Lab Medicine Conference :

Toxicology Studies

Jim Holliman, M.D., F.A.C.E.P.Professor of Surgery and Emergency MedicineDirector, Center for International Emergency MedicineM. S. Hershey Medical CenterPenn State UniversityHershey, Pennsylvania, U.S.A.

Lab Medicine : ToxicologyLecture Topics

ƒ General use of toxicolgy studiesƒ Toxicology lab methodologiesƒ Components of drug & toxin

screensƒ Clinical interpretation of drug &

toxin quantitative levelsƒ Relative costs of different lab

methods

Drug Screens :Types or Levels

ƒ Drugs of abuse–Rapid urine assay

ƒ Comprehensiveƒ Statƒ Volatiles

Drug Screens :Role in Clinical Careƒ Rarely has significant impact on

clinical careƒ 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 dataƒ Psychiatric evaluation to rule

out organic brain syndrome

Drug Screens :Caveats

ƒ Many false positivesƒ 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 negativesƒ 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 historyƒ List other suspected toxinsƒ 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 everythingƒ Expert advice is readily availableƒ 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 & reliableƒ Often used for comprehensive

screensƒ Expensive

Tox Lab Methodology : Gas Chromato-graphy 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 Most commonly employed in hospitals

ƒ Rapid screening testƒ Easy to performƒ Intermediate costƒ However inaccuracies are

common

Toxicology Lab Methodology :EMIT Immunoassay System

ƒ Specimen usually urine ; may be serumƒ First, add antibody directed against the

drug being analyzed forƒ Then add the drug linked to an enzymeƒ When active, the enzyme converts NAD to

NADHƒ NADH absorbs 355 nm wavelength lightƒ Absorbance amount correlates with

concentrationƒ 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 Similar operation theory to EMITƒ Quantity of drug-specific antibody added to

specimenƒ Then known amount of radioactive-labeled

drug addedƒ Precipitate then analyzed with gamma counterƒ Emittance is inversely correlated with drug

concentrationƒ Method is slow & expensiveƒ 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 Barbituratesƒ Cocaineƒ Amphetaminesƒ Marijuanaƒ Benzodiazepinesƒ Opiatesƒ 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 $ 100 ($ 86 to confirm)

Rapid Turnaround

50 ml urine or 14 ml blood (2 red tops)

16 to 40 hours

$ 100 to $ 260

Stat 50 ml urine & 14 ml blood(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 bloodƒ Caveats : metabolized to formate ; late

presenters may not have detectable levelsƒ 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 :Isopropanolƒ Assay : GC or GLC ; done on serumƒ 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 Glycolƒ Assay : GC with OV-1 column or mass

spectrometry ; done on serumƒ 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 / mlƒ 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/dlƒ 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 :Theophyllineƒ Assay : EMIT, HPLC ("gold standard") ;

done on serumƒ Caveat : distinguish chronic from acute

ingestionsƒ Therapeutic levels : 10 to 20 mcg/mlƒ 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/mlƒ 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 :Phenobarbitalƒ Assay : EMIT, GC, RIA ; done on

serumƒ 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 serumƒ Caveats : also metabolized to

phenobarbitalƒ 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/mlƒ 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 :Carbamazepineƒ Assay : EMIT, HPLC ; done on

serumƒ Caveat : active metabolite

carbamazepine epoxide not included as part of results in EMIT assay

ƒ Therapeutic levels : 4 to 12 mcg/mlƒ 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/mlƒ 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/mlƒ Toxicity : usually seen at > 8 mcg/ml ; EKG

and QRS widening demonstrates toxicityƒ 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/Lƒ 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 : Arsenicƒ Assay : 24 hour urine best ; also blood level by

AASƒ Caveats : check recent diet, as a seafood or

kelp meal will dramatically increase levelƒ 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 : Mercuryƒ 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 :Paraquatƒ 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 Levelsƒ 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 : ToxicologySummaryƒ Should familiarize yourself with your

lab's toxicology assay capabilitiesƒ 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 after-the-fact confirmation, even though they will not affect acute clinical management