot be read after 4 minutes.
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Q: The test line is very faint and the control line is very strong at the suggested
minute read time. What does this mean?
A: The shade of red in the test line region (T) will vary, but it should be considered
negative whenever there is even a faint pink line.
Not applicable for Specimen Validity Testing.
Q: When the specimen sample is added to the test, red/pink fluid can be seen
migrating up the membrane. Is this normal?
A: A red/pink background is normal as the sample flows up the test strip and will not affect
the test result. As long as it does not interfere with the interpretation of the line(s), the
background can be disregarded.
Q: How does one know that the test has been performed properly?
A: As an internal procedural control, a red line appearing in the control (C) region confirms
the addition of sufficient specimen volume and the performance of correct procedural
technique.
Q: A negative result is read when two distinct red lines appear, one in the control
region (C) and another in the test region (T). Do the two lines need to be of the
same intensity?
A: No. The intensity of the red color in the test line region (T) will vary. Any shade of a red
line in the test (T) region (darker than, the same color as, or lighter than the control line)
along with a red line in the control (C) region is considered a negative result.
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