Occupational Drug & Alcohol Testing Conference

1. Occupational Drug & Alcohol Testing Conference

2. POCT Testing: The Basic Principles Dr Genevieve Boshoff

3. Principles • Based on immunoassay • Reaction between an antibody and an antigen • Antigen = drug of interest • Antibody = protein produced by the immune system

4. Antigen-Antibody Binding • Antibodies bind strongly to a compound with a specific shape • Normally only binds to compound that was used to produce the antibody • Antibody produced using single compound of interest for each test • Tests kits allow us to visualise and quantify antigen-antibody binding

5. POCT Drug Test Kits • Number of different types • Dip sticks • Cassettes-testing of multiple drugs simultaneously • Cups-testing of multiple drugs simultaneously • Generally used to test urine samples • Detect use within the last week • Other test matrixes include saliva and blood

6. Elements of immunoassay • 3 essential components required: • Antigen that you would like to detect e.g. drug • Specific antibody to the drug • Method of quantifying the antigen • Number of immune complexes formed • Use detectable labels e.g. enzyme, dye or pigment, radioactive component

7. Lateral Flow Assays • Sample pad-adsorbent pad to which sample is applied • Conjugate pad- contains antibodies or antigens specific to the drug of interest conjugated with pigment or colour compound • Test strip –contain antibodies/antigen • Control strip- contains antibodies to the conjugate • Wicking pad -absorbent pad to draw sample across the test strip Test Strip Control Strip Sample pad Membrane containing test strip and control strip Wicking pad Conjugate pad

8. Assay Methods • Direct Assay • Positive result indicated by presence of test line • Competitive Assay • Positive result indicated by absence of a test line • Control line is present in both cases

9. Competitive Assay • Competition between free drug and immobilised drug (immobilised antigen) for limited antibodies • In presence of drug, free drug binds to the antibody conjugate, preventing it from binding to immobilised drug. No colour reaction. • If no drug present antibody complex binds to the immobilised drug to produce coloured line. 1. Antibody conjugated to gold or pigment (conjugate) 3. Antibodies to conjugate 2. Immobilised Drug T C Positive Result C Negative Result

10. Positive Result • Any indication of a drug above the cut-off level/threshold • Minimum concentration of the drug or metabolite that must be present in the specimen in order for the test to be reported as positive. • Vary from drug to drug and kit to kit • Negative result does not mean that the sample is drug free

11. How results from POCT kits are reported • True Positive (TP): The result of the test is positive and that drug is present in the sample at or above the threshold concentration of the test. • False Positive (FP): The result of the test is positive but the drug is not present in the sample or at concentrations below the threshold of the test. • True Negative (TN): The result of the test is negative and the drug is not present in the sample or is below the threshold concentration of the test. • False Negative (FN): The result of the test is negative but the drug is present in the sample above the threshold concentration of the test.

12. Sensitivity • Lowest concentration of analyte that can be detected number of positive samples determined by the POCT device number of positive samples determined by the comparison method • Comparison method normally GC-MS • Reasons for poor sensitivity: • Antibody affinity for the antigen • Amount of antibody or antigen used • Membrane use for producing dipstick • Storage conditions • Production methodology

13. Specificity • The ability of the kit to detect the analyte of interest and not other compounds Number of negative samples determined by the POCT devices Number of negative samples determined by the comparison method • Ability to detect true positives and few false positives • Varies between drugs • e.g. opiate/opiod tests have low specificity while cocaine tests have high specificity

14. Cross-Reactivity • Non-specific influence of substances in sample that: • Structurally resemble the analyte • Molecules have similar cross reacting epitopes or sites which bind to the antibody receptors • E.g. amphetamine assays- ephedrine/pseudoephedrine structurally similar to amphetamine • Marijuana – can produce false positives with hemp products • Leads to the production of false positives

15. Conclusions • PoCT devices offer a inexpensive screening method for drugs of abuse. • Due to the underlying method, it is always possible that a PoCT devices will report false positives or false negatives due to cross reactivity and non-specific binding. • All results should be validated using GC-MS or a similar technique. • False negative results cannot be screened i.e. just because the result is negative does not mean the drug is not present.

16. Direct Assay • At conjugate pad, sample mobilises the antibody conjugate which migrates with the analyte towards the test and control strip. As it migrates, the drug reacts with the antibody conjugate to form a drug-antibody conjugate • Drug-antibody conjugate reacts with drug antibodies in control strip-forms a coloured line – positive test • Antibody conjugate reacts with conjugate antibodies to form a coloured line Antibodies to the conjugate Antibodies to the drug Antibodies conjugated to gold or pigment (conjugate) T C Positive Result C Negative Result

18. Introduction to ‘real world’ use of PoCT devices Dr Simon Davis

19. What to consider before using PoCT devices ! • How accurate are PoCT devices?

20. Laboratory based performance of PoCT devices Taken from data collected by the College of American Pathologists Proficiency Testing Surveys over a 6 year period Modified from Melanson et al.(2010)

21. How do PoCTs perform outside a clinical environment • ROADSIDE Testing Assessment Project (ROSITA 2) project was carried out between 2003 and 2005 With 2046 subjects Observed sensitivity plots of OF PoCT devices to different classes of drug. Modified from Blencowe et al 2011.

22. Driving Under the Influence of Drugs (DRUID) project (2012) 1025 subjects PoCT sensitivity to Cocaine and Cannabis as reported in Strano-Rossi et al. 2012. Positivity criteria were based on the kit manufacturer’s recommendation (CUTTOFF KIT) and those used in the DRUID study (CUTOFF DRUID).

23. Does a PoCT device conform to your testing program criteria? • To ensure good analytical performs and reduce the risk of false positives and negatives, all testing programs have a “Threshold” of concentrations above which a positive is reported and below which a negative is reported.

24. Effect of Sample THC concentration on drug detection by a range of immunoassay techniques A range of different immunoassay techniques were compared. These were: enzyme donor immunoassay (diamonds), colloidal metal immunoassay (Triage; squares, &), enzyme immunoassay (Diagnostics Reagents Inc; triangle), enzyme immunoassay (enzyme multiplied immunoassay technique; X-shape), fluorescent immunoassay (asterisk,), fluorescence polarization immunoassay (circle), microparticle immunoassay (kinetic interaction of microparticles in solution; plus sign. Modified from Melanson (2010)

25. Why are Thresholds a problem for PoCT devices Threshold concentrations for a range of PoCT Devices commonly used compared to the SAMHSA threshold concentrations Substance Abuse and Mental Health Service Administration (SAMHSA)

26. Can a PoCT device reliably identify the drugs of abuse you wish to control? • There will always be a risk of false positive results with PoCT devices. • This risk can be mitigated by a process of screening and confirmation of all positive results by GC-MS or another gold standard technique. • There is also a risk of false negatives, this risk cannot be mitigated.

27. Sample Collection Can operatives with minimal training be used to carry out PoCT tests and interpret and record the results? • Operatives must understand and be able to identify false positives due to cross reactivity. • Operatives must be able to review a subjects medical, pharmaceutical, dietary and behavioural characteristics to identify false positives and false negatives. • Operatives must be able to identify individuals claiming to be taking a legal substance with cross reactivity to an illegal substance in an attempt to mask drug abuse.

28. Sample Collection “It is well known that in clinical settings immunoassay tests are more accurate when the results are interpreted by clinicians rather than non-technical staff (Melanson et al., 2010).

29. Can a PoCT Device Have a Legally Defensible Chain of Custody? What is a Chain of Custody? US government regulations: “[a]ll urine specimens must be collected using chain of custody procedures to document the integrity and security of the specimen…” (Bush, 2008). The World Anti-Doping Agency (WADA) considers CoC to have been appropriately conducted when: “[t]he external record is initiated at the collection site and ensures that the Samples and the results generated by the Laboratory can be unequivocally linked to the [donor].” (WADA TD2009LCOC). The EWDTS provides similar language requiring that: “…the results reported relate beyond a doubt to that specimen.” (EWDTS (2011)).

30. Can a PoCT Device Have a Legally Defensible Chain of Custody? Results from PoCT devices only remain visible as long as the sample remains aqueous. ISO 17025requires a laboratory to: “retain records of original observations, derived data and sufficient information to establish an audit trail…” (ISO 17025 2005).

31. Are you confident that a PoCT devices will provide a consistent analytical performance? • PoCT devices are not covered by any formal accreditation. • There are no regulations monitoring the manufacture, use or distribution of PoCTs within the UK or Europe. • No ISO accreditation exists for occupational testing. • Most Devices are not CE marked. • Quality, performance and accuracy vary between different providers and even between batches of devices from the same manufacturer. • Don’t forget about Thresholds!

32. Conclusions PoCT devices can only be used as a screening method and require confirmation by a gold standard method such as GC-MS. The use of PoCT devices will always run the risk of false negatives, even if used as part of a screening program. Therefore, PoCT devices should never be used in safety critical environments. Positivity criteria (thresholds) are fixed in PoCT devices, if your positivity criteria is different to that of the PoCT, you cannot use the PoCT device. The lack of accreditation means you cannot be confident of the quality of the devices you purchase. Detection limits may vary causing doubt over threshold levels. PoCTs should only be used by trained operatives. A full chain of custody is not possible with PoCT devices.

34. Case Study Michalakis Michael – LGC Ltd April 2013

35. LGC history Origins dating back to 1842 as customs laboratory protecting excise duty payable on tobacco importation into the UK Company established in 1996 on privatisation from government agency Grown organically and through strategic acquisitions Organised in to divisions – Health Sciences, Genomics, Forensics, Standards, Science and Technology

36. LGC locations FINLAND Edinburgh RUSSIA Turku St Petersburg N. IRELAND SWEDEN Belfast UK Borås Leeds Bury IRELAND Wakefield Risley Runcorn Tamworth Fordham Beijing St Neots Hoddesdon Berlin Culham Almere Luckenwalde CHINA POLAND Twickenham Teddington Lomianki NETHERLANDS Wesel Sandwich Shanghai Exeter CZECH REPUBLIC Cologne GERMANY Brno Molsheim Szentendre HUNGARY Cluj-Napoca FRANCE Manchester Istanbul USA ROMANIA Beverly Milan ITALY Lexington Denver TURKEY BULGARIA Sofia Barcelona SPAIN UAE Delhi Dubai INDIA SOUTH AMERICA Ahmadabad Mumbai Hyderabad Sao Paulo Goa Bangalore Johannesburg SOUTH AFRICA 36

37. LGC Health Sciences • Nutritional Composition • Fatty acids • Vitamins • Workplace Drug Testing • Pre-employment, random, incident & for-cause • Urine, hair, oral fluid • Banned Substance Testing • Doping control • Drug surveillance • Residue analysis • ILVs • QuEChERS/bespoke • Bioanalysis • Small Molecule • Biologicals • Sports supplement assurance schemes • Informed-Sport TM • Informed-Choice TM • Materials Science • Particle Characterisation • Physical properties • Food contaminants • Heavy metals • Vet drugs & pesticides • Product Safety • E&L • Nitrosamines • Pharmaceutical Impurities • Contamination testing • Foreign particulates • Genotoxic • Health & Wellbeing • Nutritional biomarkers • Fitness Screening

38. Drug Testing • Dedicated laboratory established for over 16 years • Urine, oral fluid, and hair • Controlled, prescribed, and ‘legal high’ drugs • Steroids and supplements • Legal, clinical, employment sectors • Experienced team • Broader scientific network within LGC

39. Case Study • Customer background • Employ over 50,000 people • 2000 pre-employment interviews per annum • Professional workforce • High specification training • Pre-employment drug testing • Random drug testing • For-cause/Incident drug testing • Total cost to recruit, train, staff downtime = £25,000 per successful candidate

40. Historic Testing Approach • Previously used a combination test approach (even spread) • urine testing (random) • urine point of care testing (pre-employment/random) • oral fluid (pre-employment) • oral fluid point of care testing (pre-employment) • Instant result • Quick • Easy • Cheap

41. Customer Review • Customer requirements • Legally defendable • Effective Programme • Simple • Cost efficient • Reliable service • Good support • Meet policy requirements

42. Oral Fluid Detection • Advantages • No specific facility (i.e. toilet) required • Observed sample • Low to moderate sample cost • Disadvantages • Short window of detection (12-24hrs) • Collection devices are all different (not universally supported) • Inconsistent approach between labs • True A and B samples questionable • Contested samples can be problematic

43. Urine Detection • Advantages • Industry standard • Low initial sample cost • Robust and proven procedures • No shortage of sample • Disadvantages • Requires specific collection facilities (toilet) • Not observed sample • Longer collection time if ‘shy bladder’

44. Hair Detection • Advantages • Easy to collect • Quick to collect • Observed sample • Easy to store and send • Long timeframe available (up to 3 months) • Disadvantages • Initial costs are high • Not suitable for ‘for-cause’

45. Point Of Care Test (POCT) • Advantages • Generally low cost device • Initial test result available in few minutes • Decision can be taken based on screen result • Disadvantages • Decision can be taken based on screen result • No detailed guidelines for performance criteria • Huge variation in performance between test devices • Less analytical flexibility (i.e. harder to change cut offs)

46. Detection Times

47. Trial Period • Hair Testing • Best for pre employment testing (longest window) • Policy easier to change as it’s pre-employment • More expensive initial outlay but expecting to be ‘cost efficient’ • Urine Testing • Random and for-cause (reasonable window) • Policy easy to appease as industry standard • Cost efficient • Oral Fluid • Historically used but dropped in favour of urine and hair • POCT • Historically used but dropped in favour of urine and hair

48. Trial Results • 3 month trial • Both hair an urine run side by side for pre-employment • Findings • 2.5% positive rate in urine (overlap with hair) • 5% positive rate in hair • Comparison to previous methods • Prior methods (POCT and Oral Fluid) found 0.5% over the previous 3 year period • Similar (some the same) sample population and conditions to historic populous

49. Finance • Background • Average of £25,000 to recruit and train • 2,000 pre-employment interviews • Hair = £90 per test • Urine = £30 per test • Oral Fluid = £30 per test • POCT = £20 per test • Overall costs at 100% selection • Hair (at 5%) = 2000*90 = £180,000 • Potential of 100 donors being recruited and £2.5M ‘lost’ (+£2.32M) • Urine (at 2.5%) = 2000*30 = £60,000 • Potential of 50 donors being recruited and £1.25M ‘lost’ (+£1.19M) • POCT (at 0.5%) = 2000*20 = £40,000 • Potential of 10 donors being recruited and £250K ‘lost’ (+£210K) • Figures are not actual but representative of cost

50. Finance 2 (NOT ACTUALS) • 100% selection not realistic therefore based on 10% selection • Hair (at 5%) = 2000*90 = £180,000 • Potential of 10 donors being recruited and £250,00 ‘lost’ (+£70K) • Urine (at 2.5%) = 2000*30 = £60,000 • Potential of 5 donors being recruited and £125,000 ‘lost’ (+65K) • POCT (at 0.5%) = 2000*20 = £40,000 • Potential of 1 donor being recruited and £25,000 ‘lost’(-£15K) • Recruitment and Training figures do not include loss due to • Absenteeism • Theft • Related accidents • Loss of productivity • Disciplinary actions • Dismissal