Colorado Department of Public Health and Environment. Laboratory Services Division Toxicology Laboratory

Transcription

1 Colorado Department of Public Health and Environment Laboratory Services Division Toxicology Laboratory Drugs of Abuse Screen, ELISA, Blood Revision 0 June 2006

2 TITLE... 1 REFERENCES... 1 METHOD... 1 PRINCIPLE... 1 SAMPLE... 1 SAFETY... 2 EQUIPMENT:... 2 REAGENTS... 2 ELISA KITS... 2 STOCK STANDARD PREPARATION:... 3 NEGATIVE BLOOD, 1% SODIUM FLUORIDE... 5 POSITIVE CONTROL PREPARATION:... 5 LOW POSITIVE CONTROL PREPARATION:... 5 HIGH POSITIVE CONTROL PREPARATION:... 5 PROCEDURE... 6 SPECIMEN SECURITY AND CHAIN OF CUSTODY... 6 SPECIMEN PREPARATION... 6 DAILY MAINTENANCE... 9 MONTHLY MAINTENANCE DATA INTERPRETATION,QUALITY CONTROL, AND REPORTING PROCEDURE NOTES AND LIMITATIONS OF METHOD POLLUTION PREVENTION WASTE MANAGEMENT... 12

3 Section: Toxicology June 2006 TITLE Drugs of Abuse Screen by Elisa - Qualitative, Blood REFERENCES 1. Immunalysis, Elisa for Forensic Matrices, Operator s Manual, (August 2004). 2. Elisa Training and General Information, Communication from Jeremy George, Applications Specialist, Immunalysis Corporation, June Immunalysis Elisa Reagent Package Inserts 4. Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society s (ACS) Department of Governmental Regulations and Science Policy, th Street NW, Washington D.C , (202) Rules and Regulations Concerning Testing for Alcohol and Other Drugs, Colorado Department of Public Health and Environment, 5 CCR METHOD ELISA - Enzyme-Linked Immunosorbent Assay PRINCIPLE The Immunalysis Direct Elisa Kits are based upon the competitive binding of enzyme labeled antigen and unlabeled antigen to an antibody (IgG), in proportion to their concentration in the reaction mixture. An aliquot of diluted unknown specimen is incubated with a dilution of enzyme (horseradish peroxidase) labeled derivative in micro-plate wells. These wells have been coated with fixed amounts of high affinity purified polyclonal antibody. After incubation, the wells are washed thoroughly to remove any unbound material and a chromogenic substrate is added. The color produced is stopped using a dilute acid stop solution and the wells read at 450 nm. The intensity of the color developed is inversely proportional to the concentration of drug in the sample. The technique is sensitive to 1 ng/ml. SAMPLE Human blood, containing sodium fluoride (NaF), preservative, and potassium oxalate, anticoagulant. Blood samples are to be drawn as stipulated in the Rules and Regulations Concerning Testing for Alcohol and Other Drugs regulated by the Colorado Department of Public Health and Environment. Any samples drawn in a manner different from these regulations will have such variations documented on the Request For Analytical Services form. Standard Operating Procedure Page 1 of 14

4 Section: Toxicology June 2006 SAFETY Use routine precautions found in the Chemical Hygiene Plan (Appendix I Safety Manual) when working in the laboratory. Follow the Bloodborne Pathogens Exposure Control Plan (Appendix G Safety Manual), when working with biological fluids or tissues. Read all Material Safety Data Sheets before handling unfamiliar reagents. EQUIPMENT: 1. Tecan Absorbance Reader (Or equivalent). 2. Tecan Washer (Or equivalent). 3. Volumetric pipettes, (Class A required for standard preparation). 4. Microliter syringes (Hamilton or equivalent). 5. Volumetric flasks. 6. Beakers. 7. Vortex mixer x 75 mm borosilicate glass disposable culture tubes REAGENTS Reagent grade chemicals are used in all tests unless otherwise indicated. It is intended that all reagents will conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficient high purity to permit its use without lessening the accuracy of the determination Elisa Kits 1. Amphetamine Direct Elisa Kit ng/ml cutoff level. 2. Methamphetamine Direct Elisa Kit 100 ng/ml cutoff level. 3. Cocaine / Benzoylecgonine Direct Elisa Kit 50 ng/ml cutoff level. 4. Cannabinoids (THC/CTHC) Direct Elisa Kit 10 ng/ml cutoff level. 5. Opiates Direct Elisa Kit 50 ng/ml cutoff level. Standard Operating Procedure Page 2 of 13

5 Section: Toxicology June Barbiturates 50 ng/ml cutoff. 7. Benzpdiazepines 50 ng/ml cutoff. 8. Methadone 50 ng/ml cutoff. 9. Propoxyphene 100 ng/ml cutoff. 10. Phencyclidine 25 ng/ml cutoff. 11. Oxycodone 50 ng/ml cutoff 12. Carisoprodol 100 ng/ml cutoff 13. Flunitrazepam 5ng/ml cutoff 14. Ketamine 50 ng/ml cutoff Each Kit contains: Component Note: All working chemical solutions, mixtures, or dilutions shall be labeled with the following information: chemical name, concentration, date prepared, analysts initials, special storage instructions, and expiration date. Stock Standard Preparation: 192 Test Kits Specific For Each Kit? 96 well Micro-plate 2 Yes Conjugate 25 ml Yes Positive Urine Std 2 ml Yes Negative Urine Std 2 ml Yes TMB Substrate 28 ml No Stop Reagent 25 ml No Cerilliant Corporation, in sealed ampules containing: Standard Operating Procedure Page 3 of 13

6 Section: Toxicology June 2006 Class Cerillian t Cat # mg/ml stock conc. Solvent Cannabinoid T MeOH Opiates M MeOH Amphetamine A MeOH Methamphetamine M MeOH Cocaine/BE B MeOH 1. Cannabinoid, 1,000 ng/ml Transfer 1 ml of the Cerilliant Cannabinoid Standard (100 ug/ml) to a 100 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than - 4 o C). Expiration date of 1 year. End concentration of 1,000 ng/ml. 2. Opiates, 1,000 ng/ml Transfer 1 ml of the Cerilliant Opiate Standard to a 10 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than-4 o C). Expiration date of 1 year. End concentration of 100 ug/ml. Transfer 1 ml of the Opiate 100 ug/ml solution to a 100 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than-4 o C). Expiration date of 1 year. End concentration of 1,000 ng/ml. 3. Amphetamine, 1,000 ng/ml Transfer 1 ml of the Cerilliant Amphetamine Standard to a 10 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than-4 o C). Expiration date of 1 year. End concentration of 100 ug/ml. Transfer 1 ml of the Amphetamine 100 ug/ml solution to a 100 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than-4 o C). Expiration date of 1 year. End concentration of 1,000 ng/ml. 4. Methamphetamine, 1,000 ng/ml Transfer 1 ml of the Cerilliant Methamphetamine Standard to a 10 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than-4 o C). Expiration date of 1 year. End concentration of 100 ug/ml. Transfer 1 ml of the Methamphetamine 100 ug/ml solution to a 100 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than-4 o C). Expiration date of 1 year. End concentration of 1,000 ng/ml. Standard Operating Procedure Page 4 of 13

7 Section: Toxicology June Cocaine, 1,000 ng/ml Transfer 1 ml of the Cerilliant Cocaine Standard to a 10 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than-4 o C). Expiration date of 1 year. End concentration of 100 ug/ml. Transfer 1 ml of the Methamphetamine 100 ug/ml solution to a 100 ml volumetric flask; bring to volume with methanol. Transfer to a storage bottle and store in freezer (less than-4 o C). Expiration date of 1 year. End concentration of 1,000 ng/ml. Negative blood, 1% sodium fluoride Place 1 gram of sodium fluoride in a 100 ml volumetric flask. Fill to mark with defibrinated sheep s blood and mix thoroughly. Blood must reach room temperature before use, to ensure proper percentage of sodium fluoride. Label and refrigerate at less than 8 C. Expiration date of 6 months. Positive Control Preparation: 1. In a 10 ml tube, place exactly 200 ul each of the Amphetamine and Methamphetamine 1,000 ng/ml standard. Add 100 ul each of the Cocaine and Opiate 1,000 ng/ml standard, and then add 20 ul of Cannabinoid 1,000 ng/ml standard. Then add 1380 ul of negative blood. 2. Aliquot into serum vials. Store frozen until ready for use. Expiration date of 6 months. Low Positive Control Preparation: 1. In a 10 ml tube, place exactly 100 ul each of the Amphetamine and Methamphetamine 1,000 ng/ml standard. Add 50 ul each of the Cocaine and Opiate 1,000 ng/ml standard, and then add 10 ul of Cannabinoid 1,000 ng/ml standard. Then add 1690 ul of negative blood. 2. Aliquot into serum vials. Store frozen until ready for use. Expiration date of 6 months. High Positive Control Preparation: 1. In a 10 ml tube, place exactly 400 ul each of the Amphetamine and Methamphetamine 1,000 ng/ml standard. Add 200 ul each of the Cocaine and Opiate 1,000 ng/ml standard in the same tube and then add 40 ul of Cannabinoid 1,000 ng/ml standard. Then add 760 ul of negative blood. 2. Aliquot into serum vials. Store frozen until ready for use. Expiration date of 6 months. Note: The calculations made to determine the amount of each analyte to add to the controls are at the end of the method. These calculations may change if the detection limit for an assay Standard Operating Procedure Page 5 of 13

8 Section: Toxicology June 2006 changes. If this occurs new calculations will be attached to the method. Note: You may make 3 levels of controls (LPC, PC, HPC) or one level and do appropriate dilutions. PROCEDURE Specimen Security and Chain of Custody Accurate specimen identification is required at all times with zero exceptions. The analyst must assure accuracy of specimen identification at all times including before analysis, during analysis, after analysis, and during storage. Discrepancies must be noted and resolved before reporting results. Specimen Preparation 1. Remove the Elisa Kits, PBS Buffer, and controls from refrigerated storage. Allow to reach room temperature before use. Note: The procedures in the package inserts are not specific for our detection limits. The amount of analyte we add to each well was calculated based on manufacturer specifications. Also, Positive Reference Standard and Negative control received with each kit are made with urine and thus not acceptable for use with this method because of the different matrix. 2. Obtain unknown samples to be run from refrigerated storage. Place on rocker and mix for at least 10 minutes. Note: All blood specimens are to be handles and stored as biohazardous material. Open specimens with the tubes and containers pointed away from self and others. Dispose of blood-contaminated waste in biohazard waste bags and take them to be treated in the autoclave. 3. Label 12 x 75 mm borosilicate glass disposable culture tubes with: a. NC (Negative Control) b. LPC (Low Positive Control) c. PC (Positive Control) d. HPC (High Positive Control) And a tube for each sample to be screened. Standard Operating Procedure Page 6 of 13

9 Section: Toxicology June Make a 1:10 dilution with PBS buffer and the control or sample to be analyzed. a. Usually, add 900 ul of PBS Buffer to disposable tube. Then add 100 ul of control or sample. b. Vortex sample until well mixed. Note: Volume of sample may change depending on quantity of sample received. 5. When specimen preparation is complete, the opened tube of blood and the unopened tube are placed in a secure refrigerator for storage. Temperature is to be less than 8 C. Temperature is top be checked once per regularly scheduled working day. The temperature is to be recorded and initialed on the appropriate form kept on the refrigerator. 6. Store tubes in numerical (Lab ID number) order for at least one year. Disposal of specimens will be recorded in the specimen disposal log. (Initial and date all disposals.) Some specimens must have the appropriate District Attorney s approval before disposal. These are stored separately with a copy of the letter requiring special storage. 7. Add 100 ul of conjugate to the 1:10 sample solution. Note: Each bottle of conjugate is lot specific. Do not use different lots in the same run. 8. Incubate for 1 hour in the dark and at room temperature. Place plate in a drawer or other location where dust cannot fall into the wells. 9. Wash plates with DI water 6 times. a. For using the Tecan washer. i. Turn washer on in the morning you are running. ii. Follow the questions on the screen to set up the washer. 1. You should rinse? No The rinse should have been run at the end of the day as part of the daily maintenance. If it was not then the system should be rinsed. 2. Run 1:Elisa. Yes Elisa is the first method (and only method) programmed in the washer and the one we will be running. 3. Set Rows. Keep the row Y if you have set up wells in that row to be Standard Operating Procedure Page 7 of 13

10 Section: Toxicology June 2006 washed. Change the Y to n if you do not have any rows in that position. You must run a full strip of wells in a row. 4. Prime the instrument. This must be done each morning before use to remove any air bubbles from the tubing. a. Select No b. Channel 1? Yes c. Prime solution? Yes or No The Prime solution is the same as the washing solution - DI water. d. Waste Bottle ok? If the waste bottle needs to be emptied then you can do that here. Once emptied press yes. 5. Repeat Steps Channel 1 Primed? Yes 7. Plate inserted? Yes? 8. Waste bottle ok? Yes This should start the washer. It will wash the plate 6 times and beep when it is finished. 9. When the washer is finished tap out any residual water onto a paper towel. 10. Add 100 ul of TMB substrate and incubate for 30 minutes. Place plate in a drawer or other site where dust cannot fall into wells. 11. Add 100 ul acid stop. Note: The TMB substrate and the Acid Stop solution are not unique for each kit. You may use TMB substrate and Acid Stop solution from different kits as long as the lot is the same. The TMB substrate will start the color development. You should see the more negative wells turning a darker blue as the color is inversely proportional to the concentration of analyte. The Acid Stop solution will stop the color development. 12. Read the plate on the Tecan reader. a. Turn on the Tecan reader, then turn on the computer. b. Open the Magellan3 software. This will cause the reading tray to open. Make Standard Operating Procedure Page 8 of 13

11 Section: Toxicology June 2006 sure that nothing is blocking the front of the reader. c. Click on the File menu at the top of the screen. Select New. Double-click on or select the Sample ID List option. d. Select the one sample IDs per well option and select the 96 well plate option. e. Enter control and sample IDs. f. Click on the File menu at the top of the screen. Select Save. Save the Sample ID List as the day in the following format: MMDDYY. For example, if the day was June 26, 2006 the format would read g. Click on the File menu at the top of the screen. Select New. Double-click on or select the Workspace option. h. Click on the Insert menu at the top of the screen. Select Method. Double-click on or select the ELISA.mth option. i. Click on the Insert menu at the top of the screen. Select Strip Test. Load the assay you are running. Then select the control row and as many sample rows as necessary. Repeat selecting the assay and control/sample rows for as many assays as needed. j. Click on the Insert menu at the top of the screen. Select the Sample ID list. Double-click or select the Sample ID list that was made on step f. k. Place the plate on the tray of the reader with the corner that is not square at the top left. l. Press the GO button at the top of the screen. This will cause the tray to move inside the reader and for the reader to read the plate. m. After reading is completed, remove the plate and perform any daily or monthly maintenance required. Note: Never leave a plate containing stop solution in the reader for a long period of time as the stop solution is a corrosive and can damage the instrument. DAILY MAINTENANCE Record in Maintenance Log. 1. Wipe down the washer and reader with dilute bleach or isopropyl alcohol. Standard Operating Procedure Page 9 of 13

12 Section: Toxicology June Perform the rinse on the washer at the end of each run before the instrument is left to stand or switched off at the end of each day. MONTHLY MAINTENANCE Record in Maintenance Log. 1. Remove the manifold of the washer (page 6-2 of Operating Manual) and clean thoroughly. a. Carefully push the supplied cleaning needles into the aspirating and dispensing needles. i. The small needle is for the dispensing needles and the larger cleaning needle is for the aspirating needles. 2. Remove the filter slide from the reader. a. Carefully clean the lenses with a kimwipe and re-insert slide in the reader. DATA INTERPRETATION,QUALITY CONTROL, AND REPORTING 1. Check the Validation Criteria of the controls. The negative control (NC) should have a greater absorbance reading than the low positive control (LPC). The LPC should have a greater absorbance reading than the positive control (PC), and the PC should have a greater absorbance reading than the high positive control (HPC). The Tecan plate reader automatically performs this internal validation. If all these criteria are met the printout will read TRUE. If the internal validation criteria are not met then the printout will read FALSE. In this case, re-run the controls and the samples associated with those tests. Note: Do not run more than 9 sets of controls on a plate. There is an error in the Magellan3 software that makes it so that the internal validation criteria are not met and thus a FALSE statement is printed. This does not affect the absorbance reading of the plate, just the calculation for the reader s internal validation. This was confirmed by Immunalysis technical support and our method validation. 2. Check the variation coefficient (CV) of the raw data (column 5 of the raw data printout). The CV between duplicate samples and controls should be less than 10. If a value of greater than 10 is achieved, a comment is made on the Corrective Action Log and approved by the Toxicology Supervisor based on a review of the entire run. 3. Record all quality control results in the Quality Control Logbook. 4. Assemble a data package containing the ELISA Sample Position page, raw data, qualitative Standard Operating Procedure Page 10 of 13

13 Section: Toxicology June 2006 reports for all controls and samples. Submit the data package for review by the Toxicology Supervisor or designee for approval in accordance with the Toxicology Data Review, Approval, and Reporting Instruction. PROCEDURE NOTES AND LIMITATIONS OF METHOD 1. Specificity: Was shown to be 86% or greater for all assays. 2. L.O.D. (Limit of Detection) (estimated): The LOD was established by analyzing 10 negative specimens. The mean and standard deviation was calculated. LOD was determined by multiplying the standard deviation by 3. The Limit of Detection (L.O.D.) (estimated) Amphetamine = rate units. Methamphetamine = rate units. Cannabinoids = rate units. Cocaine = rate units. Opiates = rate units. 3. Carryover: Sample carryover may be observed as a falsely elevated result when a sample follows another sample of higher analyte content. Acceptable performance yields no carryover that is clinically significant. Clinically significant carryover is defined as any carryover that would cause a change in determination of results reported. 4. For all chemical (Working Standards, Calibration Standards and Reagents) prepared fresh and on the day of use, an acceptable performance of the positive and negative controls constitutes acceptability of those chemicals. 5. ELISA and other antigen/antibody screening techniques have been used effectively to screen blood samples for various drugs of abuse. A negative screen is presumed to be negative. However, a positive immunoassay screen requires confirmatory testing. False-positive, resulting from cross-reactivity of similar molecules to the "specific" antibodies have plagued the immunoassay screening technique. The package inserts contain the cross-reactivity data for different analytes with a particular kit. Since there is a chance that an analyte has crossreacted with an assay, all positive screens must be confirmed by a different analytical technique. GC/MS offers the appropriate sensitivity and specificity to confirm the presence specimens that screen postive. POLLUTION PREVENTION 1. Pollution prevention encompasses any technique that reduces or eliminates the quantity or toxicity of waste at the point of generation. Numerous opportunities for pollution prevention exist in a laboratory operation. The United States Environmental Protection Agency (US EPA) has established a preferred hierarchy of environmental management techniques that Standard Operating Procedure Page 11 of 13

14 Section: Toxicology June 2006 places pollution prevention as the management option of first choice. Whenever feasible, laboratory personnel should use pollution prevention techniques to address waste generation. When wastes cannot feasibly be reduced at the source, the US EPA recommends recycling as the next best option. 2. The quantity of chemicals purchased should be based on expected usage during the shelf life and disposal cost of unused material. Actual reagent preparation volume should reflect anticipated usage and reagent stability. 3. For information about pollution prevention that may be applicable to laboratories, consult Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society s (ACS) Department of Governmental Regulations and Science Policy, th Street NW, Washington D.C , (202) WASTE MANAGEMENT 1. The US EPA requires that laboratory waste management practice be consistent with all applicable rules and regulations. Excess reagents, samples and method process wastes should be characterized and disposed of in an acceptable manner. The agency urges laboratories to protect the air, water, and land by minimizing and controlling all releases from hoods and bench operations, complying with the letter and spirit of any waste discharge permits and regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste identification rules and land disposal restrictions. For further information on waste management consult the Waste Management Manual for Laboratory Personnel, available from The American Chemical Society at the address listed above. 2. Dispose of all consumables contaminated by blood in appropriate biohazard waste containers for autoclaving. 3. Contact the Chemical Hygiene Office for disposal recommendation of other chemicals or solutions. Standard Operating Procedure Page 12 of 13

15 Section: Toxicology June 2006 Written by: Jennifer Richardson Procedure Author Approved by: Cynthia Burbach Section Supervisor Laurie Peterson-Wright Program Manager David Sikes QA Officer David A. Butcher LSD Director Effective Deleted From Service Standard Operating Procedure Page 13 of 13

16 Section: Toxicology June 2006 REVISIONS File Name: Revision: Approved by: Edited by: Section Supervisor: Program Manager: Quality Assurance Officer: Division Director: Date File Name: Revision: Approved by: Edited by: Section Supervisor: Program Manager: Quality Assurance Officer: Division Director: Date File Name: Revision: Approved by: Edited by: Section Supervisor: Program Manager: Quality Assurance Officer: Division Director: Date File Name: Revision: Approved by: Edited by: Section Supervisor: Program Manager: Quality Assurance Officer: Division Director: Date Standard Operating Procedure Page 14 of 13