Oral fluid testing device

Abstract

CTLS as part of the collaborative xxxxx Trading Ltd for validating One Step Multi-Drug Oral Fluid Drug Testing Device. Also the DOA test device registered with medicines and healthcare products.

Validation study of one step multi device was performed in CTLS by two ways.

Abbreviations

µg/L - Microgram per litre
QA - Quality Assurance
POCT - Point of Care Test
MHRA - Medicines &Healthcare products Regulatory Agency
UKNEQAS - United Kingdom National External Quality
CAP - Clinical Pathology Accreditation
CTLS - Clinical Trials Laboratory
AMP - Amphetamine
BENZO - Benzodiazepines
COC - Cocaine
THC -Marijuana
MAMP - Methamphetamine
MOR -Morphine
OF - Oral Fluid
GC - Gas Chromatography
GC/MS - Gas Chromatography Mass Spectrometry
LC - Liquid Chromatography
CE - European Conformity
DDS - Digital data storage
0C - Degree centigrade
SOP - Standard Operating Procedure
IVDMD - In-Vitro Diagnostic Medical Device


Validation method 1

Validation method (1) was conducted from June 2009 through to July 2009. Seven samples were collected from UKNEQAS, which were offered pilot surveys for drugs of abuse in oral fluid and tested using a one step multi device. Three samples were detected by the one step multi device. The one step multi drugs testing device test result was compared to UKNEQAS analyst report.

Validation method 2

Validation method (2) started July 2009 and was completed in August 2009, eighteen urine and saliva sample were collected from volunteers and tested for drugs of abuse and tested using one step multi and Dipro Drug lab Multi panel Drug Test devices. During the test, three samples were providing positive results. The test results were compared with each method, and test result would be kept confidential so that client’s information would not be disclosed for any other purpose.

Chapter: 1

1     Introduction

The goal of this project at CTLS was to validate the Drug Abuse Point of Care Testing Kit which will have immense value to the public as well as to the medical research. The validation of testing the kit will certainly in turn become a reliable resource for definitive screening tool in the future. Our aim was to establish clinical cross validation trials necessary to prove drugs of abuse point of care testing kit for human saliva. It could be used as an advanced, accurate and handy clinical laboratory technology to evaluate individual’s drug abuse level. Our validation was a team effort to achieve the goal to use human saliva as a non-invasive-bio fluid.

The saliva determines the status of a human body. In other words, it is a mirror of the body. The human saliva is easy to collect and can be obtained at low cost in sufficient quantities for analysis. Saliva based tests are more accurate, have a quicker turnaround time and less expensive, particularly as a home drug testing kit.

One Step Multi-Drug Oral Fluid Test kit is ideal for qualitative determined level of Amphetamine, Benzodiazepines, Cocaine, Marijuana, Methamphetamine and Morphine in human saliva. This kit provides only preliminary screening of the results within three (3) minutes, and provides the amount of all the drugs present in the human body. If positive result occurs, confirmation must be obtained by more specific chemical method utilizing GC/MS or LC/MS. The drug of abuse of POCT kit will detect, whether any drug is present in oral fluid below the cut-off limit.


1.1  One Step Multi-Drug Oral Fluid Test kit Cut-off Concentrations.

 

Symbol

 

Test

 

Calibrator

Point of Care kit Cut-off level(ng/ml)

AMP

Amphetamine

D-Amphetamine

50

BENZO

Benzodiazepines

Benzodiazepines

30

COC

Cocaine

Cocaine (parent)

20

THC

Marijuana

∆9-THC(parent)

50

MAMP

Methamphetamine

D-Methamphetamine

50

MOR

Morphine

Morphine

40

1.2  The following are the advantages of saliva-based drug of abuse point of care testing kit:

  • Reliable diagnostics and quick turnaround time.
  • Easier than other test methods, which mean no additional equipment and operator training is required.
  • Safer to use, and cheaper than current oral fluid used methods. For example other Oral Fluid methods: GC/MS, ELISA, Point of Care lab tests.
  • Testing kit can be kept at room temperature.
  • Testing provides an accurate and reliable assessment. This test detects most of the common drug abuses.

1.3  Reasons for using the Saliva based Drug Testing Kit:

  • For individual who can check for themselves if there are any drugs presents in their circulation system.
  • Testing can help the employers to determine if any of their current or potential employees abuse any of the banned (illegal) substances.
  • Non-invasive.[19]
  • Sample cannot be adulterated.
  • CE mark. (European Conformity)

1.4  POCT method of DOA test compared with other DOA Test methods

Method

POCT Oral Fluid

ELISA

POCT urine

GC/MS

Sample

Oral Fluid

Oral Fluid/Urine

Urine

Oral Fluid, Urine, Blood, Hair

Format

Lateral Flow

ELISA

Lateral Flow

GC /MS

Test time

5min

48-72 hours

5min

3-7days

Additional Equipment Required

None

Incubator ,ELISA reader

None

Spectrometer

Storage

RT

2-8 0C

RT

NA

Shelf Life

18 months

NA

24 months

NA

Advantage

Accurate & reliable Easy to use at any time and any where Non-invasive, No additional equipment required, cheap

Accurate reliable
High specificity

Less expensive

Instrument-based
testing ,Accurate

Disadvantage

More expensive than rapid urine testing

Expensive, Complex method

Sample can be easily adulterated

Very expensive


1.5  Oral Fluid

Oral fluid is referred to as “saliva”. The saliva is excreted primarily by 3 glands namely, parotid, sub maxillary and sublingual. It has low protein content and can vary in flow rate from zero to several milliliters depending upon influence from various factors including emotional state and hunger. Spitting or expectoration provide neat saliva but is relatively viscous, and can be difficult to work within the clinical laboratory. It may also be contaminated with food and other particles from the mouth and will therefore require centrifugation. Some of the collection methods used by various companies are those indicated in Table :( 1.8.5) and they use some forms of proprietary diluent to mix with collected saliva. The absorbent pad or foam is used to collect saliva comparatively there is no one type of collection device that is better than other when it comes to ease of use.

However, there are continuous developments of new products. [6] Plasma electrolytes such as bicarbonate, chloride, potassium and sodium and other plasma elements such as enzyme, immunoglobulin and DNA are present in saliva produced from three major and several minor salivary glands. Plasma electrolytes such as bicarbonate, chloride, potassium and sodium and other plasma elements such as enzyme, immunoglobulin and DNA are present in saliva. Most of drugs can be detected in saliva; including amphetamine, barbiturates, benzodiazepine cannabinoids cocaine methamphetamine ethanol and heroin. [1]

However many factors may affect drug transfer into saliva, such as degree of protein binding, pKa, physical size and lipophilicity of drug.

 

1.6  Advantages of saliva as drug testing matrix

Nowadays saliva matrix is more popular for screening of drugs of abuse .The advantages of using saliva matrix are provided below.

  • Saliva collection is easier than other biological fluid. Also direct collection is not embarrassing and significantly reduces an influence of interference enter into sample collection process. Blood collection is a highly invasive method.
  • Testing is more reliable and adaptable for roadside testing.
  • Samples are analysed directly without any further pre-treatment and test results are provided immediately, for example the new brand of Cozard® DDS system can provide results within ninety seconds for two major classes of drugs and less than six minutes for six other major classes of drugs. This can help to initiate law enforcement process with sufficient data.
  • An interpretation of drugs in saliva can be used to determine pharmacokinetic limitation. Therefore a low analyte concentration can be easily detected.

1.7  Disadvantages of saliva as drug testing matrix

Concentration of drug can be changed by pH during the sample collection. Therefore Saliva and Plasma ratio may be changed. [8]

1.8  Saliva collection methods

Production of saliva is influenced by a variety of methods such as stimulating and non-stimulating collection of saliva. [9, 4]

1.8.1           Stimulating method

Saliva can be swabbed from mouth using absorbent materials such as sour coated cotton balls. The absorbent material of cotton ball becomes soaked with saliva, which can be removed from the mouth and the saliva is separated by centrifugation or applying pressure to the cotton ball. [3, 4].However, saliva collection method by stimulation is connected with several potential problems, for example the absorbent material can react or absorb some drugs. Therefore actual amount of drug concentration can be varied in saliva.

Also some salivary stimulate material (sour flavor) may change the saliva pH level. For example, the level of codeine present in saliva collected after stimulation, which can be decreased by an average of 3.6 times of non-stimulated method of collection. [5].Also non-stimulated saliva contained substantially more drugs than stimulated [8].

1.8.2           Non-stimulating method

Saliva can be collected from mouth into a collection device without using any salivary stimulate material .However, some physical techniques are used to produces saliva ,such as tongue, cheek and lip movements. [8]

1.8.3           Problems with Sample collecting device of saliva

Most of sample collection devices contain a form of an inert absorbent pad for saliva collection [9]. However, different amount of saliva is absorbed during the sample collection process by different devices. Collection of sample is not assured by most of sampling devices. Therefore many of devices have a colour changing indicator. An absorbent pad made from different materials. This material must be chosen carefully because some materials may absorb some of the drug. This can lead to errors. Different buffers /preservatives which used in different devices may be incompatible with hyphenated MS techniques for a confirmation of drug abuse levels [4].

Some of the most common problems:

  • ion suppression in LC-tandem MS
  • long term contamination of columns and ion source in GC

1.8.4           Advantages of sample collecting devices

  • The point of care saliva collector uses an optimal method for obtaining full saliva.
  • Saliva is easily obtained by chewing on a roll-shaped saliva collector and Very Cheap.
  • The chewable kit are coated with citric acid .This can be stimulated more saliva.

1.8.5    The list of saliva sample collection devices and characteristics

 

Collection Device & Made in

 

Method of collection

 

Control of, saliva volume

 

Buffer /diluents

 

Stimulated collection

Cozart®oral swab. UK

Absorbent swab

Yes

Yes

No

Intercept®
Device. USA

Salt impregnated pad

No

Yes

Yes

Drug Wipe. Germany

Absorbent pad

Yes

No

No

Greiner Bio-One. Austria

Saliva extraction solution

Yes

Yes

No

Saliva Twist device Drug Test. UK

Sponge swab

No

Yes

No

Oralab 6. USA

Absorbent pad

Yes

No

No

Oraline. USA

Collection cup

Yes

No

No

Oral Stat. USA

Sponge swab

No

Yes

No

Oratect. USA

Absorbent pad

Yes

No

No

VerOFy.USA

Absorbent pad

Yes

No

No

Saliva Screen. Germany

Absorbent pad

Yes

Yes

Yes, acidic available
& mechanical

Smart clip multidrug. Germany

Absorbent pad

No

Yes

Yes, mechanical

Quantisal. USA

Absorbent pad

Yes

Yes

No

Salivette. Germany

Cotton wool/polyester swab

No

No

Yes, acidic available &mechanical

Source: young scientist journal June 2009 and the kit manufacture’s web sites


1.9  Literature Reviews

(1) Biermann et al., 2004 did research on “On-site testing of illicit drugs using Toxiquick”. The experiment was carried out for 18 months. During the periods 751 individuals were tested at road site with immunochemical test device “Toxiquick” for drug abuse such as amphetamine, MDMA, MDE, cannabis, cocaine, heroin and morphine. 302 individuals’ oral and blood samples were taken during the periods. This was tested with immunochemical and non immunochemical method (GC-MS) and the test results were compared. In general 75% Toxiquick results were agreed with GC-MS results. However, cannabinoids test were shown to have negative values. This can be caused by metabolise.

(2)Vanna De Rose et.at(2004) wrote a review report on “drug testing in the work place”, and the summary of their review report is given below directly quoted from their work. Drug testing in the work place: ‘There are various methods of approaches to identify drugs of abuse for staff ’. For example urine, blood, oral fluid, hair and sweat can be used to test. Four fundamental reasons concern the author, for drug testing at work place; the cost of drug testing at work place, how reliable are the results? Cut off levels and strength of drug testing device.

Safety: drugs of abuse can increase safety risk to own self and co-workers at work place, for example, where employees are working at higher sensitivity working area such as air traffic controllers, Pilots and

Ambulance drivers etc.

Organizational efficiency: drugs of abuse can increase low productivity, high staff turnover and absenteeism. Therefore drugs testing can increase the productivity and reduce the cost of new employees and training them.

Reputational risk: When an employee takes an illegal drug, the reputation of the organization can be damaged.
Employee welfare: Drug test can improve employee health and would help to identifying staff that need to be sued for drug intake.
The cost of drug testing at work place can be influenced by three major factors.

  • Higher financial costs are involved for drug test.
  • Drug testing can be damaged relationship between staff.
  • Employment of staff and human resource service are a significantly higher cost.

Testing device can detect drugs other than illicit drugs. For example over-the-counter painkillers can be detected and have produced a “false positive result” because of the opiate.
This problem can cause discrimination against a person who is accused of false impairment.
The problem can be resolved by changing of identification cut off limits for specific level of illicit drugs.
Most of drug testing device can indicate only drug metabolite activity in body but not the level of intoxication.

(3)E.J. Cone. et.al (2007) in their paper on “Interpretation of oral fluid test for drugs of abuse” The article reviewed several aspects of oral fluid testing, detection times, application of oral fluid testing and interpretation of test result .They also reported the influence of chemical, physiological and pharmacological factors that are involved in oral fluid testing result.

(4)Prof Olaf H Drummer. Clinical Biochemist Reviews (2006, August 27(3): 147-159)
In The Clinical Biochemist Journal the application of method of testing for drugs in saliva such as LC-MS, HPLC with MS are compared with road site drug testing kit. The Advantages of testing kit were described, in the Journal, to be a level of accuracy and efficiency.

(5)Kato et al .J Anal Toxicology 1993 Oct: 17(6):338-41 “Cocaine and metabolite excretion in saliva under stimulated and non-stimulated conditions” .The collections of saliva sample were carried out by stimulated or non-stimulated condition from six healthy volunteers who were given cocaine during the test session. The presence of cocaine level and metabolites were analysed by GC-MS .They reported that the non-stimulated saliva contained more cocaine than stimulated. This can be caused by levels of pH present in saliva.

(6) Dr.Graham Facile. “Evaluation of the Drager Drug Test® 5000Test System”. (2008): Drager Drug Test 5000 test system has been used for detecting drugs in human-saliva /oral fluid. During the period of testing, 503 individual tests were done .The test results obtained were within minutes utilizing Drager Drug Test ® Test System .Also this device can detect the trace level of drugs present in saliva, for example, Cocaine (20ng/ml), Opiates (20ng/ml), Benzodiazepine (15ng/ml), Delta -9-THC (25 ng/ml) and Amphetamine (50 ng/ml).The results were compared with drugs of abuse point of care kit cut- off values. The Drager Drug Test ® 5000 Test System was found as more powerful immunochemical based diagnostic medical device.

(7) N.Fucci et.al “SPME-GC analysis of THC in saliva sample collected with “EPITOPE” device. (2001).The article mentioned several factors about oral fluid analysed with different techniques, method of collection device and oral fluid reflection for drug concentration. They reported that the drug concentration in saliva is directly reflected to plasma concentration in body. They detected a higher level of cannabinoid in saliva. So, this could be due to the fact that cannabinoid smoke is directly saturated in the oral cavity. They used an ‘EPITOPE’ device, which has an absorbent pad or foam used to collect saliva.

(8)O.R.Idowu and B.Caddy “A Review of the use of Saliva in the forensic detection of drugs and other chemicals” (2008).In their review they reported the saliva sample to be useful for drug detection in the forensic analysis. Concentration of drug level in Plasma can be reflected in saliva.

(9)Schramm.W et.al “Drugs of abuse in saliva” Journal of Analytical Toxicology (1992) This article reviewed various aspects of drugs of abuse in saliva. Analytical methods were used for the detection of drugs, including gas chromatography with mass spectrometry, thin layer chromatography and immunoassay. The study included that the level of concentration of cocaine and phencyclidine is correlated with saliva and plasma. They also found that the drug concentration of tetrahydrocannabinol (marijunana) can be reflected in saliva and blood.

(10)Dennis .J.Crouch et.al“An Evaluation of innovation Sweat Based Drug Testing Techniques for use in Criminal Justice Drug Testing” April 2002
The article discussed the acceptable drug/metabolites confirmations techniques such as GCMS / LCMS and LC/MS/MS. However, the researcher pointed out that the cannabinoid (THC) drug analysed with LC/MS/MS, can cause some interference, such as, electron spray ionization due to lack of a functional group ionized made electron spray.

(11) Creative Research Systems. http://www.surveysystem.com/sscalc.htm.
National Statistical Service. http:// www.abs.gov.au
These articles provide the relationship between the sample numbers, confidence level and the confidence interval.


Chapter: 2

2     Validation Method

This study of saliva matrix based on drugs of abuse point care testing kit [DAPOCTK] validation was performed in CTLS using two methods.

2.1  Validation Method: 1

A validation of Drug Abuse Testing Point of Care Kit [DAPOCTK] was performed by participating in accordance with United Kingdom National External Quality Assessment Scheme [UKNEQAS] for oral fluid Drugs of Abuse.

 

2.1.1           Role of United Kingdom National External Quality Assessment Scheme [UKNEQAS]

The UKNEQAS can offers a number of proficiency testing schemes for monitoring drug assays in serum, pilot surveys for drugs of abuse in saliva toxicological investigations and drugs of abuse testing in urine. Also a quality assurance service with strong educational knowledge is provided.

2.1.2           How does the Pilot Surveys Scheme work?

Known coded samples will be distributed quarterly and drug analysis will be performed for 4 weeks. The analytical performance will be informed to participated laboratories.


2.2  Validation Method: 2

The validation purpose of this study, urine and saliva specimens were required from volunteers and tested for drugs of abuse with one step multi and Dipro Drug lab Multi panel Drug Test devices.


2.2.1           Outcome of Comparison of two POCT aid Validation:

Both CE marked POC testing kit are ideal for qualitative determined level of drugs of abuse in human bodies using human biological specimens. Saliva matrix based on POC testing kit results correlate with urine matrix POCT kit result. The Saliva matrix based on POC testing kit will be validated by urine matrix POCT kit result.


2.2.2           The voluntary participation with CTLS for validation of POCT kit purposes:

The identities of participants will not be disclosed or divulged to the scientist / other staff members of CTLS who receive sample of oral-fluid /saliva. The purpose of ‘Drug of Abuse Information and Consent Form’ [Appendix II] is to provide information pertaining to voluntary participants of drugs-abuse and CTLS for research purposes as to the nature of the role and the legal and ethical standards to be met.

2.2.3           Scope of the Legalities:

To protect the confidentiality of the identities of the voluntary participants any information about the participants will not be revealed.
Steps will be taken to protect the confidentiality of voluntary participant’s data including code numbers, database, and storage of research data in CTLS.


Chapter: 4

4     Registered a point of care testing kit with Medicines and Healthcare products Regulatory Agency.

4.1  Medicines and Healthcare products Regulatory Agency.

The Medicines and Healthcare products Regulatory Agency (MHRA) is United Kingdom government agency of the Department of Health.

4.2  Role of MHRA

  • MHRA is responsible for making sure that medicine and medical devices work with safety regulation in UK 2002.
  • Medicines and medical devices information are provided so that they are publicly available.
  • MHRA is monitoring medicines and medical devices in market and encourage the public to inform them of any problem with a medicines and medical device.
  • If there are any problems with medicines and medical devices, then they are removed from market, so that they can be investigated and the necessary action needed can be taken against manufacturer.

4.3  In - Vitro Diagnostic Medical Devices Register with the MHRA

  • CE marking declaration certification [Appendix III]
  • Registration Form RG3
  • Registration fees

The required information for registration process and other pertinent information can be found in the MHRA Web site.
http://www.mhra.gov.uk/Howweregulate/Devices/Registrationofmedicaldevices/CON009810

Chapter: 5

5     Method Validation Plan:

Title: Validation of Drugs of Abuse Point of Care Testing kit
Objective: To validate Drug Abuse Testing Point of Care Kit [DAPOCTK] by participation of the United Kingdom National External Quality Assessment Scheme for oral fluid drugs of abuse.

  • The POCT kit will be validated using UKNEQAS oral fluid samples in Clinical Trials Laboratory Services Ltd.
  • The POCT kit will be validated by comparison with Volunteer urine samples screening DOA test in Clinical Trials Laboratory Services Ltd.

5.1              Validation of Analytical Method
5.1.1           Reference Compound, Apparatus and POCT devices
5.1.2           Reference Compound of Oral Fluid UKNEQAS

Reference Item will be analysed for validation.

Identity:                  Oral Fluid
Provide by:             United Kingdom National External Quality Assessment Schemes
Batch No:                OF/1008/October 08, OF/0609/June 09,
Oral Fluid:              (OF)

5.1.3           Apparatus

Micro Pasteur pipette (10µl)

5.1.4           Different matrices of POCT devices

5.1.4.1        Saliva matrix based on a point of care test kit.

Trade name of kit: XXXX Oral Fluid Test kit.
Manufacture: XXXXX.Ltd in China
Batch No: W650690510.
Expiry date: 05/2011.
Drug tests for: COC/Amp/mAmp/THC/MOP/BZO.
CE marked by Qarad European Regulatory Service in Belgium.
XXX Oral Fluid Test kit contained: Saliva Test Panel, Saliva Collection tube, Saliva Sponge Collector.

5.1.4.2        Urine matrix based on a point of care test kit.

Trade name of kit: XXXXXX Multi panel Drug Test
Manufacture: XXXXX in Austria.
Batch No: DOA8110062.
Expiry date: 01/2010.
Drug tests for: COC/Amp/mAmp/THC/MTD/MOP/PCP/BAR/BZO/TCA
XXXX Multi panel Drug Test kit contained: Urine Test Panel

5.2  Number of sample determination:

For determinations of sample size the following factors should be considered.

  • How accurate the result needs to be?
  • The level of confidence limit of the test results.
  • Available budgets for sampling and analysis.

The number of samples needed for validation can be determined by two calculation methods.

5.2.1           Calculation method: 1

S = (z/e) 2
S -The sample size, Z -The degree of confidence (95%, 1.96)

5.2.2           Calculation method: 2

S = [Z2 x (p) x (1-p)] / C2

S -The sample size, Z -The degree of confidence (95%, 1.96), C - Accepted error value as proportion, P - An estimate of the proportion of numbers of samples falling into the group of representing the population.
A larger number of samples truly reflect the population size. However, the experiment will be conducted with a minimum number of samples. Therefore the confidence interval percentage will be chosen as +/-19.6 and number of samples found as 25 at confidence level 95% [2, 10].In this validation of POCT kit experiment, the calculation method (1) will be used for determination of number of samples.

5.3  Experimental Pathways:

This validation of this experiment can be carried out by two different experimental pathways. However, the total number of samples will be maintained. (n= 25)

5.3.1           Experiment Pathway: 1

Saliva based on drugs of abuse POCT kit validation process can be carried out by UKNEQAS oral fluid samples .Oral Fluid will be directly introduced in to POC testing panel, where a sample introduction place [Appendix 1 (4)]. This validation process can be taken over a period of one year.


5.3.2           Experiment Pathway: 2

Saliva based on drugs of abuse POCT kit validation process can be carried out by urine based on drugs of abuse POCT kit. This validation process will be completed in a short time frame. The short period of time will depend on availability of sample.

A portion of the saliva and urine sample will be introduced into appropriate matrix of POC testing panel, where a sample introduction place. [Appendix 1 (4)]

 

5.4              Description of the Validation Procedure:

5.4.1           Sample collection methods:

Saliva or oral fluid and urine samples will be collected from healthy volunteers or from patients taking known medication.

5.4.1.1        Saliva sample:

Saliva directly collected from the mouth using saliva sponge collector. Then the saliva saturated sponge will be squashed in to saliva collector device. [Appendix: I (2, 3)]

5.4.1.2        Urine sample:

Urine sample directly collected from volunteers.

5.4.1.3        UKNEQAS sample:

UKNEQAS Oral fluid samples will be collected from volunteers or patients taking known medication.

5.4.2           Sample pre-treatment:

UKNEQAS Oral fluid samples will be diluted with oral fluid free of all abused drug groups. The UKNEQAS oral fluid samples concentration will be adjusted for investigation of laboratory performance levels. The UKNEQAS oral fluid samples will be preserved by heating at 60 oC for 90 minutes and addition of bronidox 0.05%, gentamicin 0.1g/L and penicillin 0.1 g/L.[UKNEQAS Oral Fluid pilot surveys for drugs of abuse in saliva guide line 2009]

5.4.3           Handling procedure for POCT device and sample:

  • All POCT devices will be operated in accordance with manufacture’s instruction.
  • After use POCT device will be disposed in accordance with applicable law.
  • All POCT devices will not used beyond their expiration dates.
  • All biological specimen samples should be considered as potentially hazardous.
  • All used POCT device and biological specimen can transfer infections.

5.4.4           Composition of Validation Run:

Each biological specimen will be performed twice for validation run. A test result is indicated by colour or faint band in the Test Valid Window of POCT kit.

5.4.5           Sensitivity:

The drug of abuse of POCT kit will detect if any drug is present in oral fluid above the cut-off limit.

5.4.6           One Step Multi-Drug Oral Fluid Test kit Cutoff Concentrations

 

Symbol

 

Name of Drug

 

Calibrator

POCT kit Cut-off level(ng/ml)

AMP

Amphetamine

D-Amphetamine

50

BENZO

Benzodiazepines

Benzodiazepines

30

COC

Cocaine

Cocaine (parent)

20

THC

Marijuana

∆9-THC(parent)

50

MAMP

Methamphetamine

D-Methamphetamine

50

MOR

Morphine

Morphine

40

5.4.6           Specificity:

A stimulating saliva collection method is connected with several potential problems, for example, the absorbent material can react or absorb some drugs. Therefore, the actual amount of drug concentration can vary in saliva. A salivary stimulate material (sour flavor) may change the saliva pH level. For example, the level of codeine present in saliva collected after stimulation can be decreased by an average of 3.6 times of non-stimulated method of collection. [4].Therefore, non-stimulated saliva contains a substantially higher amount of drugs than stimulated saliva. [8]Testing devices can detect other than illicit drugs. For example, over-the-counter painkillers can be detected and have produced a “false positive result” because of the opiate [15]

A higher level of cannabinoid is detected in the saliva. This could be due to cannabinoid smoke being directly saturated in the oral cavity. [9]
The level of concentration of cocaine and phencyclidine is correlated with saliva and plasma. Also it was found that tetrahydrocannabinol (marijuana) drug concentration can be reflected in saliva and blood. [13]

5.4.7           Limitation of Procedure:

This One Step Multi-Drug Oral Fluid testing kit is designed for detection of drugs in human saliva only. A positive result will be visibly obtained on test window in the kit. Therefore only the presence of a drug is found in testing sample. However, the test result can indicate a false result due to technical or procedural error. This testing kit cannot be measured or indicate the intoxication level in the body and distinguish between drug of abuse and medicines.

5.4.8           POCT device and Sample Storage:

A drug of abuse testing kit will be kept at room temperature (15-30oC) in the original foil pouch. Saliva or oral fluid will be stored at 4oC in refrigerator for up to 2 days or frozen at -20 oC until use.

5.4.9           Interpret Test Validity and Test Result:

Test validity and result will be interpreted within 5 minutes after initiating the test and result confirmation will be reported within 10 minutes.

5.4.9.1        Interpret Test Validity:

The test validity is indicated by formation of a colour band in the POCT device “Test Valid window”. However, the development of colour bands may require up to 15 minutes in the Test Valid Window. This can be caused by the high viscosity and variability of saliva sample.

5.4.9.2        Interpretation of Test Result:

The test result will be interpreted as either negative or positive immediately. However, the random error of visual test result interpretation will be determined by three analysts. If three individuals’ visual test results will be found as significantly different, confirmation must be obtained by more specific chemical method using GC/MS or LC/MS.


5.4.9.2.1     Positive Test Result:

There is a positive result when the coloured band cannot be observed in test valid window next to the specified drug name. Therefore specified drugs can be found in saliva above the cut –off limit. However, one coloured band observed in the control valid window, has to be present for the results to be valid.


5.4.9.2.2     Negative Test Result:

Two coloured band can be observed. One colour band can be observed in test valid window next to the specified drug name and the other colour band can be observed in control valid window.

5.4.9.2.3     Invalid Test Result:

Coloured band cannot be observed in control valid window.

Chapter: 6

6             Result:
6.1          Table R: 1                    Results for volunteer saliva and urine samples screened with POCT.


Sample

Saliva Sample Screening with POCT Result

Urine Sample Screening with POCT Result

ID

AMP

BENZO

COC

THC

mAMP

MOP

AMP

BENZO

COC

THC

mAMP

MOP

0330661

N

N

N

N

N

N

N

N

N

N

N

N

0330662

N

N

N

N

N

P

N

N

N

N

N

P

0330644

N

N

N

N

N

P

N

N

N

N

N

P

0330670

N

N

N

N

N

N

N

N

N

N

N

N

0330675

N

N

N

N

N

N

N

N

N

N

N

N

0330691

N

N

N

N

N

N

N

N

N

N

N

N

0330692

N

N

N

N

N

N

N

N

N

N

N

N

0330701

N

N

N

N

N

N

N

N

N

N

N

N

0330702

N

N

N

N

N

N

N

N

N

N

N

N

Note: AMP: Amphetamine, BENZO: Benzodiazepine, COC: Cocaine, THC: Marijuana, mAmp: methamphetamine,
MOP: Morphine, P = positive, N = negative, U=uncertain or numeric result. OF: oral fluid

 

6.2    Table R: 2 Results for UKNEQAS oral fluid (Oct 2008) sample screened in CTLS with One Step Multi-drug POCT and UKNEQAS Analyst Report.

Sample

Oral fluid sample Screening in CLTS with POCT

UKNEQAS  analyst result  report

ID

AMP

BENZO

COC

THC

mAMP

MOP

AMP

BENZO

COC

THC

mAMP

MOP

OF11008

P

N

P

N

N

N

P

N

N

N

N

N

OF21008

N

N

N

N

N

N

N

N

N

N

N

N

OF3 1008

N

N

N

N

N

N

N

N

N

N

N

N

OF4 1008

N

N

P

N

N

N

N

N

N

N

N

N

Note: AMP: Amphetamine, BENZO: Benzodiazepine, COC: Cocaine, THC: Marijuana, mAmp: methamphetamine,
MOP: Morphine, P = positive, N = negative, U=uncertain or numeric result. OF: oral fluid

 

6.3    Table R: 3                Results for UKNEQAS oral fluid (June 2009) sample screened in CTLS
                                           with One Step Multi-drug POCT and UKNEQAS Analyst Report.

Sample

Sample Screening Result in CLTS

UKNEQAS  analyst result

ID

AMP

BENZO

COC

THC

mAMP

MOP

AMP

BENZO

COC

THC

mAMP

MOP

OF10609

N

N

N

N

N

N

N

N

N

P

N

N

OF20609

N

N

N

N

N

N

N

P

N

N

N

N

OF30609

N

N

N

N

P

N

P

N

N

N

N

N

Note: AMP: Amphetamine, BENZO: Benzodiazepine, COC: Cocaine, THC: Marijuana, mAmp: methamphetamine,
MOP: Morphine, P = positive, N = negative, U=uncertain or numeric result. OF: oral fluid


Chapter: 7

7     Discussion

During POCT validation process, there were a number of difficulties encountered in collection samples to obtain valid data.
One of the problems identified was poor sampling transportation. Therefore “cannabinoid” group and “Delta-9-THC” drugs initial concentration can be decomposed or metabolized by directly contact with plastic vials as such long hours.

Second problem was that when using a One Step Multi –drug POCT kit detect over-the-counter painkillers, “false positive” result was provided due to the opiate.
Volunteers’ saliva and urine sample were tested with saliva matrix POCT kit and urine matrix POCT kit for drugs of abuse. The results were shown table R1.

Two out of six (6) sample results had morphine in them 0330662 and 0330644.One of the two test results was confirmed by volunteer’s medical history. The other result was not confirmed. This is because the volunteer did not disclose any information. Also the saliva and urine POCT kit did not detect for Amphetamine, Benzodiazepine, Cocaine, Marijuana and Methamphetamine in saliva. This could have been due to the presence of lower level of drug concentration in the oral fluid and urine specimens, which was below the device designated cut-off limit. UKNEQAS sample batch (2008) of oral fluid samples were tested with One Step Multi –drug POCT kit in CTLS lab for specific six drugs namely, Amphetamine, Benzodiazepines, Cocaine, Marijuana, Methamphetamine and Morphine. The results were shown table R2.

Amphetamine and Cocaine drugs were correctly identified by employing an immunoassay based on One Step Multi –drug POCT kit in oral fluid OF1 1008, OF4 1008 respectively. In the following UKNEQAS (Nov 2008) report, ‘eighteen out of the twenty-two laboratories tested had positive results of amphetamine in oral fluid OF11008 was reported. Ten laboratories correctly identified the amphetamine using hyphenated mass spectrometry techniques. Two laboratories results showed the detection of amphetamine, using ELISA method and the results were confirmed by GCMS techniques. Additionally, a UKNEQAS analyst was informed, 52.2µg/L of amphetamine was spiked in oral fluid OF1 1008.The One Step Multi –drug POCT kit cut-off value for amphetamine was reported as 50 µg/L by manufacturer. Also Cocaine was detected in oral fluid OF11008 and OF4 1008 respectively by one step multi drug POCT in CTLS. In addition, eight out of twenty-two laboratories’ results were reported to contain cocaine in oral fluid OF4 1008. However, the UKNEQAS analyst report (Nov 2008) mentioned “ no correct report has been entered for the “cocaine & metabolite” group represented by the presence of benzoylecgonine at a concentration above that for the single analyt (8µg/L) but not that for the group (20µg/L)”.

UKNEQAS sample batch (2009) oral fluid samples were tested with One Step Multi –drug POCT kit in CTLS lab for six particular drugs namely, Amphetamine, Benzodiazepines, Cocaine, Marijuana, Methamphetamine and Morphine. The results were shown table R2.

The One Step Multi-Drug test device gave more false negative results than valid results for cannabinoid group, benzodiazepine group and amphetamine group, from UKNEQAS oral sample batch June 2009.
There were possible reasons:

7.1  Cannabinoid group:

Initial, the oral sample was spiked with 19.8 ng/ml of delta-9 tetrahydrocannabinol. The sample arrived two days later by post. Therefore “cannabinoid” group and “Delta-9-THC” drugs initial concentration can be decomposed or metabolized by direct contact with plastic vials during such a long period of time. Also One Step Multi-Drug test device cut-off limit for delta-9 tetrahydrocannabinol was reported as 20ng/ml.

7.2  Benzodiazepine group:

Initial, the oral sample was spiked with 11.5ng/ml of Benzodiazepine group. However, One Step Multi-Drug test device cut-off limit for Benzodiazepine group was reported as 30ng/ml.

7.3  Amphetamine group:

Initial, the oral sample was spiked with 52.3ng/ml of MDMA (3, 4-methyl enedioxymethylafetamineand) 17.6 ng /ml of MDA.  (Methyl ene dioxymethylafetamine)
However, One Step Multi-Drug test device was made for amphetamine immunoassay only.


Chapter: 8

8     Conclusion
The One Step Multi-Drug test device was sensitive enough to detect the THC and term of amphetamine group, in UKNEQAS external quality assessment scheme for drugs in oral fluid (June 2009) as reported in results.

The outcomes of the Validation of POCT device raise several interesting analytical problem that could resolve in further code known sample analysis.

Appendix II            Consent Form

Drugs of Abuse Information and Consent Form

Information and Consent Form for volunteers to donate Saliva, and Urine samples to Clinical Trials Laboratory Services for research purposes.Fresh human Saliva and Urine are required for research purposes by Clinical Trials Laboratory Service’s (CTLS) clients. These clients use the Saliva and Urine to help with basic research into various drugs of abuse. Please note that genetic research (DNA genotyping studies) will NOT be performed using these bio samples. Any remaining unused saliva and urine product will be discarded once a specific piece of work has been completed. However, in a few instances processed samples may be stored for later subsequent research.

You are being asked to give consent for your saliva and urine to be used by scientists for research purposes. To protect your privacy under the Data Protection Act your sample will be labeled (or “coded”) only with a subject number, not your name or any other personal identifier. Some information (e.g. donor gender and age) may also be supplied with your sample in a similarly coded fashion to the scientists. Only CTLS’s authorized Clinical Study Unit staff will have access to the link between your subject number and your name. In this way, your identity will not be divulged to the scientists who receive your saliva and urine sample.
Your participation is voluntary; you are free to withdraw consent and leave the Volunteer Panel at any time without explanation.

After each donation you will be reimbursed for your inconvenience and time.
The results of any research might be valuable for commercial and/or intellectual property purposes (patenting for example). If you decide to become a volunteer, you are giving your sample to CTLS’s clients who will retain sole ownership of any such research results and of any use or development of the research records (including your sample) consistent with this consent. You will not receive any financial benefit that might come from the research results.

Consent
My signature below indicates that:

  1. I have read and understand this form and all the information given to me.
  2. I have been given the opportunity to discuss the donation and ask questions.
  3. I am satisfied with the answers.
  4. I wish to be part of the CTLS Saliva and Urine Donation Volunteer Panel.
  5. I understand that my Saliva and Urine will be used for non-genetic research purposes.
  6. I understand that I will not be able to receive the results of any research.
  7. I will have no ownership of the research results or the research records. I agree that CTLS’s clients may apply for and use patents relating to the research results, records and developments.

I do give consent to take _______________ ml of saliva.
I do give consent to take _______________ ml of urine.

Volunteer’s name (Please print)            Signature of Volunteer                 Date
________________________                _____________________             _________
Volunteer is fit for blood, urine and saliva donation.
Screening Staff: ___________________Signature: ________________
Witness Name: ____________________Witness Signature: _____________________
Contact no:  _________________E-mail ID: ___________________________

A.IV.3   Saliva and URINE SAMPLE test result

CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse

Result sheet: Study No: 339
Date: 07/07/2009
Lab Ref No: 0330661


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

P

P

Date:           07/07/2009                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine .P=Positive, N= Negative.

CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse
Result sheet: Study No: 339
Date: 08/07/2009
Lab Ref No: 0330662


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

N

N

Date:           08/07/2009                                                                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine .P=Positive, N= Negative.


CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse
Result sheet: Study No: 339
Date: 08/07/2009
Lab Ref No: 0330664


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

P

P

Date:           08/07/2009                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine. P=Positive, N= Negative.


CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse
Result sheet: Study No: 339
Date: 13/07/2009
Lab Ref No: 0330670


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

N

N

Date:           13/07/2009                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine, P=Positive, N= Negative.


CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse
Result sheet: Study No: 339
Date: 21/07/2009
Lab Ref No: 0330675


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

N

N

Date:           21/07/2009                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine, P=Positive, N= Negative.


CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse
Result sheet: Study No: 339
Date: 28/07/2009
Lab Ref No: 0330691


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

N

N

Date:           28/07/2009                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine, P=Positive, N= Negative.


CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse
Result sheet: Study No: 339
Date: 29/07/2009
Lab Ref No: 0330692


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

N

N

Date:           29/07/2009                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine, P=Positive, N= Negative.


 

CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse
Result sheet: Study No: 339
Date: 05/08/2009
Lab Ref No: 0330701


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

N

N

Date:           21/07/2009                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine, P=Positive, N= Negative.


CLINICAL TRIALS LABORATORY SERVICES

PRE SCREENING STUDY CHECK LIST
Drugs of Abuse
Result sheet: Study No: 339
Date: 05/08/2009
Lab Ref No: 0330702


Name of Drug

Result : Saliva

Result: Urine

AMP

N

N

BENZO

N

N

COC

N

N

THC

N

N

MAMP

N

N

MOR

N

N

Date:           28/07/2009                             Analyst: S.Mayuran
Note: AMP- Amphetamine, BENZO-Benzodiazepines, COC-Cocaine, THC-Marijuana, m-Amp-Methamphetamine, MOR-Morphine, P=Positive, N= Negative.

References:

  1. Biermann. T,Schwarze B,Zedler.B and Betz.P“On-site testing of illicit drugs” Journal of Forensic Science International 143 (2004) 21-25.
  2. Creative Research Systems.411B Street Suite2 Petaluma CA 94952

Tel: (707)765-1001 http://www.surveysystem.com/sscalc.htm online. 30th June 2009

  1. Dennis .J. Crouch, Dr. Royer F Cook, Dr James V.Trudea,David C.Dove “An Evaluation of Innovation Sweat Based Drug Testing Techniques for use in Criminal Justice Drug Testing” U.S. Department of Commerce, Office of Law Enforcement Standard.2002,206825.http://www.eeel.nist.gov. Accessed 30th June 2009
  2. Dennis J.Crouch et.al “Oral fluid collection: The neglected variable in oral fluid testing”. Journal of Forensic Science International 150 (2005) 165-173.
  3. Dr.Graham Facile Cope, Honorary Senior Research Fellow. “Evaluation of the Drager Drug Test ® 5000Test System”. University of Birmingham 2008 November.
  4. Edward J. Cone and Marilyn A Huestis “Interpretation of Oral Fluid Tests for Drugs of Abuse”. Ann NY Acad Sci.2007 March 1098, 51-103.
  5. Jayne E. Thatcher. “ROSITA II Project: Evaluation of On-Site Saliva Drug Testing Devices in Washington State”.2007
  6. Kato K,Hillsgrove M,Weinhold L,Gorelick DA,Darwin WD,Cone E J “Cocaine and metabolite excretion in Saliva under Stimulated and non-Stimulated conditions”J Anal Toxicology 1993 Oct: 17(6):338-41
  7. N.Fucci,N.De Giovanni,M.Chiarotti,S.Scarlata.“SPME-GC analysis of THC in saliva sample collected with EPITOPE” device. Journal of Forensic Science International 2001 Volume 119, Issue 3,318-321.
  8. National Statistical Service, Australian Bureau of Statistical, PO Box 10 Belconnen ACT 2616, Austrialia. http:// www.abs.gov.au accessed 03rd July2009
  9. O.R.Idowu and B.Caddy “A Review of the use of Saliva in the forensic detection of drugs and other chemicals” (1982) Journal of Forensic Science Society Volume 22, Issue 2,123-135. http:// www.icadts2007.org/print/80rosita_salivascreen.pdf Accessed 03rd July 2009
  10. Prof Olaf H Drummer. “Drug Testing in Oral Fluid” Clinical Biochemist Reviews (2006) August 27(3): 147-159.
  11. Schramm.W.Smith RH, Craig PA“Drugs of abuse in saliva” Journal of Analytical Toxicology (1992)16:1-9.39
  12. UKNEQAS Oral Fluid pilot surveys for drugs of abuse in saliva guide line 2009]. http:/ www.heathcontrol.com Accessed 03rd August 2009.
  13. Vanna De Rosas,Marcus Roberts,Ruth Evans,Yolande Burgin“Drug testing in the work place - the report of the independent inquiry into Drug Testing at work”. the Joseph Rowntree Foundation. 2004 online pdf ISBN 185935212X, www.jrf.org.uk. Accessed 03rd August 2009
  14. Vincent Cirimele, Marion Villain, and Patrick Mura Marc.Bernard Pascal Kintz “Oral fluid testing for cannabis: On-site Oraline IV s.a.t device versus GC/MS”.Forensic Science International 16, 2006, 180-184.
  15. Walsh J.M, Flegel R., Crouch D.J, Cangianeli L, Baudys J. “An Evaluation of Rapid Point-of-collection Oral Fluid Drug-Testing Devices” Journal of analytical Toxicology, volume 27,No 7, October 2003,pp.429-439(11)
  16. Jonnne Birchall. “Sampling and samples” Market Research World. Online article: accessed on 15th July 2009. http://www.marketresearchworld.net.
  17. Dr.Rob C.A.A van Schie,Dr Mark E.Wilson “Saliva: Your Spitting Image” National Institutes of Health in Bethesda, Maryland. October 1997. Online article: accessed on 15th July 2009.http://www.nih.gov/news/pr/oct97/nidr-22.htm