TEST METHODOLOGY. ALC Alcohol. Intended Use For in vitro diagnostic use only. Vitros ALC Slides quantitatively measure ethanol

Transcription

1 TEST METHODOLOGY Vitros ALC Slides ALC Intended Use For in vitro diagnostic use only. Vitros ALC Slides quantitatively measure ethanol (ethyl alcohol) concentration in serum and plasma. Summary and Explanation of the Test Ethanol is the most commonly encountered toxic substance. The measurement of ethanol is used in the diagnosis and treatment of alcohol intoxication and poisoning. Chronic ethanol use has been linked to liver disease, high blood pressure, cardiac disease, and birth defects. Acute ethanol intoxication can result in coma and death. 1 Principles of the Procedure The Vitros ALC Slide is a dry, multilayered, analytical element coated on a polyester support. A 10 µl drop of patient sample is deposited on the slide and is evenly distributed by the spreading layer to the underlying layers. Ethanol in the sample is oxidized by alcohol dehydrogenase (ADH) to acetaldehyde. The coenzyme nicotinamide adenine dinucleotide (NAD ) is transformed into its reduced form (NADH). Tris(hydroxymethyl)aminomethane (TRIS) buffer in the reaction layers traps the acetaldehyde to drive the reaction to completion. Reaction Sequence The concentration of ethanol in the sample is determined by measuring the increase in NADH concentration at 340 nm after a five-minute incubation at 37 C. Test Type Colorimetric Wavelength 340 nm Assay Time and Temperature Approximately 5 minutes at 37 C ethanol NAD acetaldehyde TRIS ADH acetaldehyde NADH acetaldehyde-tris complex 11/96. Supersedes 5/95. Part No. MP of 10

2 Reagents Slide Ingredients Reactive ingredients are alcohol dehydrogenase (yeast, E.C ); nicotinamide adenine dinucleotide; and tris(hydroxymethyl)aminomethane. Other ingredients include pigment, binders, stabilizer, surfactants, protein, and cross-linking agent. Slide Diagram Slide Labeling The cartridge s outer carton is labeled with the test name, slide lot number, expiration date, and required storage temperature. Slide Cartridge Handling Upper slide mount Spreading layer (BaSO 4 ) NAD Reagent layer alcohol dehydrogenase TRIS buffer, ph 8.75 Support layer Lower slide mount CAUTION: Protect the inner wrapper from damage before opening. Do not drop a case of cartridges. Do not cut into the inner wrapper with a sharp instrument when opening the case. Slide Storage Unopened slide cartridges: Store at or below 18 C (0 F). Cartridges in the analyzer s slide supply: Leave in the slide supply for no more than one week, then replace with a fresh cartridge. Leave in the slide supply when the analyzer is turned off for up to two hours. Verify performance with control materials: If the analyzer is turned off for more than two hours After reloading cartridges that have been removed from the slide supply and stored for later use Slide Stability Vitros ALC Slides are stable until the expiration date on the carton when they are stored and handled as specified. Slide Preparation Remove slide cartridges from storage. The slide cartridge must reach room temperature, C (64 82 F), before it is unwrapped and loaded into the slide supply. Allow the cartridge to warm up at least 60 minutes after removing from the freezer. Remove the inner wrapper and immediately load into the slide supply. NOTE: Load the cartridges within 24 hours after they reach room temperature. 2 of 10 Part No. MP /96. Supersedes 5/95.

3 Specimen Collection and Preparation Patient Preparation Do not cleanse the sample draw site with alcohol or other volatile disinfectants. Use only aqueous disinfectants. Recommended Specimen Types Serum; heparin and sodium fluoride/potassium oxalate plasma. 2 Special Precautions Do not use mercuric chloride as a preservative. Monitor and eliminate alcohol from all outside sources, if present. Postmortem or immediately antemortem specimens may produce false positive results. Refer to Limitations of the Procedure. Specimen Collection and Preparation Collect specimens using standard laboratory procedures. 3,4 Refer to the operator s manual section on sample handling for recommended minimum specimen volumes for your system. Centrifuge specimens and remove the serum from the clot within 3 hours of collection. 5,6 Analyze samples at room temperature when testing. Handling and Storage Conditions Handle specimens as biohazardous material. Handle and store specimens in stoppered containers to avoid contamination and evaporation: Cap all sample cups. If a test needs to be repeated, use a freshly filled cup. Handle specimens in tightly stoppered containers with a minimum of dead air space. Storage requirements: 7,8 Store at room temperature up to 3 hours Refrigerate at 2 8 C (36 46 F) up to one week Freeze for storage longer than one week; frozen specimens are stable for at least six months Testing Procedure Materials Required But Not Provided The following items are required to perform the test for ALC: Vitros Calibrator Kit or Vitros Chemistry Calibrator Kit 8 Quality-control materials, such as Vitros Performance Verifiers For dilution, Vitros 7% BSA Operating Instructions Refer to the operator s manual for complete instructions on operation of your system. Sample Dilution If samples show alcohol concentrations that exceed the system s reportable (dynamic) range, follow this procedure. 1. Dilute sample with Vitros 7% BSA. An initial twofold dilution is recommended. 2. Reanalyze. 3. Multiply the results by the dilution factor to obtain the original sample s alcohol concentration. 11/96. Supersedes 5/95. Part No. MP of 10

4 Calibration Required Calibrators Vitros Calibrator Kit or Vitros Chemistry Calibrator Kit 8 NOTE: For the calibration of ALC on the Vitros 950 Chemistry System, use only Vitros Chemistry Calibrator Kit 8 Calibrator Preparation, Handling, and Storage Refer to the calibrator package insert for information about reconstitution and use of the Calibrator Kit. Keep vials capped tightly until use to avoid evaporation of ethanol. Calibration Procedure Refer to the calibration section of your operator s manual. When to Calibrate Calibrate when the slide lot number changes. Calibrate when critical analyzer parts are replaced due to service or maintenance. If quality-control results are consistently outside acceptable limits, calibration might be required. Refer to your operator's manual for more detail. Calibrate when government regulations require. In the U.S., CLIA regulations require calibration or calibration verification at least once every six months. Reference Method Calibration is traceable to a head space gas chromatography method 9 (modification of the Dubowski method). 10 Calibration Model End-point colorimetry (described in your operator s manual). Quality Control Procedure Recommendations Handle quality-control materials as biohazardous material. Analyze quality-control materials in the same manner as patient samples, before or during patient sample processing. Analyze control materials at least once per day to verify system performance. Choose control levels that check the clinically relevant range. Refer to the quality control section in your operator s manual for additional information on quality-control procedures for Vitros Systems. Refer to Internal Quality Control Testing: Principles and Definitions for general quality-control recommendations. 11 Quality-Control Material Selection Vitros Performance Verifiers are specially formulated for use with Vitros Systems. Other control materials may show a difference when compared with other alcohol methods if they: Depart from a true human serum/plasma matrix Contain high concentrations of preservatives, stabilizers, or other nonphysiological additives Do not use control materials stabilized with ethylene glycol. Quality-Control Material Preparation and Storage Refer to the manufacturer s product literature for preparation, storage, and stability information. 4 of 10 Part No. MP /96. Supersedes 5/95.

5 Expected Values and Reporting Results Reference Interval 12 Conv. (mg/dl) SI (mmol/l) Alternate (g/l) Negative < 10 < 2 < 0.1 Toxic Depression > 100 > 22 > 1.0 of CNS Fatalities reported > 400 > 87 > 4.0 Reporting and Unit Conversion Conventional mg/dl SI mmol/l (0.217 x mg/dl) Alternate g/l (0.01 x mg/dl) Limitations of the Procedure Known Interfering Substances Postmortem or immediately antemortem specimens may produce false positive results because of extremely high levels of lactate dehydrogenase and lactate. Positive results on these specimens should be confirmed by gas chromatography (GC). Table of Known Interfering Substances The Vitros ALC method was screened for interfering substances. The following substances, when tested at the concentrations indicated, caused the bias shown. * Acetaldehyde, acetone, and methanol do not interfere. Further information about the specificity of the assay is summarized in a table in the Specificity section. Other Limitations Interferent Concentration Conventional (mg/dl) Analyte Concentration Some drugs and patient conditions are known to alter ethanol concentrations in vivo. A compilation of this information is available in the literature. 13,14 Average Bias Interferent Concentration SI (mmol/l) Analyte Concentration Average Bias Interferent* Metronidazole Methotrexate * It is possible that other interfering substances may be encountered. These results are representative; however, your results may differ somewhat due to test-to-test variation. The degree of interference at concentrations other than those listed might not be predictable. Cross-Reactivity Related alcohols may cross-react. The following results were obtained when the alcohols indicated were added to an ethanol-free serum pool. Conventional (mg/dl) SI (mmol/l) * Concentration Average Bias Concentration Average Bias Ethylene glycol n-propanol Isopropanol n-butanol /96. Supersedes 5/95. Part No. MP of 10

6 Performance Characteristics Reportable Range (Dynamic Range) Conv. SI Alternate (mg/dl) (mmol/l) (g/l) Refer to Sample Dilution under Testing Procedure for out-of-range samples. Sensitivity The lower limit of the reportable (dynamic) range is 10 mg/dl (2.2 mmol/l). Precision Precision was evaluated with quality-control materials on Vitros 250, 700, and 950 Chemistry Systems following NCCLS Protocol EP5-T2. 15 These results are guidelines. Variables such as instrument maintenance, environment, slide handling/storage, control material reconstitution, and sample handling can affect the reproducibility of test results. ALC Precision Conventional (mg/dl) SI (mmol/l) SYSTEM Mean Conc. Within Day SD* Within Lab SD Mean Conc. Within Day SD* Within Lab SD Within Lab CV% No. Observ. No. Days Vitros Vitros Vitros * Within Day precision was determined using two runs/day with two to three replications. Within Lab precision was determined using a single lot of slides and calibrating weekly. 6 of 10 Part No. MP /96. Supersedes 5/95.

7 Performance Characteristics (continued) Accuracy The plot and table show the results of a comparison of serum specimens analyzed on the Vitros 700 System with those analyzed using a head space gas chromatography method. Testing followed NCCLS Protocol EP9-A. 16 The table also shows the results of comparisons between the Vitros 700 System and a commercially available method, and comparisons of the Vitros 250 and 950 Systems with the Vitros 700 System. ALC/Serum Vitros 700 System mg/dl mmol/l mmol/l mg/dl Head Space Gas Chromatography Method Method Comparison (Serum) y = x 700 System vs. reference method 250 System vs. 700 System 700 System vs. commercially available ADH method 950 System vs. 700 System Conventional (mg/dl) Range of Sample Concentration Intercept Sy.x SI (mmol/l) Range of Sample Concentration Intercept Sy.x Correlation n Slope Coefficient /96. Supersedes 5/95. Part No. MP of 10

8 Performance Characteristics (continued) Specificity The following substances were tested with Vitros ALC Slides and found not to interfere (bias < 1): Compound Concentration Compound Concentration Acetaldehyde 10 mg/dl Hydrochlorothiazide 2 mg/dl Acetaminophen 20 mg/dl Hydroxychloroquine Acetate 50 mmol/l Hydroxyurea 0. Acetone 450 mg/dl Hydroxyzine 0. Acyclovir 10 mg/dl Hypaque 500 mg/dl Albuterol sulfate 18 µg/dl Ibuprofen 40 mg/dl Allopurinol 20 mg/dl Imipramine Amitriptyline Isoniazid 7 mg/dl Ammonia 0.5 mmol/l Keflin 0.6 mg/dl Amphotericin B 35 µg/ml L-dopa 30 mg/dl Ampicillin 200 mg/dl Lactate 176 mg/dl Ascorbic acid 6 mg/dl Lactose 3000 mg/dl Atenolol 2 mg/dl Lactulose 6.5 mg/dl Azathioprine Lecithin (phospholipids) 500 mg/dl Benzalkonium chloride 10 mg/dl Lidocaine 6 mg/dl 7.5% Betadine 20 µl/dl Lithium 3.5 mg/dl Bicarbonate 40 mmol/l Lovastatin 0.06 mg/dl Bilirubin (unconjugated) 20 mg/dl Mannitol 650 mg/dl 2,3-Butanediol 3.5 mmol/l Metaprolol 0.3 mg/dl 2-Butanone 3.5 mmol/l Methadone Captopril 2 mg/dl Methanol 700 mg/dl Carbamazepine 12 mg/dl N-Acetylcysteine 100 mg/dl Cefazolin 400 mg/dl Naproxen 120 mg/dl Ceftriaxone 250 mg/dl Neomycin 20 mg/dl Chloral hydrate 10 mg/dl Nifedipine Chloramphenicol 25 mg/dl Nortriptyline Chloride 170 mmol/l Nystatin 100 mg/dl Chlorpromazine D-Penicillamine 4 mg/dl Cholesterol 405 mg/dl ph 6.9 Cholic acid (bile acid) 6 µmol/l ph 8.9 Cimetidine 8 mg/dl Phenylpropanolamine 0.18 mg/dl Clonidine 37 µg/dl Pseudoephedrine 0.6 mg/dl Codeine 1.7 mg/dl Phenelzine 0.25 mg/dl Cytarabine 120 mg/dl Phenobarbital 15 mg/dl Desipramine Phenylbutazone 100 mg/dl Dexamethasone 1.35 µg/ml Phenytoin 30 mg/dl Dextran mg/dl Prednisone 0. Diazepam 2 mg/dl Procainamide 20 mg/dl Diethyldithiocarbamate 2.3 mg/dl Promethazine Digoxin 30 µg/dl Propanolol 0.5 mg/dl Diltiazem 0.2 mg/dl Propylene glycol 200 mg/dl Dipyrone 250 mg/dl Pyruvate 2 mg/dl Disulfiram 0.8 mg/dl Quinidine 5 mg/dl Dopamine 30 µg/dl Ranitidine 15 mg/dl Doxepin 2 mg/dl Salicylate 50 mg/dl Doxorubicin 0.6 µg/ml Suramin 100 mg/dl Erythromycin 20 mg/dl Streptokinase 700 U/mL Ferric gluconate 300 µg/dl Streptomycin 50 mg/dl Fluoxetine 0.7 µg/ml Theophylline 25 mg/dl Formaldehyde 35 mg/dl Thimerosal 100 mg/dl Formic acid 450 mg/dl Thioridazine 2 mg/dl Furosemide 10 mg/dl Tolazamide 55 mg/dl Geneticin disulfate 50 mg/dl Total protein 4.0 g/dl Gentamicin 12 mg/dl Total protein 9.0 g/dl Gentisic acid 25 mg/dl Triamterene 6 mg/dl Glyburide 6.4 µg/dl 1,1,1-Trichloroethane 72 mg/dl Glycerol 1 mmol/l Triglycerides 1600 mg/dl Glycolic acid 700 mg/dl Trimethoprim 25 mg/dl Glyoxylic acid 5 mg/dl Valproic acid 50 mg/dl Haloperidol 0.2 mg/dl Warfarin 10 mg/dl Hemoglobin 500 mg/dl Zephiran chloride 1 µl/ml 8 of 10 Part No. MP /96. Supersedes 5/95.

9 References 1. Freitas GG, Muiller LW (eds). Manual of Medical Therapy. ed. 23. Boston: Little, Brown; ; Clinical Laboratory Handbook for Patient Preparation and Specimen Handling. Fascicle IV: Therapeutic Drug Monitoring/Toxicology. Skokie, IL: College of American Pathologists; NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture. NCCLS Document H3-A3. Wayne, PA: NCCLS; NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Skin Puncture. NCCLS Document H4-A3. Wayne, PA: NCCLS; Winek CL, Paul LJ. Effect of Short-Term Storage Conditions on Concentrations in Blood from Living Human Subjects. Clin. Chem. 29: ; Meyer T, Monge PK, and Sakshaug J. Storage of Blood Samples Containing. Acta. Pharmacol. Toxicol. 45: ; Winek. Effect of Short-Term Storage Conditions on Concentrations in Blood from Living Human Subjects. 29: Refer to reference 5 for complete citation. 8. Meyer. Storage of Blood Samples Containing. 45: Refer to reference 6 for complete citation. 9. Mostek S, Taylor D, Nealon D. Evaluation of Automated Headspace Gas Chromatography as a Reference Method for Ethanol Quantitation in Biological Fluids [Abstract]. Clin. Chem. 39:1242; Dubowski KM. Manual for Analysis of Ethanol in Biological Fluids. U.S. Dept. of Transportation Report #DOT-TSC-NHTSA-76-4; NCCLS. Internal Quality Control Testing: Principles and Definitions. NCCLS Document C24-A. Wayne, PA: NCCLS; Tietz NW (ed). Fundamentals of Clinical Chemistry. ed. 3. Philadelphia: WB Saunders; 951; Young DS. Effects of Drugs on Clinical Laboratory Tests. ed. 4. Washington, D.C.: AACC Press; Friedman RB, Young DS. Effects of Disease on Clinical Laboratory Tests. Washington, D.C.: AACC Press; NCCLS. User Evaluation of Precision Performance with Clinical Chemistry Devices. NCCLS Document EP5-T2. Wayne, PA: NCCLS; NCCLS. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline. NCCLS Document EP9-A. Wayne, PA: NCCLS; /96. Supersedes 5/95. Part No. MP of 10

10 When this Test Methodology Sheet is replaced, sign and date below and retain as specified by local regulations or laboratory policies, as appropriate. Signature Obsolete Date Ortho-Clinical Diagnostics, Inc. 100 Indigo Creek Drive Rochester, NY Vitros is a trademark of Ortho-Clinical Diagnostics, Inc. Ortho-Clinical Diagnostics, Inc., 1996 All rights reserved. Printed in USA. 10 of 10 Part No. MP /96. Supersedes 5/95.