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Analysis of practices of post mortem toxicology of drug-related deaths cases in 28 EU countries, Turkey and Norway

Axel Heinemann, Stefanie Iwersen- Bergmann

Institute for Legal Medicine, University Medical Center Hamburg- Eppendorf Germany

EMCDDA CONTRACT CODE: CT.16.IBS.0178.1.0

Background

• Different coverage of autopsies but also of different toxicology practices are observed across countries, but also over time within a country

• Impact of thresholds and of the changes of the equipment/techniques used

• Recent developments in autopsies and post mortem toxicology techniques are changing options with regard to post mortem toxicology of suspected DRD cases • Needle Autopsy, Minimally Invasive Autopsy (MIA)

Objectives

• Analysis of international and national guidances with regard to PM investigation of suspected DRD cases • including minimum requirements, recommendations by national expert associations

regarding sampling, processing, confirmatory testing, unknown general screening, technical specifications and reporting (prioritizing in case mixed poisonings) with special reference to new psychoactive substances.

• Survey of practices: Mapping of the ‘typical’ or ‘standard’ toxicology practices in place in each country through a survey with toxicologists/NFP

• Discussion how to interpret DRD prevalence data considering the background of toxicology standards/ capacity in selected countries

Survey of practices in EU countries

Methods • Identification of 118 experts by

• Nomination by NFP • Direct communication to Public Forensic Institutes, Forensic Toxicology departments • On-topic publications • Professional associations

• Surveymonkey – based online questionnaire (54 items)

• Laboratory performance/Volume of postmortem analyses in DRD • Guidances • Analytical strategy • Potential hindrances in daily work/ for analytical advancements • Coverage of substance groups and examples for specific analytes/ NPS • Awareness of their role as gate-keeper for statistical registration of CoD

• Response rate 53% (n=63)

Does your laboratory perform analyses from biological samples in cases of DRD?

No 5%

Yes 95%

N=63 N=60

Response: 60 laboratories from 28 countries with DRD- toxicology experience

Country n %

Austria 1 1,7

Belgium 2 3,3

Bulgaria 1 1,7

Croatia 1 1,7

Cyprus 1 1,7

Czech Rep. 11 18,3

Denmark 3 5,0

Estonia 1 1,7

Finland 1 1,7

France 4 6,7

Germany 6 10,0

Greece 1 1,7

Hungary 1 1,7

Ireland 1 1,7

Italy 3 5,0

Country n %

Latvia 1 1,7

Lithuania 1 1,7

Luxembourg 1 1,7

Malta 0 1,7

Netherlands 1 3,3

Norway 2 5,0

Poland 3 1,7

Portugal 1 1,7

Slovakia 1 5,0

Spain 3 1,7

Sweden 1 5,0

Switzerland 3 1,7

Turkey 1 5,0

United Kingdom 3 1,7

Total 60 100,0

Response: Reduced sample for analysis (Czech Republic, Hungary): N=54

Country n %

Austria 1 1,9

Belgium 2 3,7

Bulgaria 1 1,9

Croatia 1 1,9

Cyprus 1 1,9

Czech Rep. 6 11,1

Denmark 3 5,6

Estonia 1 1,9

Finland 1 1,9

France 4 7,4

Germany 6 11,1

Greece 1 1,9

Hungary 0 1,9

Ireland 1 5,6

Italy 3 1,9

Country n %

Latvia 1 1,9

Lithuania 1 1,9

Luxembourg 1 1,9

Malta 0 1,9

Netherlands 1 3,7

Norway 2 5,6

Poland 3 1,9

Portugal 1 1,9

Slovakia 1 5,6

Spain 3 1,9

Sweden 1 5,6

Switzerland 3 1,9

Turkey 1 5,6

United Kingdom 3 1,9

Total 60 100,0

Laboratory Capacity for postmortem analyses

13%

43%

25%

12%

7%

Postmortem analyses: Cases / year

1-100 cases

101-500 cases

501-2500 cases

>2500 cases

unknown

43%

30%

20%

7%

Suspected DRD analyses: Cases / year

1-50 cases

51-250 cases

>250 cases

unknown

Share of nationwide DRD-analyses (estimate, %)

35%

18% 11%

12%

12% 12%

1-10 %

11-25 %

26-50 %

51-95 %

96-100 %

unknown

Share of national DRD-analyses (estimate, %): All laboratories/ Labs with single response from country (n=17)

35%

18% 11%

12%

12% 12%

1-10 %

11-25 %

26-50 %

51-95 %

96-100 %

unknown

18%

23% 12%

41% 6%

11-25 %

26-50 %

51-95 %

96-100 %

unknown

N= 17 N= 60

Share of national DRD-analyses (estimate, %): All laboratories/ Labs with single response from country (n=17)

18%

23% 12%

41% 6%

11-25 %

26-50 %

51-95 %

96-100 %

unknown

N= 17 N= 60

Country National Share (Anal.DRD) %

Austria 25

Bulgaria 30

Croatia 50

Cyprus 70

Estonia 100

Finland 100

Greece 20

Hungary ?

Ireland 98

Latvia 100

Lithuania 100

Luxembourg 20

Netherlands 60

Portugal 100

Slovakia 50

Sweden 100

Turkey 50

Is it always a complete autopsy where samples for toxicological analyses are collected?

28%

39, 72%

No

Yes

Samples from “needle autopsies”?

28%

72%

No

Yes

Country City

needle

autopsy? Comment Belgium Liège Yes

Bulgaria Sofia Yes

Cyprus Nikosia Yes we receive blood,urine

and sometimes eye

fluid,liver,stomach

content

Czech Rep. Prague Yes

France Strasbourg Yes

Paris Yes

Grenoble Yes

Versailles Yes

Germany Hamburg Yes 40-60 % of the DRD

cases

Munich Yes very few

Dresden Yes

Italy Rome Yes

Luxembourg Luxembourg Yes

Netherlands The Hague Yes

Norway Oslo Yes Very few cases

Poland Lublin Yes

Turkey Istanbul Yes

United Kingdom London Yes boold samples

occasionally

Glasgow Yes Rarely, but it does

happen with certain

pathologists in certain

circumstances

Is the objective of your analytical strategy in cases of poisonings…(Multiple choice)…

„Yes“

…the detection and quantification of the drug/ drugs that was/were directly causing death?

100%

…analysis for further concomitant drugs/substances not relevant for the death?

81,5%

Do you experience limitations concerning the toxicological analyses in DRD cases?

„Yes“

…budgetary restraints? 33%

…lab equipment? 35,2%

…reference substances? 42,6%

Analytical strategy: Pretests

1, 2%

53, 98%

Do you use chromatographical methods as screening tests?

N.a.

Yes

38, 70%

16, 30%

Do you use routine immunological screening tests

related to drugs of abuse?

yes

no

Which kind of specimen in use for pretests?

Labs with Immunol. Screening Mentions of „Other matrices“:

• Cerebrospinal fluid

• Vitreous humor

• Bile

• Liver

• Stomach contents

• Pericardial fluid

• Muscle

• Kidney dialysates

94.7

39.5

50

21.1

0

10

20

30

40

50

60

70

80

90

100

Urine Serum Whole Blood other Matrix

Subsample of labs with application of immunological screening:

97%

3% 0% 0%

Since when do you perform immunological screening or other

screening tests?

>10 years

5-10 years

2-5 years

prev. 2 years

97%

3%

Do you routinely confirm positive immunological screening or other

screening test results?

yes

no

Please indicate if you use immunological screening or other pretests for a specific substance group (if yes, introduced last 5 years?):

97.4

2.5

97.4

4.4

94.7

7.9

73.7

7.9

97.4

7.9

26.3

2.6

36.8

5.3

60.5

7.9

13.2

5.3

0 20 40 60 80 100 120

ALL

<5 YEAR

S

Oxycodone Barbiturates Trizykl Antid. LSD Cannabinoids Ecstasy Amphetamine Cocain Opiate

Please indicate if you use immunological screening or other pretests for a specific substance group (if yes, introduced last 5 years?):

13.2

5.3

18.4

4.4

65.8

5.3

57.9

7.9

89.5

7.9

2.6

0

15.8

5.3

0 10 20 30 40 50 60 70 80 90 100

ALL

<5 YEAR

S

GHB Zolpidem Benzodiazepines Buprenorphine Methadone Fentanyl Tramadol

If you do not use immunological screening… Is your strategy for analysis in suspicious DRD cases always "general unknown"?

50

18.8

25

6.3

0

10

20

30

40

50

60

yes, always "gen. unknownscr."

no, focused/case- dependent always "general unknown"AND focused/case-dependent

n.a.

%

N=16

Development of analytical technologies in Forensic Toxicology

10-15 years ago:

Shift from gas chromatography–mass spectrometry (GC/MS) to liquid chromatography–mass spectrometry (LC/MSMS),

• Progress in instrument technology • Polarity and low volatility of many new relevant substances and of metabolites

Last 5-10 years:

• High-resolution mass spectrometry (HRMS) enables mass measurement at high resolving power and very high accuracy

• Elemental composition can be determined directly • Time-of-flight mass spectrometry (TOF) • Ion traps like Orbitrap Fourier-transform mass spectrometry.

• Ultrahigh-performance LC (UHPLC), combined with TOFMS: Additional selectivity, sensitivity, and speed resulting from increased chromatographic resolution

• Comprehensive screening

10-15 years ago: Shift from gas chromatography–mass spectrometry (GC/MS) to liquid chromatography–mass spectrometry (LC/MS),

• Progress in instrument technology • Polarity and low volatility of many new relevant substances and of metabolites

Last 5-10 years:

• High-resolution mass spectrometry(HRMS) enables mass measurement at high resolving power and very high accuracy

• Elemental composition can be determined directly • Time-of-flight mass spectrometry (TOF) • Ion traps like Orbitrap Fourier-transform mass spectrometry.

• Ultrahigh-performance LC (UHPLC), combined with TOFMS: Additional selectivity, sensitivity, and speed resulting from increased chromatographic resolution

• Comprehensive screening

Development of analytical technologies in Forensic Toxicology

Analytical strategies in Forensic toxicology

• Targeted screening • Immunoassays do not important substances, e.g.newer antidepressants, antipsychotics,

pregabalin

• Comprehensive screening • important for revealing also NPS, rare drugs and metabolites

• Confirmation • Screening in Forensic toxicology should be accompanied by a confirmation step either

simultaneously within the same analytical run or separately.

• Identification based on metabolite patterns, qualifier ions or fragmentation information (spectra acquired after collision-induced dissociation)

• Reference standards must be available for producing the appropriate reference for comparison

• Libraries of accurate mass data and molecular formulae, existing for more than 7.500 toxicologically relevant substances

Availability of lab instrumentation

25.9

42.6

59.3

87

77.8

33.3

81.5

35.2

50

4

0

10

20

30

40

50

60

70

80

90

100

% available

Analytical strategies: Classification

Classification n %

1 Multimodal mixture of methods: ImmS AND Multi-target CS,

including LCMSMS or HPLC-TOF, UHPLC-HRMS

23 42,6

2 Multi-target CS., including LCMSMS or HPLC-TOF, UHPLC-

HRMS

14 25,9

3 Primarily targeted ImmS, based on GC-MS/HS-FID, confirmed

by diverse Chromatogr./MS methods

14 26,0

4 Others 3 5,6

Gesamt 54 100,0

ImmS: Immunological Screening CS: Chromatographical Screening

Since when have you been equipped with this technique?

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

Previous 2 years since 2-5 years since 5-10 years > 10 years unknown

GCMS LCMS GCMSMS HPLC-TOF HS-FID HS-MS

%

When questions arise about substances you cannot analyse in your laboratory: Do you have the opportunity to send samples to a specialised lab?

5%

28%

67%

n.a.

No

Yes

When questions arise about substances you cannot analyse in your laboratory: Do you have the opportunity to send samples to a specialised lab?

Labs with conventional methods Labs with multi-target methods

5% 21%

74%

f

n.a.

No

Yes

7%

46%

47%

Spalte1

n.a.

No

Yes

Level of routine quantitative determination

„Yes“

Do you perform a quantitative determination of the major drug(s) that caused death?

92,6%

Do you perform a quantitative determination of the other drugs that might have contributed to death?

87,0%

Do you perform a quantitative determination of additional findings e.g., cannabinoids?

64,8%

Routine quantitative determination: Labs with conventional methods/ multi- target methods

64.1

86.7

86.7

66.7

87.2

94.9

0 10 20 30 40 50 60 70 80 90 100

Do you perform a quantitative determination of additionalfindings e.g., cannabinoids?

Do you perform a quantitative determination of the otherdrugs that might have contributed to death?

Do you perform a quantitative determination of the majordrug(s) that caused death?

%

Labs applying multi- target methods Labs applying conventional methods

Does your final report include…

71%

25%

2% 2%

…quantitative results?

Always

Frequently

Never

Sometimes

68%

12%

10% 10%

interpretation of results?

Always

Frequently

Never

Sometimes

Are incidental/ additional findings which are rated as being irrelevant for the cause of death listed in your report?

60% 20%

2%

18%

Always

Frequently

Never

Sometimes

Final report: Labs with conventional methods/ multi- target methods

46.7

33.3

60

80

59

76.9

71.8

71.8

0 10 20 30 40 50 60 70 80 90

Are incidental/ additional findings which are rated as beingirrelevant for the cause of death listed in your report?

Does the report include interpretation of results?

Does your final report include...quantitative results?

Does your final report include...qualitative results?

%

Labs applying multi- target methods Labs applying conventional methods

What is your level of satisfaction regarding the exchange of case-related information between institutions (police/hospitals/ forensic pathologists) and your laboratory?

13%

37% 29%

17%

4%

Very satisfied

Satisfied

OK

Dissatisfied

Very dissatisfied

Are you aware whether results of your toxicology reports are in fact used to do the codification of the causes of death?

19%

27%

54% No

Not sure/ don´t know

Yes

What does your analytical strategy cover…? (Routinely/ on Request)

96

.3

1.9

96

.3

1.9

98

.1

98

.1

96

.3

1.9

92

.6

5.6

55

.6

38

.9

7.4

66

.7

25

.9

59

.3

1.9

57

.4

83

.3

11

.1

ROUTINELY ON REQUEST

%

Opiates Cocaine Amphetamine Methamphetamin

MDMA, MDA, MDEA Nat.Cannabin. Volatiles Gases(Butane,Prop.)

Solvents Poppers Prescr.opoids


92

.6

5.6

74

.1

16

.7

74

.1

22

.2

92

.6

5.6

37

16

.7

98

.1

77

.8

16

.7

ROUTINELY ON REQUEST

%

Methadone Buprenorphine Fentanyl Tramadol Tilidin Benzodiazepines Z-drugs

ROUTINE analytical strategy covers…: Labs with routine GUS versus Labs without

10

0

89

.7

93

.3

66

.7

10

0

64

.1

10

0

89

.7

26

.7

41

10

0

97

.4

93

.3

71

.8

LABS WITH ROUTINE GUS LABS WITHOUT ROUTINE GUS

%

Methadone Buprenorphine Fentanyl Tramadol Tilidin Benzodiazepines Z-drugs


64.8

2.5

94.4

4.4

35.2

51.9

0

10

20

30

40

50

60

70

80

90

100

routinely on request

Pregabaline Ketamine GHB

Analytical strategy covers… according to Lab classification

60

66.7

94.9 93.3

13.3

43.6

0

10

20

30

40

50

60

70

80

90

100

Labs using conventional methods Labs using multi- target analyses

%


Analytical strategy covers… according to Lab classification

66.7 64.1

94.9 93.3

40

33.3

0

10

20

30

40

50

60

70

80

90

100

Labs with routine GUS Labs without routine GUS

%


Analytic strategy covers: NPS?

64.8

81.5

61.1

50

64.8

74.1

18.5

0

10

20

30

40

50

60

70

80

90

% „

yes“

Synth.Cannabinoids Synth.Cathinones Designer-opioids Designer-Benzod.

Piperazines Phenylethylamines Designer Cocaines

Analytic strategy covers: NPS?- according to lab classification

46.7

71.8

60

89.7

26.7

74.4

26.7

59

40

74.4

53.3

82.1

0

25.6

0

10

20

30

40

50

60

70

80

90

100

Labs using conventional methods Labs using multi-target methods

% „

yes“



Analytic strategy covers: NPS?- according to lab classification

66.7 64.1

93.3

76.9 80

53.8 53.3 48.7

86.7

56.4

86.7

69.2

33.3

12.8

0

10

20

30

40

50

60

70

80

90

100

Labs with routine GUS Labs without routine GUS

% „

yes“



Suggestions by responders for improving the analytical strategy of suspected DRD

• Improve… • Networking/Communication with other stakeholders (police, justice, health

system)

• Cooperation with forensic pathologists

• Standardization of sample collection

• Technical and operational needs: Equipment, methods needed

• Funding for method development and research

• Guidelines for postmortem toxicological analyses

• Guidelines for codification of cause of death on death certificate

Preliminary conclusions (I)

• In many small and some larger countries in Europe, post mortem forensic toxicology is reserved to single or very few laboratories, run by public authorities

• Rapidly evolving technologies caused an increasing multitude of methods –The diversity of potential substances involved do not allow their inclusion in a single method (at least with adequate Limits of Detection (LOD))

• The majority of labs apply mixed analytical strategies including advanced technologies

• Conventional targeted methods like immunochemical screening as a first step are still common. This way, advantages and disadvantages of the different methods may be balanced, the credibility of results is even better safeguarded.

• A general unknown screening is meanwhile applied in about 30% of the labs.

Preliminary conclusions (I)

• In many small and some larger countries in Europe, post mortem forensic toxicology is reserved to single or very few laboratories, run by public authorities

• Rapidly evolving technologies caused an increasing multitude of methods –The diversity of potential substances involved do not allow their inclusion in a single method (at least with adequate Limits of Detection (LOD))

• The majority of labs apply mixed analytical strategies including advanced technologies

• Conventional targeted methods like immunochemical screening as a first step are still common. This way, advantages and disadvantages of the different methods may be balanced, the credibility of results is even better safeguarded.

• A general unknown screening as first step is applied in about 30% of the labs.

• (It does never include all imaginable substances but has become the method of choice for analytes with forensic relevance in very low concentrations and NPS.)

Preliminary conclusions (II)

• Potential disadvantages of targeted conventional strategies can be balanced by an unimpeded case- dependent information exchange

• …but up to 25% of the labs value the present situation regarding communication with other parties in their field as dissatisfying

• There are obvious challenges for the reliable determination of highly potent opioids (Buprenorphin, fentanyl/derivates), and NPS in laboratories without systematic GUS- strategies.

• The coverage of analytes like pregabalin, GHB, but also solvents/volatiles and poppers remains challenging for all laboratories and needs case- dependent decisions.

• There is no guidance about the objective to quantify any substance at any concentration in any specimen in DRD – regardless of whether forensic importance in the individual case can be expected.

Preliminary conclusions: NPS

• Laboratories with highly- standardized GUS- methods and advanced technology are clearly better positioned in determining relevant NPS groups.

• But even most of them cannot specialize in all substance groups exisiting and – even more difficult- upcoming substances:

• These laboratories seem to have established a quite reliable coverage for (common) piperazines, phenylethylamines and cathinones – but there is a gradually reduced coverage for synthetic cannabinoids, designer opiates –benzodiazepines and –cocaines.

• Laboratories with advanced technology seem to be more open for sending specimen to reference laboratories

• Experts emphasize that DRD could better reflect the evolution of the NPS problem if analytical strategies would better adjusted to the monitoring of groups of prevalent substances in the regional catchment area

• …. and if networking and information exchange / Early Warning Systems would include them in a more systematic way.

Limitations

Representativity:

• Private labs missing - For the private lab sector, forensic toxicology has become a more important market during last years.– most private labs are equipped with recent technology - in public labs, there should be more longstanding experience.

Professional desirability bias:

• Regarding instrumentation of the laboratories, some laboratories may have mentioned technologies that they implemented in other areas of forensic toxicology but not yet or not fully for postmortem specimen.

Preliminary conclusions: Impact?

• Impact of toxicological practices on epidemiological data • Survey represents current state of the art in European labs • Only limited back-calculation possible concerning practices 5 or 10 years ago. Death certification basing on toxicological results • Variability is in the pathologist´s decision on what to include as cause of death

in the certificate and therefore what is included in the national DRD statistics. • Guidelines addressing sufficiently this problem seem to be not at hand.

• Need for single country-related expert studies, promising particularly in countries with centralized laboratory services

Scoping study on national/ international guidances

PA: Postmortem applications considered GR: General recommendations for all fields of Forensic Toxicology

Guidances

• Following international accreditation standard ISO/IEC 17025 (General requirements for the competence of testing and calibration laboratories), guidances that represent all fields of forensic toxicology.

• They fully apply also for postmortem applications which follow the same general principles of quality assurance and methodology

• Only we address particular issues related to postmortem specimen: • Specimen collection at autopsy • Potential impact of postmortem matrix- related effects • Analytical impact: Systematic Toxicological Analysis (STA) involving "General

unknown Screening" (GUS)

• There is no specific guidance for DRD- related Forensic Toxicology available

Lack of standardization (Examples)

• Pre-analytical management: Sample collection, preservatives, storage of post mortem specimen and preparation • Only few recommendations for specific scenarios (“needle autopsy”, decomposed bodies) • Pre-autoptic sampling/ sampling at autopsy • Sampling in case of prolonged survival

• Calibration in case of postmortem specimen • Appropriate matrix-matched calibrators

• Post mortem screening and confirmation methods • Recommendations concerning minimum instrumentation

• Analytical methods/ Minimum LODs

• Interpretation and reporting of findings • Tables of therapeutic, toxic and lethal concentrations can be highly misleading • Reference concentrations in different tissues related to concentrations in postmortem blood?

• Toxicological report and Certification of DRD • “Listing the generic names of all chemical agents considered responsible for causing death”

Conclusions (scoping study)

• A high specialization level is required for the investigation of postmortem material from suspected DRD and evaluation of the findings.

• Basically, professional guidances do not address specifically these requirements

• National/ International expert recommendations tend to give high degree of flexibility

• …taking into account lack of adequate equipment with expensive technology in some laboratories and limited resources for reference standards for constantly emerging new substances appearing on the market.

• Guidelines tend to be rapidly outdated in times of rapidly emerging new trends in substance abuse as well as available technologies.

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