Occupational Drug & Alcohol Testing Conference

POCT Testing: The Basic Principles

Dr Genevieve Boshoff

Principles

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

system

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

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

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

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

Sample pad

Conjugate pad

Membrane containing test strip and control strip

Wicking pad

Test Strip Control Strip

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

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

Posi

tive

Resu

lt

Neg

ative

Re

sult

TC C

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

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.

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

Specificity

• The ability of the kit to detect the analyte of interest and not other compounds

Number of negative samples determined by the POCT devicesNumber 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

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

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.

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 conjugated to gold or pigment

(conjugate)

Antibodies to the

conjugate

Antibodies to the drug

Posi

tive

Resu

lt

Neg

ative

Re

sult

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Introduction to ‘real world’ use of PoCT devices

Dr Simon Davis

What to consider before using PoCT devices !

How accurate are PoCT devices?

Laboratory based performance of PoCT devices

Modified from Melanson et al.(2010)

Taken from data collected by the College of AmericanPathologists Proficiency Testing Surveys over a 6 year period

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.

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).

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.

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)

Why are Thresholds a problem for PoCT devices

Drug SAMHSA

EMIT

(ng/ml)

Fastect® II

(ng/ml)

Reditest

(ng/ml)

Amphetamines 500 1000 1000

Cocaine 150 300 300

Marijuana 50 50 50

Opiates 2000 300 300/2000

Phencyclidine 25 25 25

Threshold concentrations for a range of PoCT Devices commonly used compared to the SAMHSA threshold concentrations

Substance Abuse and Mental Health Service Administration (SAMHSA)

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.

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.

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).

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)).

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 17025 requires a laboratory to:

“retain records of original

observations, derived data and sufficient information to establish an audit trail…” (ISO 17025 2005).

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!

Conclusions

1. PoCT devices can only be used as a screening method and require confirmation by a gold standard method such as GC-MS.

2. 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.

3. 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.

4. 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.

5. PoCTs should only be used by trained operatives.

6. A full chain of custody is not possible with PoCT devices.

Case Study

Michalakis Michael – LGC Ltd

April 2013

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

SWEDEN

Borås

FINLANDTurku

BerlinLuckenwalde

Almere

Wesel

Cologne

Lomianki

Brno

SzentendreCluj-Napoca

Molsheim

GERMANYCZECH

REPUBLIC

POLAND

HUNGARY

ROMANIAITALY

FRANCE

BULGARIASofia

Milan

Barcelona

SPAIN

NETHERLANDS

RUSSIASt Petersburg

CHINA

Beijing

Shanghai

TURKEY

Istanbul

DubaiINDIA

UAEDelhi

Ahmadabad

Mumbai

Goa

Bangalore

Hyderabad

Edinburgh

Wakefield

LeedsBury

RuncornRisley

Tamworth

UK

Culham

ExeterSandwich

HoddesdonSt Neots

Belfast

N. IRELAND

IRELAND

Fordham

TwickenhamTeddington

BeverlyUSA

LexingtonDenver

Manchester

Sao Paulo

SOUTH AMERICA

Johannesburg

SOUTH AFRICA

LGC locations

36

LGC Health Sciences

Bioanalysis• Small Molecule• Biologicals

Sports supplement assurance schemes

• Informed-Sport TM

• Informed-Choice TM

Materials Science• Particle Characterisation• Physical properties

Pharmaceutical Impurities• Contamination testing• Foreign particulates• Genotoxic

Health & Wellbeing• Nutritional biomarkers• Fitness Screening

Residue analysis• ILVs• QuEChERS/bespoke

Workplace Drug Testing• Pre-employment, random,

incident & for-cause• Urine, hair, oral fluid

Nutritional Composition• Fatty acids• Vitamins

Product Safety• E&L• Nitrosamines

Food contaminants• Heavy metals• Vet drugs & pesticides

Banned Substance Testing• Doping control• Drug surveillance

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

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

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

Customer Review

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

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

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’

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’

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)

Detection Times

0 20 40 60 80 100

Hair

Urine

Oral Fluid

Detection Time (days) for different sample types

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

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

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

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

Case Conclusion (Historic)

• POCT/Oral fluid combination– Oral fluid gave too short detection time for pre-employment– POCT and oral fluid gave a very low (confirmed) positive rate– POCT random testing was viewed as unsuitable due to questions over the

integrity of test result and on-site POCT result not essential– POCT did not offer the flexibility of laboratory testing– Often slow collections when dealing with larger numbers of people

• Cost– Cheapest initial cost and met budgetary requirements but…– After considering all costs easy to show that it works out as the most

expensive option (on recruitment and training alone)

Customer Conclusion (New)

• Hair - pre-employment– Excellent window of detection– Excellent ‘positive return’ (5%)– Quick and easy to collect, store, and send– Most expensive initial cost but excellent overall value

(based on recruitment and training costs alone)• Urine – random and for-cause and incident

– Suitable window of detection– Industry standard– Good ‘positive return’ (2.5%)– Good value initial outlay and good overall value– Requires specific collection facilities (i.e. toilet)

• Successful programme– More efficient, cheaper overall– More effective, people aware programme is more

effective with higher calibre recruitment

mdm@lgc.co.ukLuckenwalde, Germany

Teddington, UK

Bangalore, India

Wesel, Germany

Lexington, USA

Beijing, China

Kyalami, South Africa

Bury, UK

Teddington, UK

Questions?

Point of Collection Testing – the Legal Issues

Peter FeldschreiberFour New Square, Lincolns Inn

Relevant technical issues

• Do the validity, reproducibility, precision sensitivity and specificity of the point of collection (POCT) diagnostic tests for drugs provide the degree of accuracy that such testing for both screening and diagnostic purposes requires in order to satisfy the statutory health and safety legislation ?

• Potential for false positive and false negative test results. This implies very wide confidence intervals for the statistical values of the point of collection evaluations of urine tests. Such wide confidence intervals would amplify the risk of false results in random testing.

• Technology of POCT analysis for drugs could enhance the risk of contamination and interaction of chemical analytes with potential for spurious results with different drugs.

Harm from false positive and from false negative tests (1)

• Examples of indirect harm include• misdiagnosis,• delayed diagnosis,• delayed treatment,• inappropriate treatment,• absence of treatment,• transfusion of inappropriate materials.• Indirect harm may be caused by• imprecise results,• inadequate quality controls,• inadequate calibration,• false positive or• false negative results

Harm from false positive and from false negative tests

• Employers could be liable in negligence for taking action regarding employment of examinees on the basis of both false negative and false positive tests.

• False positive test resulting in a prospective employee being refused employment on the grounds of alcohol or drug misuse, employer liable in negligence.

• False negative screening test; subsequently operates machinery under the influence and causes injury or death leading to action for substantial damages, and potentially prosecution e.g. corporate manslaughter.

Insurance

• Validity of insurance contract depends on compliance with statutory health and safety legislation.

Potential for failure to mitigate against the risk of unreliable and inaccurate testing. If a test result, originally evaluated as negative, is subsequently found to be unreliable or incorrect and the employee causes injury or death and/or damage in the course of his job, questions as to the applicability and validity of the insurance policies, regarding negligence and possibly product liability may arise.

Insurance (2)

• Validity of indemnity insurance for negligence when decisions on employment of persons who subsequently cause loss and/or damage are found to have been employed on the basis of false negative tests. Reliance on technologically flawed tests without due care or process to ensure their accuracy could result in void contracts of insurance. This could result from false negative tests, when an employee was subsequently found to be under the influence of drugs and/or alcohol and also from false positive results if an applicant claimed discrimination if not appointed.

Obligations of Confidentiality

• Disclosure of confidential and private information regarding the health and welfare status of the examinee could breach employer’s and relevant medical staff’s obligation of confidentiality.

• Current legal position is that the obligation of confidence applies where a doctor or medical professional receives information other than in connection with a professional relationship, e.g. medical screening in respect of employment.

• May have implications for training and qualifications of staff undertaking screening.

Statutory Obligations

• Type of testing, sample collection and security of the sample from contamination are specifically referred to in the European Workplace Drug Testing Society Guidelines:

‘1.1 Legally defensible workplace drug testing is a three stage process. The specimen has to be collected, analysed and finally the analytical result has to be correctly interpreted. This all has to be done in the context of ‘Chain of Custody’.

•

1.3 If any one of these three stages has flaws, then the whole process may be invalid.’

• 1.4 Where immediate test results are required, Point of Collection Tests (POCT) can be utilised, but the principles and procedures for specimen collection outlined in these guidelines still apply.’

• BUT : note the appendix referred to was never drafted!

• POCT testing is explicitly approved, with the caveat that privacy and security is ensured at the specimen collecting site, there is proper identification of the examinee, appropriate steps are taken to protect against tampering and adulteration, there is evidence of written informed consent of the individual and that disclosure of recent medication or evidence that the individual was advised of the significance of recent medication is confirmed. .

Practical Questions (1)

• POCT test fails to spot a positive and the person is then involved in an incident. If they are then tested by laboratory analysis and found positive (and if, for example there is serious injury or death on a construction site) what legal sanctions could be taken against the contractor and his Occupational Health Company?

False negatives – negligence and potential criminal issues

• If employee operates machinery under the influence drugs/alcohol and causes injury, action for substantial damages. Employer could face potential liability for gross negligence manslaughter. Employer owed duty of care, was in breach of that duty and the breach of duty must have caused the death of the victim. The nature of the breach of duty must have been so bad as to amount to gross negligence with criminal consequences.

Jury question. In a foreseeably false negative test missing a dangerous drug level the jury would have to consider whether the extent to which employer’s conduct (in failing to ensure that their testing policy was followed) departed from the proper standard of care incumbent upon them, involving a risk of death, was such that it should be judged criminal. Similarly there is a real possibility of prosecution for corporate manslaughter.

• What would be the legal implications where an employee gives a false positive test via POCT and is suspended pending laboratory confirmatory testing?

• Could be action in breach of confidence

• Although suspension is a neutral act, the reputational damage suffered by ‘an employee could cause him to feel that there had been a breakdown in trust and confidence in his relationship with his employer, leaving him no choice but to resign – constructive dismissal

• As regards the duty of confidentiality of the company towards employees in relation to drug testing results, what would be the legal situation whereby contractors designate potentially untrained administrative staff to conduct POCT testing?

• Current operating system contravenes the EWDTS guidelines. Audit trail for chain of custody sampling demands a robust administrative procedure and mechanisms to ensure privacy and security of the collection site, identification of both the individual and the samples, protection againsttampering and adulteration, evidence of informed consent and disclosure of recent medication or evidence that the examinee was advised of the significance of recent medication.In the absence of a qualified, trained and competent individual to collect the test sample, analyse and interpret the results and then have a robust administrative system to handle the test sample(s) appropriately, testing process invalid and potentially legally indefensible in actions in negligence and breach of confidence.

Point of Collection vs Chain of Custody

• Immediacy of the result and subsequent lack of a tamper proof and auditable ‘chain of custody’ procedure in POCT testing considerably reduces the protection from disclosure of medical information that is inherent in the conventional sampling and laboratory testing.

• Inadvertent disclosure of confidential information without the written express consent of the examinee may constitute negligent disclosure

PoC tests as medical devices

• Regulated under the In Vitro Devices Directive

• Must comply with essential requirements as specified by appropriate Notified Body

• Obligations to report incidents of defective performance

• Both false and negative test results constitute ‘incidents’ and may cause indirect harm; must be reported to Notified Body and potentially to competent authority such as MHRA

CORPORATE GOVERNANCE

• accurate and reliable results are paramount

• If company fails to apply sufficiently vigorous governance to regime of testing Board of Directors may be held to be negligent

CONCLUSIONS

• False positives and false negatives, coupled with unqualified staff reading tests and making employment and/or operating decisions could render employer liable in negligence.

• Potential insurance issues• Potential criminal liability• Serious doubts over use of PoC tests as

employment screening tools

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