Lab Medicine Conference : Toxicology Studies Jim Holliman, M.D., F.A.C.E.P. Professor of Surgery and Emergency Medicine Director, Center for International Emergency Medicine M. S. Hershey Medical Center Penn State University Hershey, Pennsylvania, U.S.A. Lab Medicine : Toxicology Lecture Topics ƒ ƒ ƒ ƒ General use of toxicolgy studies Toxicology lab methodologies Components of drug & toxin screens Clinical interpretation of drug & toxin quantitative levels ƒ Relative costs of different lab methods Drug Screens : Types or Levels ƒ Drugs of abuse –Rapid urine assay ƒ Comprehensive ƒ Stat ƒ Volatiles Drug Screens : Role in Clinical Care ƒ Rarely has significant impact on clinical care ƒ Should be confirmatory –Most diagnoses made from history, physical, (& in some cases) other lab tests ƒ Rx is often required before the information is available Drug Screens : Indications ƒ Comatose patient with many potential diagnoses or etiologies ƒ Part of final brain death verification ƒ Academic manuscript data ƒ Psychiatric evaluation to rule out organic brain syndrome Drug Screens : Caveats ƒ Many false positives ƒ Cross-reactions between substances with similar structures –Most commonly : over-the-counter sympathomimetics react like amphetamines ƒ Information about false positives is available from the company or lab Drug Screens : Caveats (cont.) ƒ Many false negatives ƒ Many screens are simply not comprehensive & may not screen for the drug that is suspected ƒ Drug can be present but below detection limit –Dilute urines may have concentration below detection limit ƒ Rapid acting toxins (e.g., TCA's) may present before significant amounts are excreted into urine Drug Screens : Importance of Communication with the Laboratory ƒ Request form should list confirmed ingestants ƒ List patient's medication history ƒ List other suspected toxins ƒ Lab can then prioritize screening to look for specific drugs first, & shorten turnaround time Toxicology Screens : Role of the Poison Control Center ƒ Interpretation is everything ƒ Expert advice is readily available ƒ Many information systems accessed Toxicology Lab : Quantitative Analysis ƒ Often critical to patient management –Need for antidote administration –Need for extracorporeal elimination procedures –Serial measurements help anticipate therapies and proper setting for care ƒ Be careful about measurement units when interpreting results Toxicology Lab : Methodology ƒ Thin - layer chromatography (TLC) –e.g., Toxi-Lab –Glass plate coated with absorbent (silicic acid or aluminum oxide) –Solvent system (varies by lab) sits at base –Specimen (usually urine) is added –Interaction of drugs with sorbent & solvent leads to partitioning –Plate is dried & developed Toxicology Lab : Methodology for TLC (cont.) ƒ Patterns are characteristic of toxins, drugs, & their metabolites ƒ Requires pattern recognition by technician –Experience = better results ƒ Time consuming (typically 4 hours) ƒ Cost : generally low for materials & labor Toxicology Lab Methodology : Gas Chromatography (GC) ƒ A volatile substance is dissolved in a solvent (e.g., n-propanol) ƒ Specimen injected into chromatograph & then subjected to heat vaporization ƒ Specimen is carried thru a column packed with a substance (Carbowax or adiponitrile) that alters the retention time ƒ A detector (flame ionization, electron capture, or nitrogen phosphorus) plots the emergence Toxicology Lab Methodology : Gas Chromatography (cont.) ƒ Standard patterns compared for recognition of substance ƒ Area of the plot also correlates with quantification ƒ Use of GC : is primary method for toxic alcohols (methanol, ethylene glycol, isopropanol) ƒ Problems : –Set-up time & standardization required –Not readily available at all hospital labs –Expensive Toxicology Lab Methodology : High Pressure Liquid Chromatography (HPLC) ƒ Methods & principles same as for GC, but not restricted to volatiles ƒ High pressure pump (1000 to 6000 psi) facilitates movement thru the column ƒ Accurate & reliable ƒ Often used for comprehensive screens ƒ Expensive Tox Lab Methodology : Gas Chromatography Mass Spectrophotometry (GC - MS) ƒ Same as GC, but then linked to a mass spectrophotometer as emerges from column ƒ Like getting a fingerprint of a toxin ƒ This is the "gold standard" for drug screening ƒ Is the confirmatory test for legal specimens Toxicology Lab Methodology : Enzyme Mediated Immunoassay Techniques (EMIT) ƒ ƒ ƒ ƒ ƒ Most commonly employed in hospitals Rapid screening test Easy to perform Intermediate cost However inaccuracies are common Toxicology Lab Methodology : EMIT Immunoassay System ƒ Specimen usually urine ; may be serum ƒ First, add antibody directed against the drug being analyzed for ƒ Then add the drug linked to an enzyme ƒ When active, the enzyme converts NAD to NADH ƒ NADH absorbs 355 nm wavelength light ƒ Absorbance amount correlates with concentration ƒ Spectrophotometer then records & quantifies Toxicology Lab Methodology : EMIT (cont.) ƒ If no drug is in specimen, the antibody will bind the drug - linked enzyme & inactivate this reaction ƒ If drug is present, it will bind the antibody, leaving some drug - enzyme complexes active to convert the NAD to NADH ƒ Note : if low concentrations of drug are present, only a small amount of NADH is produced ; this yields result below detection cut-off & will be reported as negative Toxicology Lab Methodology : Radioimmunoassay (RIA) ƒ ƒ ƒ ƒ ƒ Similar operation theory to EMIT Quantity of drug-specific antibody added to specimen Then known amount of radioactive-labeled drug added Precipitate then analyzed with gamma counter Emittance is inversely correlated with drug concentration ƒ Method is slow & expensive ƒ Good for low concentration drugs (Digoxin, LSD, paraquat) Toxicology Lab Methodology : Atomic Absorption Spectrophotometry ƒ Most accurate & usual method for inorganic agents ƒ Used for metals : –Iron –Lead –Arsenic –Mercury –Thallium –Cadmium Toxicology Lab : H.M.C. Drugs of Abuse Screen ƒ ƒ ƒ ƒ ƒ ƒ ƒ Barbiturates Cocaine Amphetamines Marijuana Benzodiazepines Opiates At other labs, PCP is often added Toxicology Lab : Drug Screens ƒ Rapid turnaround, comprehensive, & stat tests (by American Medical Labs) by class : –Narcotics –Amphetamines –Phenothiazines –Tranquilizers –Hypnotics –Antidepressants –and 60 other drugs Toxicology Lab : Drug Screens Turnaround Time & Costs Test Specimens Turnaround Cost Drugs of Abuse 50 ml urine 2 hours Rapid Turnaround 50 ml urine or 14 ml blood (2 red tops) 50 ml urine & 14 ml blood 16 to 40 hours $ 100 ($ 86 to confirm) $ 100 to $ 260 Stat (1 red, 1 grey) 4 to 10 hours $ 440 Toxicology Lab : Heavy Metal Screens ƒ Includes : mercury, arsenic, lead –Also available : thallium, cadmium –Others may be available on request ƒ Specimen is 24 hour urine collection in special metal-free container ƒ Spot urine can be used in emergency situations ƒ Symptoms & clinical presentation guide therapy, since turnaround times are long Toxicology Lab : Analysis for Drugs or Toxins in Substances ƒ Analysis for drugs or toxins (liquid or solid) contained in other substances can be performed by some labs ƒ Used for checking for adulterants in food products or health store herbal medicines Toxicology Lab Quantifications : Ethanol ƒ Gas chromatography is the "gold standard" ƒ Enzymatic oxidative - reductive tests include the alcohol dehydrogenase technique –Interfering substances include methanol, isopropanol, formaldehyde, & fluoride ƒ Breathalyzers give result based upon blood ethanol to breath ethanol ratio of 2100 : 1 ƒ Plasma or serum levels are 16 to 18 % higher than whole blood levels Toxicology Lab : Ethanol (cont.) ƒ Comparison of different unit equivalents reported for legal intoxication level : –0.10 % weight / volume –100 mg / deciliter or 100 mg % –1.0 gram / liter Toxicology Lab Quantifications : Methanol ƒ Assay : GC ; done on blood ƒ Caveats : metabolized to formate ; late presenters may not have detectable levels ƒ Toxicity : serum bicarbonate & / or formate level (by reference lab) provide best correlation ƒ Interventions : Begin ethanol infusion if strong history or osmolal gap evident while awaiting level ; for levels > 20 to 50 mg/dl, start ethanol infusion, hemodialysis, & 4-methylpyrazole Toxicology Lab Quantifications : Isopropanol ƒ Assay : GC or GLC ; done on serum ƒ Caveat : metabolized to acetone ; persistent acetone levels contribute to prolonged CNS effects ƒ Toxicity : > 50 mg / dl ; is twice as potent as ethanol ƒ Interventions : supportive care ; rarely need hemodialysis Toxicology Lab Quantifications : Ethylene Glycol ƒ Assay : GC with OV-1 column or mass spectrometry ; done on serum ƒ Caveat : late presenting cases (after metabolism to glycolate & oxalate) may not have detectable levels –Glycolate levels also available at reference labs ƒ Toxicity : serum bicarbonate level may be better correlation (glycolate level may also be better correlate) ƒ Interventions : consider ethanol infusion when toxicity is suspected while awaiting level –For levels > 20 to 50 mg/dl : use ethanol infusion, also hemodialysis, and consider 4-methylpyrazole Toxicology Lab Quantifications : Acetaminophen ƒ Assay techniques : EMIT, GC, HPLC, Fluorescence polarization immunoassay ; done on serum –Colorimetric & other immunoassays may be unrelable ƒ Therapeutic level : 5 to 20 micrograms / ml ƒ Toxicity : plot on Rumack - Matthew nomogram –Based on 150 mcg/ml toxic at 4 hours & then a 4 hour half life ƒ Antidote : n-acetylcysteine –Begin Rx while awaiting level if > 8 hours from ingestion Toxicology Lab Quantifications : Salicylates ƒ Assay : Old method is colorimetric (Trinder) ; now use GC, HPLC, Fluorescence polarization ; done on serum ƒ Caveat : chronic ingestions may have serious toxicity at levels > 40 mg/dl ƒ Therapeutic levels : 10 to 20 mg/dl ƒ Toxicity : is time - dependent ; > 100 mg/dl is life threatening ; clinical symptoms are best measure & more useful than Done nomogram ƒ Interventions : activated charcoal, alkaline diuresis, hemodialysis if life - threatening Toxicology Lab Quantifications : Theophylline ƒ Assay : EMIT, HPLC ("gold standard") ; done on serum ƒ Caveat : distinguish chronic from acute ingestions ƒ Therapeutic levels : 10 to 20 mcg/ml ƒ Toxicity : potentially life - threatening at : > 100 mcg/ml if acute, > 40 mcg/ml if chronic ƒ Interventions : activated charcoal, but is difficult to control emesis ; hemoperfusion or dialysis needed for life - threatening toxicity Toxicology Lab Quantifications : Phenytoin ƒ Assay : GLC, HPLC, EMIT, RIA ; done on serum ƒ Caveats : For therapeutic drug monitoring, the free drug level may be most useful ƒ Therapeutic levels : 10 to 20 mcg/ml ƒ Toxicity : cardiovascular toxicity when given intravenously is actually due to the diluent propylene glycol ; acute oral poisoning does not require cardiac monitoring ƒ Interventions : supportive care & activated charcoal Toxicology Lab Quantifications : Phenobarbital ƒ Assay : EMIT, GC, RIA ; done on serum ƒ Caveats : tolerance & enhanced enzyme metabolism affect toxicity & its duration ƒ Therapeutic levels : 15 to 40 mcg/ml ƒ Toxicity : occurs if non-tolerant at 40 to 60 mcg/ml ƒ Interventions : ventilatory & cardiovascular support, multiple doses activated charcoal, alkaline diuresis, +/hemodialysis Toxicology Lab Quantifications : Primidone (Mysoline) ƒ Assay : GLC ; done on serum ƒ Caveats : also metabolized to phenobarbital ƒ Therapeutic level : 5 to 15 mcg/ml, with 10 to 20 mcg/ml phenobarbital ƒ Toxicity : same problems as for phenobarbital ƒ Interventions : supportive care Toxicology Lab Quantifications : Valproate (Depakote, etc.) ƒ Assay : GC-MS, GC, GLC, EMIT ; done on serum ƒ Caveats : distinguish between immediate & delayed release products ƒ Therapeutic levels : 50 to 100 mcg/ml ƒ Toxicity : Beyond 100 mcg/ml increasing levels correlate with worsening CNS depression ƒ Interventions : activated charcoal ; anecdotal reports of benefits from extracorporeal removal Toxicology Lab Quantifications : Carbamazepine ƒ Assay : EMIT, HPLC ; done on serum ƒ Caveat : active metabolite carbamazepine epoxide not included as part of results in EMIT assay ƒ Therapeutic levels : 4 to 12 mcg/ml ƒ Toxicity : good correlation with level ; clinically apparent when > 20 mcg/ml ƒ Interventions : activated charcoal ; rarely need to consider hemoperfusion Toxicology Lab Quantifications : Digoxin ƒ Assay : RIA, EMIT, Fluorescence polarization assay ; done on serum ƒ Caveat : spurious results with digoxin - like immunoreactive substances (DLIS) or after digoxin Fab (antibody fragment ) Rx ƒ Therapeutic levels : 0.8 to 2.0 ng/ml ƒ Toxicity : good correlation with chronic toxicity or with acute toxicity after 6 hours from overdose (after the slow alpha distribution phase) ƒ Interventions : use digoxin antibody fragments (do not wait for level if dysrhythmias are present) Toxicology Lab Quantifications : Procainamide ƒ Assay : EMIT, HPLC, Fluorometric ; done on serum ƒ Caveats : metabolite N-acetylprocainamide (NAPA) contributes to toxicity, especially in renal insufficiency ƒ Therapeutic levels : < 16 mcg/ml procainamide –5 to 25 mcg/ml total procainamide + NAPA ƒ Interventions : activated charcoal & hemodialysis Toxicology Lab Quantifications : Quinidine ƒ Asay : Fluorometric, EMIT, HPLC, GC-MS ; done on serum ƒ Caveat : quinine interferes with HPLC method ƒ Therapeutic levels : 2 to 6 mcg/ml ƒ Toxicity : usually seen at > 8 mcg/ml ; EKG and QRS widening demonstrates toxicity ƒ Interventions : consider serum alkalinization with sodium bicarbonate +/- induced respiratory alkalosis if intubated (acidified urine would enhance elimination, but may aggravate toxicity) Toxicology Lab Quantifications : Lithium ƒ Assay : spectrophotometric or emission flame photometry ; done on serum (reported as meq/liter) ƒ Caveats : Has very slow distribution phase, & so levels do not always correlate with acute toxicity ; levels correlate with neuromuscular symptoms ƒ Therapeutic levels : 0.5 to 1.2 meq/L ƒ Toxicity : acute when sustained > 3.0 meq/L ; chronic toxicity may persist despite undetectable level ƒ Interventions : IV fluid replacement, hemodialysis if acute > 4.0 meq/L, consider if chronic > 1.5 to 2.0 meq/L Toxicology Lab Quantifications : Iron ƒ Assays : Atomic absorption spectrometry (AAS) is "gold standard" ; colorimetric & RIA also available ; done on serum ƒ Caveats : After deferoxamine, AAS is only reliable assay for free iron ƒ Toxicity : > 300 to 500 mcg/dl six hours after ingestion ; 500 to 1000 mcg/dl is serious, while > 1000 mcg/dl is potentially lethal ; symptoms correlate well with levels ƒ Interventions : chelation with deferoxamine ; chelation should begin before level is returned in patients with cardiovascular or CNS symptoms ƒ Total iron binding capacity (TIBC) is no longer considered a useful test Toxicology Lab Quantifications : Heavy Metals : Lead ƒ Assay : whole blood lead is best by AAS ; free erythrocyte protoporphyrin (FEP) is a good screening test for severe lead exposure (> 60 mcg/dl) ƒ Toxicity : see effects at > 10 mcg/d ƒ Interventions : should do chelation if child level > 25 mcg/dl, if adult > 25 to 40 mcg/dl –Monitor & consider chelation with DMSA at > 15 mcg/dl ; if encephalopathy, use BAL & EDTA, & consultation recommended Toxicology Lab Quantifications : Heavy Metals : Arsenic ƒ Assay : 24 hour urine best ; also blood level by AAS ƒ Caveats : check recent diet, as a seafood or kelp meal will dramatically increase level ƒ Toxicity : > 100mcg/Liter after acute exposure, but toxicity may be present after chronic or severe acute exposure with lower levels ƒ Interventions : Chelators British Anti-Lewisite (BAL) parenterally & oral dimercaptosuccinic acid (DMSA) or D-penicillamine –Consultation recommended –Repeat assays monitor response to chelation Toxicology Lab Quantifications : Heavy Metals : Mercury ƒ Assay : whole blood (especially inorganic & elemental) by AAS or also on 24 hour urine ƒ Toxicity : blood level > 1 to 3 mcg/dl, urine level > 50 mcg/Liter ƒ Interventions : chelation with BAL, DMSA, +/- penicillamine ; hemodialysis often required –Consultation recommended Toxicology Lab Quantifications : Paraquat ƒ Assay : RIA ; done on serum ; –Urine screen : Add 1 ml 1% sodium dithiorite in 2N sodium hydroxide to 10 ml urine blue color implies paraquat, blue-green color implies diquat ƒ Toxicity : Level & time post-ingestion correlate with probability of survival ƒ Interventions : hemoperfusion if < 8 hours from ingestion Toxicology Lab Quantifications : RBC & Plasma Cholinesterase Levels ƒ Assays : Michel, Ellman, Delta pH, & micro-Michel methods used –Specimen is 10 ml heparinized tube & must separate plasma within 1 to 2 hours ƒ Caveats : Not useful in acute management ; clinical symptoms & findings dictate therapy ƒ Monitoring serum levels : useful technique to assess for occupational exposure & return to work potential –> 50 % reduction causes mild symptoms –80 to 90 % reduction causes moderate symptoms –> 90 % reduction causes severe symptoms ƒ Interventions : Surface removal, decontamination, atropine, pralidoxime, & supportive care Lab Medicine : Toxicology Summary ƒ Should familiarize yourself with your lab's toxicology assay capabilities ƒ Consider lab results in light of clinical findings –If lab result does not fit with clinical situation, should confirm lab result with another test ƒ Lab tests will sometimes be needed for afterthe-fact confirmation, even though they will not affect acute clinical management