Importance of Quality Assurance for Forensic Toxicology in North America

1. Importance of Quality Assurance for Forensic Toxicology in North America Graham R. Jones, Ph.D. Chief Toxicologist Office of the Chief Medical Examiner Edmonton, Alberta, Canada

2. No. 2 Purpose of Talk • To tell you about the approach of the forensic toxicology profession in North America to “quality assurance”. • I am not here to tell you how to run your own laboratories, but to share some of the experiences we have had in the U.S. and Canada.

3. No. 3 Importance of Forensic Toxicology • Forensic testing is performed for a legal purpose and may affect: • Criminal trial (freedom/jail or death of accused) • Civil trial (denial of compensation/money) • Loss of a job (if drug use proven) • Loss of career (e.g. sports/athlete) • Custody of children (if drug abuse proven) • Toxicology testing must be ACCURATE!

4. No. 4 Types of Forensic Toxicology Laboratories in North America • Federal (FBI – U.S.; RCMP – Canada) • State (U.S.) / Province (Canada) • Police / Crime lab; Medical Examiner/Coroner • County / City / Local (U.S.) • Police / Crime lab; Medical Examiner/Coroner • Private (mostly in U.S.) • some have national coverage, some mainly local

5. No. 5 North American Courts - Adversarial • In the U.S. and Canada, the court system is “adversarial” • Prosecution and defense lawyers may each hire their own toxicologists who may give different opinions • Prosecution toxicologists may perform testing and give an interpretation (opinion) • Usually defense toxicologists offer only an opinion and do not usually perform analytical work on cases • The adversarial system should not affect the science, but allows more than one scientific opinion to be expressed in court

6. No. 6 Why does an adversarial court system make a difference? • Prosecution laboratory results are more likely to be challenged by defense • Prosecution toxicologist may be required to provide all details of their analysis (chromatograms, methods, chain of custody etc) • Many (good) forensic toxicologists are available to review analytical data for the defense and provide a second opinion • Prosecution toxicologists are more likely to have to defend their analytical work in court

7. No. 7 Everyone makes mistakes!!

8. No. 8 Quality Assurance Program • Every toxicologist in a laboratory will make a mistake. The problem is making sure that the mistake is detected before a result is reported • A good QUALITY ASSURANCE program will help to minimize errors and detect errors when they occur • “Quality assurance” covers every aspect of laboratory work that can affect the quality of the results reported

9. No. 9 Example of a Preventable Error • A person died in a psychiatric hospital • Her medications were dispensed by hospital staff • The pathologist determined death due to natural causes, but toxicology testing indicated an imipramine overdose! • A public inquiry was held and data of the toxicologist was reviewed – finding calculation error of 10-fold • Public disclosure of the error was very embarrassing for the toxicologist • The error could have been discovered before public embarrassment if the data had been properly peer reviewed (i.e. by a colleague in the same laboratory).

10. No. 10 Quality Assurance Includes: • Education and training • Standard Operating Procedures (SOPs) • Validation of methods; Criteria for evaluation of calibrations, controls and case data • Maintenance of equipment • Review and reporting of results • Verifying results of a colleague • Someone else should check your transcriptions and calculations • Proficiency testing • Laboratory accreditation

11. No. 11 Education and Training • Education in chemistry (and pharmacology) • Training in the laboratory • Develop good analytical skills • Learn acceptance criteria the laboratory uses for deciding which results are OK and which have to be repeated! (e.g. what is an acceptable calibration?) • Learn about the quality assurance program of the laboratory • Continuing education • Additional training, reading, courses, conferences • May be minimum requirements for personal certification

12. No. 12 Standard Operating Procedures • Important that routine analytical methods and procedures are written and should be approved by the laboratory director • Helps to ensure that all toxicologists perform the assay the same way each time • Enables the courts to know exactly which method was used to generate a result in a particular case • Makes independent review of the analytical method easier

13. No. 13 Validation of Methods • Analytical methods must be appropriately validated • Linearity (upper and lower) • Is the calibration accurate over the entire reporting range? The calibration parameters may change at the upper end (saturation) and lower end (adsorption) • Accuracy • Precision • Specificity • Is only the target compound being measured? • Mass spectrometry is increasingly preferred, but must be properly run

14. No. 14 Calibrations – What criteria? • Need to have consistent criteria for when a calibration is “acceptable” and whether controls have “passed” • Several different criteria can be used to ensure that a calibration is acceptable • Correlation coefficient – (e.g. >0.98) • Multipoint calibration (e.g. 3 – 8 points) • Each calibrator can be read against calibration line (e.g. 80 – 120% of target) • Can use linear, exponential or other curve fit • Not all assays are inherently linear • But you must use criteria of some kind…

15. No. 15 Quality Control • Used to verify the precision and accuracy of the calibration • Preferably prepared as a “pool” that is verified prior to first use • Pre-prepared and frozen for use in many assays • Analyte spiked into a similar matrix (e.g. blood) • Should be independently prepared • Can be a external supplier (purchased material) • Can be another person in the same laboratory, but the control should be made from another source

16. No. 16 Controls – What criteria? • Controls should be prepared by a person other than the toxicologist conducting the assay • The source of the standard solution should be different than that used for the calibration (e.g. different manufacturer; different lot number) • Controls should usually read within +/-20% of target • Exceptions – alcohol ±5-10% • Some difficult drugs or controls at very low concentrations may be ±25%

17. No. 17 Proficiency Test (PT) Programs • All toxicology laboratories should subscribe to one or more PT programs appropriate for their work (e.g. for alcohol and drugs) • Corrective actions must be performed when results of the lab significantly different from that expected (e.g. for drugs >±20% or ±2 SD) • It is NOT acceptable to review controls and ignore the results if they are outside the acceptable target • Some laboratories participate in 3–5 PT programs • PT programs can be internal, but external better

18. No. 18 Example of Good Proficiency Test Results Mean value 413 mg/100ml- 2 S.D. range ±10.5% (Acceptable)

19. No. 19 Example of Poor Proficiency Test Results Mean value 1021 mg/l - 2 S.D. range ±42.6% (POOR)

20. No. 20 Corrective Action • Bad control or PT results MUST be remediated • i.e. investigate why the results were out-of-range • Corrective action could be: • Review of calibration and data calculation • Review of chromatograms and integration • Re-analysis of the sample • Troubleshooting the assay (precision, accuracy) • Inter-laboratory comparison of results • Main reason for bad results: poor precision

21. No. 21 Forensically unacceptable practice • Once the calibration, controls and case samples are run: • Calibrators should not be dropped from the middle of the curve in order to improve the correlation coefficient • Failed controls should not be ignored • Failed controls should not be re-setup without at least one case sample as a check

22. No. 22 Method Validation • Analytical methods must be appropriately validated • Linearity (upper and lower) • Is the calibration accurate over the entire reporting range? The calibration parameters may change at the upper end (saturation) and lower end (adsorption) • Accuracy • Precision • Specificity • Is only the target compound being measured? • Mass spectrometry is increasingly preferred, but must be properly run

23. No. 23 Linearity • Especially important to determine if range of calibrators limited (e.g. if 1 or 3 point calibration) • Linearity may “fall off” at high end due to detector saturation • Linearity and sensitivity may decrease at low end due to adsorption to glassware and GC injection port phenomena • Important that controls run near upper AND lower reporting ranges if not covered by the calibrators

24. No. 24 Precision • Precision is important • Poor precision means that the “right answer” may only be obtained some of the time • Replicate analyses? • Running samples in duplicate can help to determine if precision is poor

25. No. 25 Professional Standards • Professional standards • Professional Standards should be used as a guide to “good laboratory practice” • e.g. SOFT/AAFS Guidelines • Can help a laboratory get the necessary resources to meet a nationally or internationally recognized professional standard • Useful to convince administrators to provide better funding (money!)

26. No. 26 Case Study – Inadequate Validation • The wife of a well-known pathologist died suddenly. He was accused or poisoning her with an unknown substance. • Prosecution thought that neuromuscular blocking agents might be a good possibility • A private laboratory developed an LC/MS/MS method • The laboratory detected succinylmonocholine (SMC) in the postmortem tissue of the wife of the pathologist • The pathologist was the charged with the murder of his wife and sentenced to life in prison

27. No. 27 Succinylcholine (SCC) • Succinylcholine (SCC) is an unstable ester of two choline molecules joined to succinic acid • Because SCC is unstable, a lab can look for the mono-ester (succinylmonocholine - SMC), but that is also unstable - but more stable than SCC

28. No. 28 Problem – Lack of Validation for Specificity • Postmortem samples have fairly high concentrations of succinic acid and choline, due to the breakdown of tissues • The defense argued that it was theoretically possible for small amounts of succinic acid and choline to combine to form SMC after death • Could be very low concentrations • Defense argued that the laboratory had not proven that SMC could not occur “naturally” in postmortem samples. They had not run cases “known to be negative” to rule-out that SMC was a postmortem artifact

29. No. 29 Prosecution Position • Prosecution toxicologists argues that hydrolysis of SCC to SMC and eventually to succinic acid and choline was in one direction only (not reversible) • Therefore, they argued that detection of SMC proved beyond any doubt that the victim must have been administered SCC (succinylcholine)

30. No. 30 Defense position • Defense toxicologists argued that hydrolysis of SCC to SMC and to succinic acid and choline was an equilibrium, even though mostly towards complete hydrolysis • Therefore, since all postmortem tissues contain high concentrations of succinic acid and choline (NOT from SCC), the combination of these compounds to for small amounts of SMC was a theoretical possibility • It was up to prosecution to run adequate negative controls (postmortem tissues) to prove that trace amounts of SMC do not exist naturally. They did not do that!!

31. No. 31 What was the outcome? • The jury trial did not take the defense argument seriously, and convicted the pathologist of murdering his wife • Several months later, the laboratory performed the specificity studies and found that SMC was formed in postmortem tissues (cases where SCC was known NOT to have been given) • Therefore, there was no scientific evidence that the pathologist murdered his wife with SCC

32. No. 32 Forensic Toxicology Accreditation • Several different programs… • Urine drug testing (workplace testing) • SAMHSA (U.S. Government - regulatory) • College of American Pathologists (CAP - voluntary) • Forensic Toxicology • American Board of Forensic Toxicology (ABFT - voluntary) • Forensic Science / Police Labs • American Board of Crime Laboratory Directors – Lab Accreditation program (ASCLD/LAB)

33. No. 33 What Does Accreditation Cover? • Administration of the laboratory • Personnel / staffing / training • Security and chain of custody • Procedure manuals / written documentation • Standard Operating Procedures (SOP) • Analytical methods • Quality control / quality assurance • Reporting of results

34. No. 34 Is Accreditation Mandatory? • Urine drug testing • Only for less than 5 – 10% of testing • Mainly for transportation related jobs • Forensic Toxicology / Science • Only in 3 states (New York State, Texas and Oklahoma • Not at all in Canada • Ideally, every laboratory should be accredited and inspected by an accreditation program that is based on accepted international or national standards (e.g. ISO 17025; SOFT/AAFS Guidelines)

35. No. 35 Are All Toxicology Labs Accredited? • NO!! • Many labs in Canada and the U.S. have no accreditation or independent assessment • The only pressure for labs to become accredited is “peer pressure”, or a serious mistake that leads to public or political pressure for accreditation • There is very little incentive for a lab manager to seek accreditation of a toxicology laboratory • It creates more work, with no promise of extra resources to address staffing and training needs!

36. No. 36 The Future? • The only way all forensic toxicology laboratories will adopt adequate and consistent QA standards - and become accredited - is if it is mandated by government legislation • Mandated accreditation can have the benefit of additional resources in order to meet the professional standards that have been set • Standards for forensic toxicology will continue to become more strict, consistent with those that already exist for the environmental and pharmaceutical industries

37. Thank you for your attention and your kind hospitality!