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

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.

No. 2

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!

No. 3

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

No. 4

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

No. 5

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

No. 6

Everyone makes mistakes!!

No. 7

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

No. 8

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).

No. 9

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

No. 10

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

No. 11

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

No. 12

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

No. 13

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…

No. 14

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

No. 15

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%

No. 16

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

No. 17

Example of Good Proficiency Test Results

Mean value 413 mg/100ml- 2 S.D. range ±10.5% (Acceptable)

No. 18

Example of Poor Proficiency Test Results

Mean value 1021 mg/l - 2 S.D. range ±42.6% (POOR)

No. 19

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

No. 20

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

No. 21

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

No. 22

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

No. 23

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

No. 24

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!)

No. 25

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

No. 26

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

No. 27

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

No. 28

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)

No. 29

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!!

No. 30

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

No. 31

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)

No. 32

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

No. 33

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)

No. 34

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!

No. 35

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

No. 36

Thank you for your attention and your kind hospitality!