Clinical Chemistry Lab - 11 Methods for Toxicology and TDM

CLINICAL CHEMISTRY LABORATORY METHODS FOR TOXICOLOGY AND THERAPEUTIC DRUG MONITORING OUTLINE • Toxicology and Laboratory • Methods in Therapeutic Drug Monitoring o Toxicity o Toxicity Test Procedures Laboratory Assays for Various Substances and Drugs of Abuse o Principle of the Tests o Drug Testing Procedures o Specimen Collection TOXICOLOGY AND LABORATORY METHODS IN THERAPEUTIC DRUG MONITORING TOXICITY TEST PROCEDURES • TOXICITY • • • • • • Toxicity can be measured by the effect the substance has on an organism, a tissue or a cell. We know that individuals will respond differently to the same dose of a substance because of a number of factors including their gender, age and body weight. Therefore a population-level measure of toxicity is often used. The probability of an outcome for a population is then related to a given individual in a population. Lethal dose (LD50) - One such population-level measure is the median lethal dose, LD50 (lethal dose, 50%). This is defined as the dose required to kill half the members of a specific animal population when entering the animal’s body by a particular route. LD50 is a general indicator of a substance’s toxicity within a short space of time. It is a measure of acute toxicity. Acute Toxicity - immediate manifestation of toxicity (death) Chronic Toxicity - small dosages may accumulate and cause some toxic manifestation after a period of time Monitoring o Effective Dose (ED50) - the concentration at which 50% of the individuals who are given a certain drug experience the benefits ▪ Minimum concentration at which a drug is considered effective ▪ Any concentration less than ED50 can’t be considered as effective; decreased chance of treating the individual o Toxic Dose (TD50) - concentration at which 50% of the population experiences toxic effects ▪ Anything in excess is considered bad ▪ Considered as the dosage limit ▪ Therapeutic dosage is between ED50 & TD50 o Lethal Dose (LD50) - concentration at which 50% of the population died ▪ Any value from LD50 and above is considered fatal of dangerous o 50 - refers to 50% of the population; determines by testing a population (clinical trial) Process for Reaching Dosage Decisions with Therapeutic Drug Monitoring • • • Principle of the test o The Diazyme Liquid Stable Enzymatic Lithium Assay is based on a lithium sensitive enzyme whose activity is lithium concentration dependent. The enzyme, a phosphatase, converts its substrate adenosine biphosphate (PAP) to hypoxanthine through a coupled enzymatic reaction to generate uric acid and hydrogen peroxide (H2 O2 ). The H2 O2 generated is then quantified by a Trinder reaction (oxidation of a reduced dye). Materials o Random blood sample o Ion-Selective Electrode (ISE) Method Procedure o The Diazyme Liquid Stable Enzymatic Lithium Assay consists of two liquid stable reagents, R1, and R2, and the assay is performed in a kinetic mode using 5 µl of serum sample and a three point calibration curve. The reaction is monitored at 550 nm and completed within 10 minutes. Results of the test o Understanding Results of Lithium Level o Reference Range &Interpretation ▪ Above 1.5 - Potentially Toxic ▪ Above 2.0 - Severely Toxic ▪ 0.6 to 1.2 - Therapeutic range (used for bipolar disorders) LABORATORY ASSAYS FOR VARIOUS SUBSTANCES AND DRUGS OF ABUSE • • The drug screen rapidly identifies a drug or drugs present in the blood, urine, or gastric contents of a patient suffering from toxicity. Neutral and basic drugs as well as drug metabolites are best detected in urine, whereas acidic drugs are best found in detectable concentrations in blood and serum. Following a positive drug screen, confirmatory methods must be used to quantitatively analyze drug levels in a patient. Confirmatory drug tests are required to confirm and quantify those drugs found in a patient’s serum or urine using drug-screening methods. Utilizes various samples o o Urine - most commonly used Some modern procedures utilize hair ▪ Metabolites can stay in the hair for months PRINCIPLE OF THE TESTS • • Screening o The drug of abuse test is an immunoassay based on the principle of competitive binding. Drugs that may be present in the urine specimen compete against their respective drug conjugate for binding sites on their specific antibody. During testing, a portion of the urine specimen migrates upward by capillary action. A drug, if present in the urine specimen below its cut-off concentration, will not saturate the binding sites of its specific antibody. The antibody will then react with the drug-protein conjugate and a visible coloured line will appear in the test line region of the corresponding drug strip. The presence of drug above the cut-off concentration in the urine specimen will saturate all the binding sites of the antibody. Therefore, no coloured line will form in the test line region. A drugpositive urine specimen will not generate a coloured line in the specific test line region of the strip because of drug competition, while a drug-negative urine specimen will generate a line in the test line region because of the absence of drug competition. To serve as a procedural control, a coloured line will always appear at the control line region, indicating that proper volume of specimen has been added and membrane wicking has occurred. o Immunoassay - ag-ab rxn ▪ Immunochromatography ▪ ELISA • Sandwich ELISA - Absorbance is directly proportional to concentration • Competitive ELISA - absorbance is inversely proportional to the concentration Confirmatory test (GC-tandem MS) - Gas chromatography and Mass spectrometer o The GC-MS is composed of two major building blocks: the gas chromatography and the mass spectrometer. The gas chromatograph utilizes a capillary column whose properties regarding molecule separation depend on the column's dimensions (length, diameter, film thickness) as well as the phase properties. The difference in the chemical properties between different molecules in a mixture and their relative affinity for the stationary phase of the column will promote separation of the molecules as the sample travels the length of the column. The molecules are retained by the column and then elute (come off) from the column at different times (called the retention time), and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass-to-charge ratio. These two components, used together, allow a much finer degree of substance identification than either unit used separately. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The o o o mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame ionization detector) cannot differentiate between multiple molecules that happen to take the same amount of time to travel through the column (i.e. have the same retention time), which results in two or more molecules that coelute. Therefore, when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically increases certainty that the analyte of interest is in the sample. Gas chromatrophy Mass spectrometry ▪ Two phases (tandem) - used in liquid chromatography ▪ One phase - used in gas chromatography ▪ Confirms retention time ▪ Ionizes eluted substance; separate molecules ▪ Analyzes each fragment producing a graph • Peak levels determine major component Determine the components based on their affinity to the mobile and stationary phase ▪ Longer retention time = higher affinity to column ▪ Every drug has different component which has different retention time DRUG TESTING PROCEDURES • • • • Materials: o 10 ml Urine sample o Drug testing kits Immunochromatography - usual method Procedure: Urine drug screen o Major metabolites of drugs of abuse (phencyclidine, benzodiazepines, cocaine metabolite, amphetamines, tetrahydrocannabinol, opiates, barbiturates) in urine. 1. Cassette test – Add 3 drops of urine to each well, for panel test dip the kit to the urine sample. 2. Recap the kit and leave in a flat surface. 3. Read results after 5 minutes. Results of the test: o o o 2 lines - negative 1 line - positive Principle: competitive binding ▪ Competitor substance (reagent component) binds to the antibody, means that target is not present in the sample o o Typical Immunochromatography ▪ Control - ab against reagent ▪ Test - ab against analyte of interest Competitive immunochromatography ▪ Changes in reagent area ▪ Has a substance similar to the target ▪ Control - ab against reagent ▪ Test - ab against target • Addition of substance and analyte will compete for the test area • Substance with higher concentration will win • Presence of line mean that similar substance binds with ab and produces a color (negative for analyte of interest) • If analyte binds with ab, it will not bind with the dye and it will not produce a color (positive for analyte for interest) SPECIMEN COLLECTION SPECIMEN COLLECTOR • • Authorized specimen collector o A trained individual who instructs, assists a donor at collection site, receives and makes an initial inspection of specimen for drug testing o Requires a special training o After collection, there is an inspection of sample ▪ Urine must be warm (indication of fresh urine) ▪ Visible contamination shall be checked o Designated specimen collector ▪ At least high school graduate ▪ With training in specimen collection • Collection protocol • Chain of custody and record keeping • Recognition of invalid specimen o Same sex will observe the patient (male = male) Who are not authorized to collect specimen? o Employer of client/patient o Crime investigator o Complainant o Owner/administrator of establishment o Note: Sample should be submitted directly to the collector COLLECTION SITE • • • • • A facility (permanent or temporary) where a donor provides a specimen for drug test May be located: o Within the laboratory o Remote colelction site Within the laboratory o Clean, adequate space (5 pxs), private o Secured area for specimen/documents o Mandatory drug testing, except criminal Remote collection site o Workplace, school. Jail, rehab, site of crime o Critically ill/disabled o Needs permit from BHFS/CHD 10 days prior Onsite specimen colelction o Specimen are collected at a designated area within the drug testing facility o For all mandatory tests except for crime scene and post accident • Remote collection o Specimen are collected at a temporary facility located at a remote site o Allowable conditions ▪ Workplace, school, jail, prison or rehab centers • Random • Follow up • Reasonable suspicion • Cause ▪ Critically ill or disabled o Secure permit from BHFS or CHD for remote collection 10 working days prior to scheduled collection o No testing/examination to be done at a remote collection site SPECIMEN CONTAINERS • • • • • • Urine: 30 or 60 mL, polyethylene bottle, wide mouth with screw cap Saliva: 30 mL, polyethylene bottle Blood: 10 mL, plain test tube Hair: self-sealed transparent bag Sweat: BFAD approved sweat patch Tissue: screw-capped plastic container TYPES OF SPECIMENS • • • • • Urine o Least expensive, most popular o Easy to do o Standardized procedures o Detects use within the week o Abstaining can produce negative reaction (drug testing are recommended to be in surprise) o Established specimen validity tests ▪ Temp, color, contaminant Hair o Expensive and tedious o 2x more specimen than urine test o Do not detect recent use o Detects chronic substance abuse o Requires 1.5 x 1.5 cm hair clump o Not affected by drug abstinence o Can determine temporal pattern Blood o Most expensive method o Most accurate o Least common method o Short detection time (recent use) o Procedure not established and standardized Saliva o Uncommon method o Easy to administer o Short detection time (recent use) o No reference standardized Sweat (patch) o Requires wearing of patch 1-2 weeks o Uncommon methods o No reference standards developed o Surface contamination can cause false positive o Can detect use for extended period of time • o Minimum quantity o Scalp hair: 100 mg or its equivalent o Saliva: 2 ml (single); 1.5 and 0.5 (split) o Sweat: DOH cleared patch worn for 7-14 days o Urine: 60 mL single; 30 mL for each split sample o Blood: 5 mL; 10 mL (PH) o COLLECTION PROCEDURE • • • • • • • Reasons for Drug Testing o Mandatory tests o Random tests o Reasonable suspicion/cause o Post accidents o Follow up, return to duty o Pre-employment Methods of Specimen Colelction o Observed collection ▪ In the presence of ASC ▪ Common practice o Unobserved collection ▪ In the absence of ASC ▪ Submitted samples ▪ Subject to validity tests Types of Specimen Collection o Single specimen collection ▪ Specimen is entirely placed in a single 60 mL bottle ▪ Common practice o Split specimen collection ▪ Specimen is collected at same time but placed in 2 separate containers at least 30 mL each Unacceptable forms of ID o Identification by a co-worker o Identification by another donor o Use of a single, non-photo ID (credit card, voter’s registration, others) Observes collection one at a time o Close attention to collection o Observe unusual behavior, if present repeat under direct observed collection Forms of tampering specimen o Dilution ▪ Internal - drinking plenty of water; diuretic like Lasix, tea, Coffee, beer ▪ External - addition of water to specimen o Substitution o Adulteration ▪ Additives • Bleach → increase pH → slower Ab-Ag rxn • Ammonia → increase pH • Liquid soap → increase pH • Table salt → increase pH • Vinegar → decrease pH • Visine eye drops → interferes with EMIT Beating the drug screens o Drug screens - chemicals taken to mask/interferes drug analysis ▪ Aspirin - NAD EMIT competition ▪ Niacin - NAD EMIT competition ▪ Zinc sulfate - bonds with THC metabolite excreted in stool • Doping the samples - spiking samples with chemicals that interfere with assay ▪ Bleach, deter gents, ammonia, table salt → increases pH, slows reaction rate ▪ H2O2 → destroys 50% of THC ▪ Visine → EMIT interference Substitution methods ▪ Catheterization ▪ Concealed container (eg. condoms) ▪ Injection into bladder ▪ Another liquid like juice, etc. ▪ Stealing the urine