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Clinical Applications: Understanding Immunoassay Drug Testing Cross-reactivity Online Training

Describe antibody preparation and specificity. State how antibody-based immunoassays are used in the detection of drugs and drug metabolites. Identify cross-reactivity issues for several drugs and drug classes. State how cross-reactivity performance data may be used to assist in drug test result interpretation.  This clinical laboratory training qualifies for continuing education units (CEU).

Welcome to the Clinical Applications: Understanding Immunoassay Drug Testing Cross-reactivity Online Training course.  The course will focus on the cross-reactivity issue in immunoassays used for drugs-of-abuse testing. Select Next to continue. Upon successful completion of this course, you will be able to: Describe antibody preparation and specificity   State how antibody-based immunoassays are used in the detection of drugs and their metabolites   Identify cross-reactivity issues for several drugs and drug classes State how cross-reactivity performance data may be used to assist in test result interpretation   Select Next to continue. Antibody Light chain Variable Constant Fc Heavy chain -S-S- -S-S- -S-S- -S-S- Fab Antibody Antigen Cross-reactants No Cross-reactivity Antigen                                            Cutoff (ng/mL)                                            Concentration (ng/mL) necessary for result equivalent to cutoff Example: ∆9-THC-COOH cutoff = 50 ng/mL 11-OH-∆9-THC (metabolite): 77 ng/mL gives result equivalent to 50 ng/mL cutoff 50 ng/mL cutoff   x  100% = 64.9% cross-reactivity 77 ng/mL for cross-reactant positive   Contribution of multiple metabolites ∆9-THC-COOH: 27 ng/mL x 100% cross-reactivity = result equivalent to 27 ng/mL ∆9-THC-COOH glucuronide: 54 ng/mL x 53% cross-reactivity = result equivalent to 29 ng/mL 11-OH-∆9-THC: 5 ng/mL x 65% cross-reactivity = result equivalent to 3 ng/mL Total immunoreactivity = 27 + 29 + 3 = 59 ng/mL = positive   Drug concentration Assay Response High control Drug-free control Cut-off "Negative" Specimen Cutoff Positive Specimen Cutoff Estimated Concentration Estimated Concentration Assay Response “… Amphetamines Assay provides only a preliminary analytical test result. A more specific alternative chemical method must be used to obtain a confirmed analytical result. Gas chromatography/mass spectrometry (GC/MS) is the preferred confirmatory method. Other chemical confirmation methods are available. Clinical consideration and professional judgment should be applied to any drug-of-abuse test result, particularly when preliminary positive results are used.” Derivatized sample   Carrier gas Column Oven Detector Gas chromatogram Mass spectrum Charge / mass separation Ionizer Ion detector Amphetamines Benzodiazepines Cannabinoids Cocaine Opiates Amphetamines Drug Cross-Reactivity d-Methamphetamine 500/500 x 100% =  100% d,l-Amphetamine 500/1,050 x 100% = 47.6% l-Methamphetamine 500/1,325 x 100% = 37.7% MDMA (“Ecstasy”) 500/9,150 x 100% = 5.5% d,l-Pseudoephedrine 500/2,600,000 x 100% = 0.0192% l-Ephedrine 500/800,000 x 100% = 0.0625% Compound 300 ng/mL Cutoff (µg/mL) 500 ng/mL Cutoff (µg/mL) 1000 ng/mL Cutoff (µg/mL) Acetaminophen 1000 1000 1000 α-Acetyl-N,N-dinormethadol (dinor LAAM), 1-α-Acetylmethadol (LAAM) 25 25 25 N-Acetylprocainamide (NAPA) 400 400 400 Acetylsalicylic Acid, Albuterol, p-Aminobenzoic Acid (PABA), Amitriptyline, Amoxicillin, Atenolol, Benzoylecgonine, Buprenorphine, Caffeine 1000 1000 1000 Carbamazepine 250 250 250 Carisoprodol, Chlorpheniramine 1000 1000 1000 Chlorpromazine 200 200 200 Cimetidine 1000 1000 1000 Clomipramine 2.5 2.5 2.5 Clonidine 1000 1000 1000 Codeine 500 500 500 l-Cotinine 100 100 100 Cyclobenzaprine 1000 1000 1000 Desipramine 300 500 800 Dextromethorphan 1000 1000 1000 Dextrorphan 280 280 280 Diphenhydramine, Doxepin, Doxylamine, l-Epinephrine, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) 1000 1000 1000 Fenoprofen 150 150 150 Fluoxetine 500 500 500 Furosemide 1000 1000 1000 Glutethimide, Haloperidol 500 500 500 Ibuprofen 1000 1000 1000 Imipramine 750 750 750 Isoxsuprine 300 500 500 Ketamine 100 100 100 Ketoprofen, Ketorolac Tromethamine 1000 1000 1000 Labetalol 750 750 750 Lidocaine 1000 1000 1000 LSD 2.5 2.5 2.5 Meperidine, Mescaline, Methadone 1000 1000 1000 Methaqualone 1500 1500 1500 d,l-Methyldopa, l-Methydopa, Monoethylglycinexylidide (MEGX), Morphine 1000 1000 1000 Nalmefene 20 20 20 Naloxone 500 500 500 Naproxen 1000 1000 1000 Nicotinic Acid 500 500 500 Noracetylmethadol (nor LAAM) 25 25 25 11-nor-Δ9-THC-9-COOH 100 100 100 Nortryptyline, Nylidrin 750 750 750 Ofloxacin 100 100 100 Oxazepam 300 300 300 Phencyclidine 1000 1000 1000 Phenelzine 50 100 100 1-Phenylcyclohexylamine (PCA) 50 50 50 Phenytoin (DPH), Phthalic Acid 1000 1000 1000 1-Piperidinocyclohexane Carbonitrile (PCC) 50 50 50 Procainamide, Promethazine, Propoxyphene, Ranitidine 1000 1000 1000 Scopolamine 500 500 500 Secobarbital 1000 1000 1000 Thioridazine 100 100 100 Tolmetin Sodium 2000 2000 2000 Tramadol, Trazodone, Trifluroperazine 1000 1000 1000 Trimethobenzamide 500 500 500 Trimethoprim, Verapamil 1000 1000 1000 Zidovudine (AZT) 2000 2000 2000 Zolpidem 100 100 100 Sympathomimetic Amines Diethylpropion, d,l-Isoproterenol 1000 1000 1000 Metaproterenol 500 500 500 3,4-Methylenedioxypyrovalerone (MDPV), 4-Methylmethcathione (Mephedrone), Methylone 100 100 100 Methylphenidate (Ritalin®) 1000 1000 1000 Phenethylamine 15 20 20 Phenylephrine 1000 1000 1000 Propylhexedrine 20 30 50 3-OH-Tyramine (dopamine) 300 300 300 Drug Usual Dose Half Life, hr Expected Peak, ng/mL Concentration needed for "Positive" result Multiples of usual dose for "Positive" l-Methamphetamine 2-7 mg/day 10-15 ? 250 1325 ng/mL 5x d,l-Pseudoephedrine 15-60 mg/4 hr 5-8 150,000 2,600,000 ng/mL 17x l-Ephedrine 24 mg/4 hr 5-7 100,000 800,000 ng/mL 8x d,l-Phenylpropanolamine 25-75 mg/4 hr 3-4 150,000 1,000,000 ng/mL 7x l-Phenylephrine 10 mg/4 hr 2-3 2,000 >1,000,000 ng/mL >500x “Positive” cutoff = 500 ng/mL Amphetamines Pseudoephedrine (30 mg)   µg/mL Phenylpropanolamine (40 mg) Ephedrine (25 mg) Hours Y. Tseng et al., 2006 Cross-Reactivity @ 500 ng/mL cutoff Drug Concentration to give "positive" result Pseudoephedrine 2,600 µg/mL Phenylpropanolamine 1,000 µg/mL Ephedrine 800 µg/mL Estimating the potential for “false positive” results Pseudoephedrine (30 mg dose): Peak concentrations: ~120,000 ng/mL Cross-reactivity: 0.02%  2,600,000 ng/mL necessary for positive @ 500  ng/mL cutoff 120,000 ng/mL x 0.0002 = Peak result equivalent to 24 ng/mL Negative @ 500 ng/mL cutoff   Legend: 1-Amphetamine 2-Dimethylamylamine DMAA Geranamine   l-Ephedrine Amphetamine Methcathinone Methamphetamine Cathinone MDMA “Ecstasy”   Methylenedioxypyrovalerone MDPV 4-Methylmethcathinone Mephedrone “Meow Meow”   Benzodiazepines Halazepam Diazepam Temazepam Camazepam Prazepam Nordiazepam Oxazepam Ketazolam Clorazepate Demoxepam Norchlordiazepoxide Chlordiazepoxide Compound Concentration (ng/mL) at 200 ng/mL Cutoff Concentration (ng/mL) at 300 ng/mL Cutoff Alprazolam 65 79 7-Aminoclonazepam 2600 8600 7-Aminoflunitrazepam 590 1400 Bromazepam 630 1400 Chlordiazepoxide 3300 7800 Clobazam 260 800 Clonazepam 580 1100 Clorazepate * * Clotiazepam 380 670 Demoxepam 1600 4000 N-Desalkylflurazepam 130 160 N-Desmethyldiazepam 110 140 Diazepam 70 120 Estazolam 90 110 Flunitrazepam 140 350 Flurazepam 190 250 Halazepam 110 160 α-Hydroxyalprazolam 100 150 α-Hydroxyalprazolam glucuronide 110 120 1-N-Hydroxyethlylflurazepam 150 150 α-Hydroxytriazolam 130 190 Ketazolam 100 140 Lorazepam 600 890 Lorazepam glucuronide >20000 >20000 Medazepam 150 210 Midazolam 130 160 Nitrazepam 320 560 Norchlordiazepoxide 2600 4900 Oxazepam 250 350 Oxazepam glucuronide >50000 >50000 Prazepam 90 130 Temazepam 140 210 Temazepam glucuronide 6900 11000 Tetrazepam 70 100 Trizolam 130 180 *Clorazepate degrades rapidly in stomach acid to nordiazepam. Nordiazepam hydroxylates to oxazepam. Cannabinoids Oxidation Oxidation Oxidation ∆9-Tetrahydrocannabinol   ∆9-THC   11-hydroxy-∆9-THC   8,11-dihydroxy-∆9-THC   ∆9-THC-carboxylic acid   ∆9-THC-carboxylic acid   ∆9-THC-carboxylic acid glucuronide   THC Metabolite Cross-reactivity ∆9-THC-COOH 50/50 x 100% = 100% ∆9-THC-COOH glucuronide 50/95 x 100% = 52.6% 11-OH-∆9-THC 50/95 x 100% = 52.6% 8-OH-∆9-THC 50/68 x 100% = 73.5% 8ß,11-diOH-∆9-THC 50/58 x 100% = 86.2% ∆9-Tetrahydrocannabinol (Dronabinol)   HU-210 Dexanabinol   Nabilone (Cesamet) anti-nausea   JWH-018 “Spice”   “K2” CB1 agonist 1995 WIN 55,212-2 CB1 agonist   ∆9-Tetrahydrocannabinol Cocaine Ecgonine methyl ester Benzoylecgonine Cocaine Compound Concentration (µg/mL) at 150 ng/mL Cutoff Concentration (µg/mL) at 300 ng/mL Cutoff Cocaine 18-53 40-119 Ecgonine 2-6 7-20   Compound Concentration Tested (µg/mL) at 150 ng/mL Cutoff Acetaminophen 1000 α-Acetyl N, N dinormethadol (dinor LAAM), L-α-Acetylmethadol (LAAM) 25 N-Acetylprocainamide (NAPA) 400 Acetylsalicylic Acid, Amitriptyline, Buprenorphine, Caffeine, Cimetidine 1000 Clomipramine 2.5 Clonidine 1000 Codeine 500 Cotinine 100 Cyclobenzaprine 1000 Desipramine 800 Dextromethorphan, Diphenhydramine, Doxepin, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), Fluoxetine 1000 Glutethimide 500 Ibuprofen 1000 Ketamine 100 Ketorolac Tromethamine 1000 Lormetazepam 1 LSD 0.01 Meperidine, Methadone 1000 Methaqualone 1500 Morphine, Naproxen, Nortriptyline 1000 Oxazepam 300 Phencyclidine, Phenytoin, Promethazine, Propoxyphene, Ranitidine 1000 Scopolamine 500 Secobarbital 1000 11-nor-Δ9-THC-9-COOH, Thioridazine 100 Tramadol 1000 Tyramine 100 Zidovudine (AZT) 2000 Zolpidem 100 Opiates Morphine Hydromorphone (Dilaudid) Oxymorphone (Numorphan) (Opana) Dihydromorphine Codeine Hydrocodone (Vicodin, Lortab) Oxycodone (Percodan, Oxycontin) Dihydrocodeine (Synalgos) Compound Concentration (ng/mL) at 300 ng/mL Cutoff Concentration (ng/mL) at 2000 ng/mL Cutoff Codeine 102-306 660-1980 Dihydrocodeine 291 1872 Hydrocodone 247 1545 Hydromorphone 498 5349 Levallorphan >5000 >120000 Levorphanol 1048 7698 Meperidine >15000 >400000 6-Acetylmorphine 435 2100 Morphine-3-Glucuronide 626 6167 Nalorphine 5540 >100000 Naloxone 360000 >350000 Oxycodone 1500 23000 Oxymorphone 9300 >100000 . Opiate Cross-Reactivity Morphine 300/300 x 100% = 100% Morphine-3-glucuronide 300/626 x 100% = 47.9% 6-Acetylmorphine 300/435 x 100 = 69% Codeine 300/(102–306) x 100% = 294–98% Hydrocodone 300/247 x 100% = 121% Hydromorphone 300/498 x 100% = 60.2% Oxycodone 300/1,500 x 100% = 20% Oxymorphone 300/9,300 x 100% = 3.2% Morphine Buprenorphine Naltrexone (ReVia,Vivitrol) Methadone Steps to distinguish innocent ingestion of poppy seeds from an opiate drug or heroin: Examine claimed consumption (positives unlikely for >1 day) Eliminate seeds from diet, collect another specimen after 3 days Test for 6-monoacetylmorphine (presence proves heroin use) Measure total morphine levels (>5000 ng/mL unlikely for seeds) Measure morphine/codeine ratio (morphine > codeine for seeds) Look for evidence of opiate use (needle tracks, needles, behavior) Heroin Morphine 6-acetylmorphine Antibodies are large proteins with binding sites that recognize antigens based on their molecular structures Antibodies are developed to detect an analyte but may also detect other molecules with similar molecular shape Detection of multiple drug metabolites may be a desired feature Percent cross-reactivity can be calculated for metabolites that are not detected with the same sensitivity as the target analyte Metabolites contribute to overall assay response in proportion to their concentrations Total test result is the sum of the responses for each of the drugs and metabolites present Amphetamines, cannabinoids, benzodiazepines, cocaine, and opiates are all drug classes for which there are immunoassay tests

  • drugs
  • abuse
  • crossreactivity