September 2006
    Environmental Technology
    Verification Report


    QTL BlOSYSTEMS LLC
    QTL BIOSENSOR
            Prepared by
             Battelle

            Balfelle
           //it? Business of Innovation
         Under a cooperative agreement with


           U.S. Environmental Protection Agency
ET1/ET1/ET1/

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                  THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
                                          PROGRAM
                                      ET
                rKAlVl £

                V
      U.S. Environmental Protection Agency
                                                                         Baireiie
                                                                        Business of Innovation
                        ETV Joint Verification Statement
       TECHNOLOGY TYPE:   IMMUNOASSAY TEST KITS
       APPLICATION:
DETECTING ANTHRAX AND RICIN
       TECHNOLOGY NAME: QTL Biosensor

       COMPANY:             QTL Biosystems, LLC
       ADDRESS:
       WEB SITE:
       E-MAIL:
2778 Agua Fria Street         PHONE   877-785-2461
Santa Fe, New Mexico 87507   FAX:     505-424-8679

www.qtlbio.com
qtlinfo® qtlbio.com
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology Verification (ETV)
Program to facilitate the deployment of innovative or improved environmental technologies through performance
verification and dissemination of information. The goal of the ETV Program is to further environmental protection
by accelerating the acceptance and use of improved and cost-effective technologies. ETV seeks to achieve this goal
by providing high-quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies. Information and ETV
documents are available at www.epa.gov/etv.

ETV works in partnership with recognized standards and testing organizations, with stakeholder groups (consisting
of buyers, vendor organizations, and permitters), and with individual technology developers. The program evaluates
the performance of innovative technologies by developing test plans that are responsive to the needs of
stakeholders,  conducting field or laboratory tests (as appropriate), collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality assurance (QA) protocols
to ensure that data of known and adequate quality are generated and that the results are defensible.

The Advanced Monitoring Systems (AMS) Center, one of six technology areas under ETV, is operated by Battelle
in cooperation with EPA's National Exposure Research Laboratory. The AMS Center evaluated the performance of
immunoassay test kits used to detect anthrax and ricin in water. This verification statement provides a summary of
the test results for the QTL Biosystems Biosensor.

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VERIFICATION TEST DESCRIPTION

The verification test for the QTL Biosensor was conducted at Battelle between November 2005 and March 2006
according to procedures specified in the Test/QA Plan for Verification of Immunoassay Test Kits for the following
parameters: contaminant presence/absence; false positive/false negative response to interferents, drinking water
(DW) matrix effects, and cross-reactivity; consistency; method detection limit; field portability; ease of use; and
sample throughput. The ability of the QTL Biosensor to detect various concentrations of anthrax and ricin was
evaluated by analyzing performance test (PT) and DW samples. PT samples included American Society for Testing
and Materials Type II deionized (DI) water fortified with the target contaminant, an interferent, both, or only a
cross-reactive species. Target analytes were added to DI water at lethal dose concentrations as well as at several
concentrations selected based on the vendor-stated limit of detection (LOD). The effect of interferents was
evaluated by analyzing two types of interferent solutions. The first type contained both humic and fulvic acids in DI
water, and the second type contained magnesium (Mg) and calcium (Ca) in DI water. Both types of interferent
solutions were prepared with and without the addition of the contaminants at a single concentration level (10 times
the vendor-stated LOD). In addition, specificity was evaluated by exposing the QTL  Biosensor to Bacillus
thuringiensis, a potentially cross-reactive compound for anthrax, and lectin from soybean, a potentially cross-
reactive compound for ricin. PT samples were analyzed in triplicate (with the exception of samples used to
determine the method detection limit for which seven replicates were analyzed). DW samples were collected from
four water utilities that use a variety of treatment methods. DW samples, both unconcentrated and concentrated by a
factor of 400, were analyzed in triplicate both with and without the addition of anthrax and ricin at a concentration
of 10 times the vendor-stated LOD. In addition to the PT and DW samples analyzed, method blank (MB) samples
consisting of DI water were analyzed to confirm negative responses in the absence of any contaminant and to
ensure that no sources of contamination were introduced during the analysis procedures.

QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted a technical
systems audit and a data quality audit of 10% of the test data. This verification statement, the full  report on which it
is based, and the test/QA plan for this verification are all available at www.epa.gov/etv/centers/centerl.html.

TECHNOLOGY DESCRIPTION

The following description of QTL Biosensor was provided by the vendor and was not verified in this test.

The QTL Biosensor is a handheld device that is similar to standard immunomagnetic sandwich assays and is
capable of detecting anthrax spores and ricin toxins in samples. The sample is added  to the QTL Biosensor
cartridge which contains sensing reagents.  The sensing reagents are composed of two materials: a magnetic
component and a fluorescent component. Receptors for the biological agent(s) of interest are contained in both
sensing reagents. Upon mixing the sample  with the reagents, the magnetic and fluorescent components form a
complex with the biological agent(s) for which they are specific. A magnetic field is then applied. This separates all
magnetic materials (including any complexes containing the biological agent) from the solution, which contains
excess fluorophore. A wash is performed to remove all excess reagents materials from the sample chamber. Then,
an excitation wavelength of light is exposed to the magnetic pellet comprised of the biological agent complexes,
and the resulting fluorescence indicates the presence of the biological agent.

The QTL Biosensor contains both positive and negative controls to ensure the validity of results and proper
functioning of the QTL Biosensor. Both liquid and solid samples can be analyzed using the QTL Biosensor. Results
are displayed as a millivolt (mV) and percent of full scale (%FS) reading. The QTL Biosensor also has red/green
warning lights that can be set to a pre-determined mV threshold reading to indicate a positive/negative response
where a green light indicates no toxin below the mV threshold, and a red light indicates the presence of a toxin
above the mV threshold.

The QTL Biosensor includes an integrated bar code reader (use is optional), and a starter kit comes with 20 test
cartridges. Test cartridges are shipped with a sample collector, bio-hazard bag, and a bar code label. The cartridge
is single-use, self contained, self sealing, and includes all necessary reagents. It is 1.9 inches wide by 1.7 inches

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high by 0.5 inch deep. The QTL Biosensor is 11 inches long by 10 inches wide by 5 inches high and weighs 6
pounds. Its battery life is 16 hours of continuous operation, and it has a recharge cycle of 2 to 4 hours.

The QTL Biosensor has a liquid crystal display screen with a four-button user interface. The bar code reader
captures the cartridge type and serial number. Date, time, location, user identification, and serial numbers
(Biosensor and cartridge) are stored with each result. There is on-board data storage for the last 100 samples and
results can be uploaded electronically to a laptop computer (not included with the QTL Biosensor). The QTL
Biosensor pricing starts at $11,500, and the price of each cartridge is $21.

VERIFICATION OF PERFORMANCE

The tables that follow summarize the performance of the QTL Biosensor in detecting anthrax and ricin,
respectively.

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Anthrax Summary Table
Parameter
Contaminant-only
PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Method Detection
Limit
Other Performance
Factors
Sample Information
DI water
0.5 mg/L humic and fulvic
2.5 mg/L humic and fulvic
50 mg/L Ca and Mg
250 mg/L Ca and Mg
Unconcentrated CA
Concentrated CA
Unconcentrated FL
Concentrated FL
Unconcentrated NY
Concentrated NY
Unconcentrated OH
Concentrated OH
1 x 106 spores/mL
Bacillus thuringiensis
Anthrax Concentration
(spore/mL)
200 (lethal dose)
1 x 105 (vendor-stated
limit of detection)
5xl05
IxlO6
5xl06
unspiked
unspiked
IxlO6
IxlO6
unspiked
Positive Results out
of 3 Replicates
0
1
3
3
3
0
0
3
3
3
3
2
3
1
2
0
2
3
3
3
3
2
3
2
3
3
3
2
3
0
False positive results occurred in Ca and Mg interferent samples as well as the
Unconcentrated water from CA, FL, and NY and all concentrated drinking
water samples.
False negative results occurred only in the Unconcentrated CA, FL, and OH
drinking water samples.
Results were consistent (i.e., produced positive or negative results without
variation among replicates) in 21 out of 29 sets of replicates or 72%.
The method detection limit was determined to be the concentration generating
a 65 mV response. It was between IxlO5 spores/mL (vendor-stated limit of
detection) and 5xl05 spores/mL.
Long term storage of the test cartridges should be at 2-8 °C, but cartridges may
be kept at room temperature for up to six months. Analysis software was user-
friendly. The QTL Biosensor uses electricity or rechargeable batteries and
includes a rugged carrying case. Test cartridges and detector were used inside
and outside a laboratory by trained operator as well as non-technically trained
operator; sample throughput was 12 samples per hour.
Shading indicates results for unspiked sample.

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Ricin Summary Table
Parameter
Contaminant-only PT
samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Method Detection
Limit
Other Performance
Factors
Sample Information
DI water
0.5 mg/L humic and fulvic
2.5 mg/L humic and fulvic
50 mg/L Ca and Mg
250 mg/L Ca and Mg
Unconcentrated CA
Concentrated CA
Unconcentrated FL
Concentrated FL
Unconcentrated NY
Concentrated NY
Unconcentrated OH
Concentrated OH
0.5 mg/L
Lectin from soybean
Ricin Concentration
(mg/L)
0.05 (vendor-stated limit of
detection)
0.25
0.5
2.5
15 (lethal dose)
unspiked
unspiked
0.5
0.5
unspiked
Positive Results out
of 3 Replicates
0
3
3
3
3
0
0
0
0
0
0
1
1
0
0
0
0
3
3
3
1
3
3
3
3
3
3
3
3
0
False positive results occurred in the Unconcentrated and concentrated FL
drinking water samples.
False negative results occurred only in the 250 mg/L Ca and Mg interferent
sample.
Results were consistent (i.e., produced positive or negative results without
variation among replicates) in 26 out of 29 sets of replicates or 90%.
The method detection limit was determined to be the concentration generating a
76 mV response. It was between 0.05 mg/L (vendor stated limit of detection)
and 0.25 mg/L.
Long term storage of the test cartridges should be at 2-8 °C, but cartridges may
be kept at room temperature for up to six months. Analysis software was user-
friendly. The QTL Biosensor uses electricity or rechargeable batteries and
includes a rugged carrying case. Test cartridges and detector were used inside
and outside a laboratory by trained operator as well as non-technically trained
operator; sample throughput was 12 samples per hour.
Shading indicates results for unspiked sample.

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 Original signed by Gregory A. Mack      10/26/2006  Original signed by Jonathan G. Herrmann   11/12/2006
Gregory A. Mack                          Date       Jonathan G. Herrmann                      Date
Vice President                                        Director
Energy, Transportation, and Environment Division       National Homeland Security Research Center
Battelle                                               U.S. Environmental Protection Agency
      NOTICE: ETV verifications are based on an evaluation of technology performance under specific, predetermined
      criteria and the appropriate quality assurance procedures. EPA and Battelle make no expressed or implied
      warranties as to the performance of the technology and do not certify that a technology will always operate as
      verified. The end user is solely responsible for complying with any and all applicable federal, state, and local
      requirements. Mention of commercial product names does not imply endorsement.

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                                     September 2006
Environmental Technology Verification
                  Report

   ETV Advanced Monitoring Systems Center

          QTL Biosystems LLC
              QTL Biosensor
                     by
                  Mary Schrock
                  Ryan James
                  Amy Dindal
                 Zachary Willenberg
                  Karen Riggs

                   Battelle
                Columbus, Ohio 43201

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                                       Notice

The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program described
here. This document has been peer reviewed by the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation by the EPA for use.
                                          11

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                                       Foreword

The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
nation's air, water, and land resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, the EPA's Office of Research and Development provides data and science support that
can be used to solve environmental problems and to  build the scientific knowledge base needed to
manage our ecological resources wisely, to understand how pollutants affect our health, and to
prevent or reduce environmental risks.

The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols developed with input from major stakeholders and customer groups
associated with the technology area. ETV consists of six environmental technology centers.
Information about each of these centers can be found on the Internet at http://www.epa.gov/etv/.

Effective verifications of monitoring technologies are needed to assess environmental quality and
to supply cost and performance data to select the most  appropriate technology for that assessment.
Under a cooperative agreement, Battelle has received EPA funding to plan, coordinate, and
conduct such  verification tests for "Advanced Monitoring Systems for Air, Water, and Soil" and
report the results to the community at large. Information concerning this specific environmental
technology area can be found on the Internet at http://www.epa.gov/etv/centers/centerl.html.
                                           in

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                                  Acknowledgments

The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. We sincerely appreciate the
contribution of drinking water samples from the Metropolitan Water District of Southern
California (Paul Rochelle and Melinda Stalvey), the New York Department of Environmental
Protection (Virginia Murray), and Orange County Utilities, Orlando, Florida (Theresa Slifko and
Liza Robles). Also, thank you to the Metropolitan Water District of Southern California for
concentrating each drinking water sample. We would also like to thank Karen Bradham, U.S.
EPA National Exposure Research Laboratory; Steve Allgeier, U.S. EPA Office of Water; Ricardo
DeLeon, Metropolitan Water District of Southern California; and Stanley States, Pittsburgh Water
and Sewer Authority, for their careful review of the test/QA plan and this verification report.
                                           IV

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                                       Contents

                                                                                   Page
Notice	ii
Foreword	iii
Acknowledgments	iv
List of Abbreviations	vii
Chapter 1  Background	1
Chapter 2  Technology Description	2
Chapters  Test Design	4
      3.1  Test Samples	5
           3.1.1  Performance Test Samples	6
           3.1.2  Drinking Water Samples	7
           3.1.3  Quality Control Samples	8
      3.2  Test Procedures	8
           3.2.1  Laboratory Testing	8
           3.2.2  Non-Laboratory Testing	9
           3.2.3  Drinking Water Characterization	9
Chapter 4  Quality Assurance Quality Control	11
      4.1  Quality Control of Stock Solution Confirmation Methods	11
           4.1.1 Anthrax testing solutions	11
           4.1.2 Ricin testing solutions	12
           4.1.3 Interferent solutions	13
      4.2  Quality Control of Drinking Water Samples	13
      4.3  Technical Systems Audit	14
      4.4  Audit of Data Quality	14
      4.5  QA/QC Reporting	14
      4.6  Data Review	14
Chapter 5  Statistical Methods and Reported Parameters	16
      5.1  Qualitative Contaminant Presence/Absence	16
      5.2  False Positive/Negative Responses	16
      5.3  Consistency	16
      5.4  Method Detection Limit	17
      5.5  Other Performance Factors	17
Chapter 6  Test Results	18
      6.1  Qualitative Contaminant Presence/Absence	18
           6.1.1  Anthrax	18
           6.1.2  Ricin	18
      6.2  False Positive/Negative Responses	18
           6.2.1  Interferent PT Samples	21
           6.2.2  DW Samples	21
           6.2.3  Cross-Reactivity PT Samples	25

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       6.3 Consistency	25
       6.4 Method Detection Limit	25
       6.5 Other Performance Factors	26
          6.5.1 Ease of Use	26
          6.5.2 Field Portability	27
          6.5.3 Throughput	27
Chapter 7  Performance Summary	28
Chapters  References	31


                                        Figures

Figure 2-1. QTL Biosystems Biosensor	2
                                        Tables
Table 3-1. Lethal Dose and Source of Contaminants	5
Table 3-2. Performance Test Samples	6
Table 3-3. Drinking Water Samples	6
Table 3-4. Water Quality Characterization of Drinking Water Samples	10
Table 4-1. Anthrax Enumeration Data for PT Samples	12
Table 4-2. Anthrax Enumeration Results for Fortified Interferent and Drinking Water Samples. 13
Table 4-3. Summary of Data Recording Process	15
Table 6-la. Anthrax Contaminant-Only PT Sample Results-Contaminant Presence/Absence
Evaluation	19
Table 6-lb. Ricin Contaminant-Only PT Sample Results-Contaminant Presence/Absence
Evaluation	20
Table 6-2. Interferent PT Sample Results- False Positive/Negative Evaluation	22
Table 6-3. DW Sample Results-False Positive/Negative Evaluation	23
Table 6-3. DW Sample Results- False Positive/Negative Evaluation (continued)	24
Table 6-4. Potentially Cross-Reactive PT Sample Results	25
Table 6-5. Method Detection Limit	26
Table 7-1. Anthrax Summary Table	28
Table 7-1. Anthrax Summary Table (Continued)	29
Table 7-2. Ricin Summary Table	30
                                          VI

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                              List of Abbreviations

%FS             percent full scale
AMS            Advanced Monitoring Systems
ATEL           Aqua Tech Environmental Laboratories, Inc.
Ca               calcium
CDC            Centers for Disease Control and Prevention
cfu               colony-forming units
CO A            certificate of analysis
DI               deionized
DW             drinking water
EPA             U.S. Environmental Protection Agency
ETV             Environmental Technology Verification
L                liter
LD               lethal dose
LOD            limit of detection
MB              method blank
MDL            method detection limit
Mg              magnesium
jaL               microliter
mg/L            milligram per liter
mL              milliliter
mV              millivolt
PD               percent difference
PT               performance test
QA              quality assurance
QC              quality control
QMP            quality management plan
TSA             technical systems audit
USAMRIID      U.S. Army Medical Research Institute of Infectious Diseases
                                         vn

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                                      Chapter 1
                                     Background


The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental
technologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use of
improved and cost-effective technologies. ETV seeks to achieve this goal by providing high-
quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.

ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative
technologies by developing test plans that are responsive to the needs of stakeholders,
conducting field or laboratory tests (as appropriate), collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality
assurance (QA) protocols to ensure that data of known and adequate quality are generated and
that the results are defensible.

The EPA's National Exposure Research Laboratory and its verification organization partner,
Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center
recently evaluated the performance of the QTL Biosystems Biosensor immunoassay test
cartridges and detector system. Immunoassay test kits were identified as a priority technology
category for verification through the AMS Center stakeholder process.

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                                      Chapter 2
                               Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of
environmental monitoring technologies for air, water, and soil. This verification report provides
results for the verification testing of the QTL Biosensor. Following is a description of the QTL
Biosensor, based on information provided by the vendor. The information provided below was
not verified in this test.

The QTL Biosensor, shown in Figure 2-1, is a handheld device that is similar to standard
immunomagnetic sandwich assays and is capable of detecting anthrax spores and ricin toxins in
samples. The sample is added to the QTL Biosensor cartridge which contains sensing reagents.
The sensing reagents are composed of two materials: a magnetic component and a fluorescent
component.  Receptors for the biological agent(s) of interest are contained in both sensing
                                            reagents.  Upon mixing the sample with the
                                            reagents, the magnetic and fluorescent
                                            components form a complex with the biological
                                            agent(s) for which they are specific. A magnetic
                                            field is then applied. This separates all magnetic
                                            materials (including any complexes containing
                                            the biological agent) from the solution, which
                                            contains excess fluorophore. A wash is
                                            performed to remove all excess reagents
                                            materials from the sample chamber. Then the
                                            magnetic pellet comprised of the biological
                                            agent complexes is exposed to an excitation
                                            wavelength of light. The resulting fluorescence
                                            indicates the presence of the biological agent.

                                            The QTL Biosensor contains both positive and
                                            negative controls to ensure the validity of results
                                            and proper functioning of the QTL Biosensor.
                                            The Negative Control Cartridge for each of its
                                            tests can be used to rule out false positives.  This
                                            cartridge contains  a sensor that is prepared with
                                            antibodies that are not directed towards an
Figure 2-1. QTL Biosystems Biosensor       antigen that is found in human samples. These
                                            cartridges have two primary uses. First, they
can be used to confirm the results of the test after a positive result is obtained with a test
cartridge.  If a negative control cartridge gives a positive result with the same sample matrix,

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then the results from that test cartridge are ambiguous. However, if the negative control gives a
negative result, it confirms the original positive result. The second use would be to determine
whether or not the QTL test can be used reliably on a given sample matrix or with a particular
interferent before any additional testing is performed. For example, if the QTL Negative Control
Cartridge  for the anthrax assay always gives positive results on water samples from a particular
water system, then the assay cannot be used in that water system.

Liquid samples are sampled directly into the sampling syringe while a hydrated swab is provided
to collect samples from surfaces. The swab sample is transferred to a sample buffer tube and
drawn into a sample syringe. After a washing syringe is inserted into the QTL Biosensor
cartridge,  the sampling syringe is inserted. The cartridge is shaken by hand for one minute and
inserted into the detector. After a three minute automatic pellet formation step, the washing
syringe is  pressed and the read button is pushed to obtain results. Results are displayed as a
millivolt (mV) and percent of full scale (%FS) reading. The QTL Biosensor also has red/green
warning lights that can be set to a pre-determined mV threshold reading to indicate a
positive/negative response where a green light indicates no toxin below the mV threshold, and a
red light indicates the presence of a toxin above the mV threshold.

The QTL Biosensor includes an integrated bar code reader (use is optional), and a starter kit
comes with 20 test cartridges. Test cartridges are shipped with a sample collector, bio-hazard
bag, and a bar code label. The cartridge is single-use, self contained, self sealing, and includes all
necessary  reagents.  It is 1.9 inches wide by 1.7 inches high by 0.5 inch deep. The QTL Biosensor
is 11 inches long by 10 inches wide by 5 inches high and weighs 6 pounds. Its battery life is 16
hours of continuous operation, and it has a recharge cycle of 2 to 4 hours.

The QTL Biosensor has a liquid crystal display screen with a four-button user interface. The bar
code reader captures the cartridge type and serial number. Date, time, location, user
identification, and serial numbers (biosensor and cartridge) are stored with each result. There is
on-board data storage for the last 100 samples and results can be uploaded electronically to a
laptop computer. The QTL Biosensor pricing starts at $11,500, and the price of each cartridge is
$21.

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                                       Chapter 3
                                      Test Design


The objective of this verification test was to evaluate the ability of the QTL Biosensor and test
cartridges to detect specific biological toxins and agents in water samples and to determine
whether the QTL Biosensor is susceptible to interferents in drinking water (DW).

During this verification test, the QTL Biosensor and test cartridges were subjected to various
concentrations of anthrax and ricin in American  Society for Testing and Materials Type II
deionized (DI) water. Table 3-1 shows the contaminants, the vendor-stated limit of detection
(LOD), the lethal dose (LD) concentrations, and the contaminant source. It should be recognized
that there is a wide range of LD concentrations in the literature. In selecting an LD level for use
in verification testing, literature oral LD50 values were reviewed and included in the test/QA
plan and amendments.(1)  In addition to reviewing the LD values in the literature, two factors
were taken into consideration in selecting the final LD concentration for use in testing:
       1) Consistency with the LD concentrations used in the first round of ETV immunoassay
          technology evaluations.
       2) Applicability of the LD concentration level to the participating technologies'
          expected limits of detection.
In some instances this resulted in an LD level being selected that was on the high end of the
literature values reported.  Given the range of LD concentrations that are available in the
literature, it is recommended that all readers evaluate the LD concentrations used for verification
testing with respect to their particular LD requirements. The lethal dose concentration was
determined using a 250 mL ingestion volume.

The QTL Biosensor also was used to analyze contaminant-fortified DW samples that were
collected from four water utilities that use a  variety of treatment methods.  The effect  of
interferents was evaluated by analyzing two types of interferent solutions.  The first type
contained both humic and fulvic acids in DI water and the second type contained magnesium
(Mg) and calcium (Ca) in DI water.  Both types of interferent solutions were prepared with and
without the addition of the contaminants.  In addition, specificity was evaluated by exposing the
QTL Biosensor to a potentially cross-reactive compound or spore for each target contaminant.
Bacillus thuringiensis was used to test the anthrax cartridge and lectin was used to test the ricin
cartridge.

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Table 3-1. Lethal Dose and Source of Contaminants
Contaminant
Bacillus anthmcis
Ames Strain (anthrax)
Ricinus communis
Agglutinin II (ricin)
Vendor-Stated
LOD
IxlO5
spores/milliliter
(mL)
0.05 milligrams/
liter (mg/L)
Lethal Dose
Concentration'3'
200 spores/mL
15 mg/L
Source of Contaminant
Independent lot prepared at Battelle
from U.S. Army Medical Research
Institute of Infectious Diseases
(USAMRIID) stock
Vector Laboratories, Inc.
(Burlingame, California)
   The lethal dose of each contaminant was determined by calculating the concentration at which 250 mL of water
   would probably cause the death of a 154-pound person, based on human mortality data and as outlined in the
   Test/QA Plan for Verification of Immunoassay Test Kits Amendment Number 5 (1l

The verification  test for the QTL Biosensor was conducted from November 2005 through March
2006, according  to procedures specified in the Test/QA Plan for Verification of Immunoassay
Test Kits including amendments 1-5.(1) This test was conducted at Battelle in West Jefferson,
Ohio. Aqua Tech Environmental Laboratories, Inc. (ATEL) of Marion, Ohio, performed
physicochemical characterization for each DW sample to determine the following parameters:
turbidity; concentration of dissolved and total organic carbon; specific conductivity; alkalinity;
concentration of Mg and Ca; pH; hardness; and concentration of total organic halides, trihalo-
methanes, and haloacetic acids. Battelle confirmed the presence of anthrax spores using plate
enumeration. The QTL Biosensor was evaluated for the following parameters:

•  Contaminant presence/absence
•  False positive/false negative response
        Interferents
        DW matrix effects
        Cross-reactivity
•  Consistency
•  Method detection limit
•  Other performance factors
     -   Field portability
     -   Ease of use by technical and non-technical operators
         Sample throughput.
3.1 Test Samples

Tables 3-2 and 3-3 summarize the samples analyzed for each contaminant. The ability of the
QTL Biosensor to individually detect various concentrations of anthrax spores and ricin was
evaluated by analyzing performance test (PT) and DW samples. PT samples included DI water
fortified with the target contaminant, an interferent, both, or only a cross-reactive species. DW
samples were analyzed using the QTL Biosensor with and without the addition of each target
contaminant. Note that test cartridges specific to anthrax only were tested with anthrax and
cartridges specific to ricin only were tested with ricin.

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3.1.1  Performance Test Samples
The contaminant-only PT samples (shown in Table 3-2) were prepared in DI water using
standards of anthrax and ricin. Anthrax concentrations were verified using a plate enumeration
technique. Reference methods were not available for quantitative confirmation of ricin test
solutions so certificates of analysis (COA) and QA oversight of solution preparation were used to
determine their concentrations.

Table 3-2. Performance Test Samples
Type of PT
Sample
Contaminant
Interferent
Cross-reactive
species
Sample Characteristics
Anthrax
Ricin
Contaminants in 50 mg/L Ca and
50 mg/L Mg
Contaminants in 250 mg/L Ca
and 250 mg/L Mg
Contaminants in 0.5 mg/L humic
acid and 0.5 mg/L fulvic acid
Contaminants in 2.5 mg/L humic
acid and 2.5 mg/L fulvic acids
Bacillus thuringiensis
(anthrax analogue)
Lectin from soybean
(ricin analogue)
Approximate Concentrations
200 to 5 x 106 spores/mL
0.05 to 15 mg/L
Anthrax- 1 x 106 spores/mL
Ricin - 0.5 mg/L
Anthrax- 1 x 106 spores/mL
Ricin - 0.5 mg/L
Anthrax- 1 x 106 spores/mL
Ricin - 0.5 mg/L
Anthrax- 1 x 106 spores/mL
Ricin - 0.5 mg/L
1 x 106 spores/mL
0.5 mg/L
Table 3-3.  Drinking Water Samples
          Drinking Water Sample Description
Water Utility
Metropolitan Water
District of Southern
California (CA)
New York City, New
York (NY)
Columbus, Ohio
(OH)
Orlando, Florida
(FL)
Water
Treatment
Filtered
chloraminated
Unfiltered
chlorinated
Filtered
chlorinated
filtered
chlorinated
Source
Type
surface
surface
surface
ground
Cone. /
Unconc.
both
both
both
both
Anthrax
unspiked and
1 xlO6
spores/mL
Ricin
unspiked and
0.5 mg/L
Approximate Contaminant
     Concentrations

-------
The contaminant only PT samples were solutions of the contaminant in DI water at the vendor-
stated LOD, the lethal dose, and approximately 5,10, and 50 times the LOD. These solutions
were used to evaluate contaminant presence/absence and  method detection limit.

The interferent PT samples consisted of samples of humic and fulvic acids isolated from Elliott
Soil (obtained from the International Humic Substances Society) and Ca and Mg (prepared from
their chlorides with concentrations based on metals only), each spiked into DI water at two
concentration levels. These solutions were analyzed both with and without the target
contaminant. In addition, because the commercially available ricin contained a preservative
(sodium azide), a preservative blank sample consisting of 0.16 mg/L sodium azide was prepared
in DI water. This sodium azide solution represents the concentration of the preservative that
would be found in the most concentrated contaminant solution (50 x LOD). This preservative
blank was analyzed along with the contaminant solutions to ensure that the preservative did not
have a significant effect on the performance of the QTL Biosensor during ricin testing. The
interferent PT samples were used to evaluate whether interferences which are commonly found
in water have the potential to cause false positive or negative results with the QTL Biosensor.

The last type of PT sample was a cross-reactivity check sample to determine whether the QTL
Biosensor produced false positive results in response to similar analytes. Bacillus  thuringiensis
(for anthrax) and lectin from soybean (for ricin) are biologically or chemically (respectively)
similar to the specified targets. Solutions of these were prepared in  DI water at concentrations ten
times greater than the vendor-stated LOD for the specified targets and analyzed to  evaluate
cross-reactivity interference.

Three replicates of each PT sample were analyzed except for the method detection limit sample
for  which seven replicates were analyzed. The results provided information about how well the
QTL Biosensor detected the presence of each contaminant at several concentration levels,  the
consistency of its responses, and its susceptibility to interferents.

3.1.2 Drinking Water Samples

The DW samples were collected from four geographically distributed municipal sources
(Table 3-3). These samples were unique in terms of their source, treatment, and disinfection
process. All collected samples were finished DW either ready for the distribution system or from
within the distribution system.

Approximately 175 liters (L) of each of the DW samples  were collected in pre-cleaned low-
density polyethylene containers. One hundred twenty-five liters of each DW sample were
shipped to the Metropolitan Water District of Southern California and dechlorinated with sodium
thiosulfate. Out of this, 100L was concentrated using ultra-filtration techniques to a final volume
of 250 mL. This concentration  factor was selected because it is the  goal of an EPA on-site ultra-
filtration sample concentration method that is being developed to increase the concentration of
insoluble microbiological species in a water sample so they may be detected by available
detection technologies. Concentrated water samples were included in the test/QA plan due to
stakeholder interest in this technique and because the large concentration factor could affect the
amount of potential interferences in various types of water compared to testing only with
unconcentrated water. Twenty-five liters of each water sample was shipped to ATEL for water

                                            7

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quality analysis. The remaining 25L of each sample was shipped to Battelle where the sample
was dechlorinated with sodium thiosulfate. Each DW sample (unconcentrated and concentrated)
was analyzed without adding any contaminant, as well as after fortification with individual
contaminants at a single concentration level.
3.1.3  Quality Control Samples

In addition to the PT and DW samples analyzed, method blank (MB) samples consisting of DI
water were analyzed to confirm negative responses in the absence of any contaminant and to
ensure that no sources of contamination were introduced during the analysis procedures.  A
positive control cartridge provided by QTL Biosystems LLC was analyzed to ensure that the
QTL Biosensor was operating properly. Analysis frequencies for the MB and positive control
samples are discussed in Section 4.2.
3.2 Test Procedures
3.2.1  Laboratory Testing

Each day, fresh samples were prepared from standards or stock solutions in either DI water, an
interferent matrix, or a DW matrix. Each sample was prepared in its own container and labeled
with a sample identification number that was recorded on a data sheet. To test a liquid sample for
the presence of anthrax or ricin, the procedure described below was used.

The QTL Biosensor case was opened, plugged into a power source, and turned on. Test
cartridges (specific to either anthrax or ricin) were provided in individually sealed foil bags that
contained one test cartridge, a small sample syringe, a large wash syringe, and a wet sampling
tube.  The QTL Biosensor was set for analyzing samples following the Biosensor's Sample
Wizard, which prompts the operator through the sample analysis using on-screen instructions,
by pressing the button labeled "SMPL". The foil bag was opened and the test cartridge, which
must be used immediately after opening the bag, was removed. For each individual sample
analyzed, approximately 300 |iL of sample were drawn into the small sample syringe and then
added to the wet sampling tube which was then capped and manually shaken for 15 seconds.
After shaking, approximately 150 |iL of this solution were withdrawn from the wet sampling
tube back into the small sampling syringe. Then the large wash syringe and the small sample
syringe were seated firmly into the test cartridge without pressing either plunger. While holding
the test cartridge at eye-level, the small sample syringe plunger was pressed slowly and
consistently until liquid filled the cartridge detection chamber, being careful not to overfill the
chamber. The "NEXT" button then the "START" button were pressed. This initiated a 60
second countdown timer.  During this 60 seconds, the test cartridge with syringes in place was
shaken up and down. After the 60 second shaking period, the "NEXT" button was pressed and
the test cartridge was placed into its port in the Biosensor. The "NEXT" button was pressed
again  starting a three minute countdown during which the cartridge was left undisturbed in the
Biosensor.  After the three minute period, the large wash syringe plunger was pressed completely
in a slow uniform manner.  The "NEXT" button on the Biosensor was pressed to activate the
detection process. Results were displayed as a mV and %FS reading. For this verification test,
mV values were manually recorded and used for data interpretation which is described in Section
5.1. The Biosensor also has red/green warning lights that could be set to a pre-determined mV

                                           8

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threshold reading to indicate a positive/negative response; however, this feature was not used in
verification testing.
3.2.2  Non-Laboratory Testing

Because of the toxic nature of ricin and anthrax, only MB samples and the vendor-provided
positive control cartridges were analyzed at a non-laboratory location in order to evaluate field
portability. The non-laboratory location was a well-lit shipping/receiving area where there was a
power source. The temperature and relative humidity were ambient (20 +/- 2 °C and 40-50%,
respectively). Both a trained technician and a non-technical, untrained first-time user performed
analyses at the non-laboratory location.

3.2.3  Drinking Water Characterization

An aliquot of each DW sample, collected as described in Section 3.1.2, was sent to ATEL to
characterize the water samples based on the water quality parameters  shown in Table 3-4.
The table lists the methods used as well as the characterization data for the four water samples
collected as part of this verification test. Water quality parameters were characterized upon
sampling in June 2005, while the QTL Biosensor was tested with DW in November and
December 2005. The time delay between collection and testing was due to the fact that the water
samples were collected for use during a separate ETV test conducted prior to this one.  Because
of this, an aliquot of each DW was tested by ATEL again in January 2006 to verify some of the
parameters with the most potential to change over time.  Note that dissolved organic carbon was
not retested as this result was verified by the total organic carbon results, additionally the total
organic halides  and calcium and magnesium were not verified as there was no reason to expect a
change in these parameters. The concentrations of most water quality parameters were similar;
however, there was a decrease in levels of volatile compounds such as trihalomethanes and
haloacetic acids over this time-period.

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Table 3-4. Water Quality Characterization of Drinking Water Samples
Parameter
Alkalinity (mg/L)
Specific conductivity
(umho)
Hardness (mg/L)
pH
Total haloacetic acids
Total organic carbon
(mg/L)
Dissolved organic
carbon (mg/L)
Total organic halides
Total trihalomethanes
Turbidity (NTU)
Calcium (mg/L)
Magnesium (mg/L)
Method
SM 2320 B(2)
SM2510B(2)
EPA 130.2(3)
EPA 150.1(3)
EPA 552.2(5)
SM5310B(2)
SM5310B(2)
SM 5320B(2)
EPA 524.2(4)
SM2130B(7)
EPA 200.7 (6)
EPA 200.7 (6)
Columbus,
Ohio
2005
40
572
118
7.6
32.8
2.1
2.1
220
74.9
0.1
33
7.7
2006
44
602
107
7.4
<6.0
2.3
NA
NA
16.6
0.6
NA
NA
Metropolitan
Water District
of Southern
California
2005
71
807
192
8.0
17.4
2.5
2.9
170
39.2
0.1
45
20
2006
97
812
182
7.9
<6.0
2.7
NA
NA
24.1
0.2
NA
NA
New York City,
New York
2005
14
84
20
6.9
39.0
1.6
1.1
82
39.0
1.1
5.6
1.3
2006
12
78
26
6.8
<6.0
4.1
NA
NA
23.1
1.3
NA
NA
Orlando,
Florida
2005
142
322
143
8.5
34.6
1.7
1.6
300
56.4
0.5
8.8
43
2006
125
325
130
7.6
<6.0
2.1
NA
NA
41.8
0.1
NA
NA
NTU = nephelometric turbidity unit
NA = not retested
                                           10

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                                      Chapter 4
                        Quality Assurance Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the
quality management plan (QMP) for the AMS Center(8) and the test/QA plan(1) for this
verification test.
4.1 Quality Control of Stock Solution Confirmation Methods
4.1.1 Anthrax testing solutions

Solutions of Ames strain Bacillus anthracis prepared at Battelle from a USAMRIID stock was used for
testing. The concentrations of the anthrax testing solutions were confirmed by a plate
enumeration method (Battelle Standard Operating Procedure MREF X-054, Enumeration ofBL-
2 and BL-3 Bacteria Samples Via the Spread Plate Technique) within 24 hours of any solution
being analyzed. As an example, for a 109 spores/mL sample to be enumerated, the method
requires that the sample be diluted to at least 103 spores/mL so 100 microliters ((^L) of sample
would provide a countable number of spores on a culture plate. The number of counted colony
forming units multiplied by the dilution factor results in the enumeration confirmation
concentration. It should be noted that while all units for anthrax are listed as spores/mL in this
verification report, colony forming units are counted during plate enumeration. Because of
colony clumping, this could cause the reference results (in spores/mL) to be slightly
overestimated.

                                       E - A\
                          PD(%} =	=—!- x 100
Table 4-1 provides the results of all plate enumerations performed throughout the verification
test on anthrax solutions prepared in DI water. The expected concentration, the actual
concentration, and the percent difference (PD) between the two are given in the table. PD is
determined using the following equation, where E is the expected concentration and A is the
actual concentration as determined by the enumeration.

With PD values all less than 24%, the enumeration data confirm that the PT samples containing
the Battelle-prepared spores from USAMRIID stock were prepared accurately at various
concentration levels.
                                           11

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Table 4-1. Anthrax Enumeration Data for PT Samples

Battelle-prepared
solution
Date
11/7/05
11/8/05
11/8/05
11/8/05
11/8/05
11/8/05
12/5/05
Spore
Solution
Description
stock
50 x LOD
lOxLOD
5xLOD
LOD
LD
10 x LOD
Expected
Concentration
(spores/mL)
IxlO9
5xl06
IxlO6
5xl05
IxlO5
200
IxlO6
Actual
Concentration
(spores/mL) (a)
0.95 x 109
5.2 xlO6
0.82 x 106
5.2 x 10s
0.87 x 10s
247
0.83 x 106
PD(%)
5
3
18
4
13
24
17
LOD= vendor-stated limit of detection.
LD = lethal dose concentration.
(a) The value reported is the average of three plates developed for each enumeration. The uncertainty about the
  average is approximately 8%.
Table 4-2 gives the enumeration data for all of the interferent PT and DW samples that were
spiked with anthrax spores. Interferent and DW samples had similar percent differences between
expected and actual concentration as compared to the contaminant PT samples, with the
exception of the 250 mg/L calcium and magnesium solution (PD = 59%). In the Chapter 6
tables, only the expected concentration  of the test samples is given along with the results from
the QTL Biosensor. No correction for the enumeration data was performed since the QTL
Biosensor test is qualitative and not quantitative.

The stock solution containing Bacillus thuringiensis (analogue of anthrax) was confirmed by the
same enumeration method used for anthrax. The expected concentration was IxlO7 spores/mL
and the actual concentration was 4.93 x 107 spores/mL. Based on this, the stock solution was
diluted to create a  1 x 107 spores/mL solution, which was then diluted by a factor of ten to make
the testing solution of 1 x 106 spores/mL.
4.1.2 Ricin testing solutions

The COA for ricin was provided by the supplier. Because standard reference methods for the
determination of ricin do not exist, the concentration of ricin was not independently confirmed.
The COA stated that the ricin standard (Vector Laboratories, Burlingame, CA) had a
concentration of 5.0 mg/niL. Test  samples containing these contaminants were prepared by
diluting aliquots of this stock solution with DI water. All records pertaining to stock solution
dilutions were reviewed as part of the technical systems audit (TSA).
                                            12

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Table 4-2. Anthrax Enumeration Results for Fortified Interferent and Drinking Water
Samples
Test
Solution
Interferent
DW
Sample
Description
0.5 mg/L each humic/fulvic
acids
2.5 mg/L each humic/fulvic
acids
50 mg/L each Ca/Mg
250 mg/L each Ca/Mg
Cone. CA DW
Unconc. CA DW
Cone. OH DW
Unconc. OH DW
Cone. NY DW
Unconc. NY DW
Cone. FL DW
Unconc. FL DW
Date
11/11/05
11/11/05
11/11/05
11/11/05
11/16/05
11/16/05
11/16/05
11/16/05
11/16/05
11/16/05
11/16/05
11/16/05
Expected
Concentration
(spores/mL)
IxlO6
IxlO6
IxlO6
IxlO6
IxlO6
IxlO6
IxlO6
IxlO6
IxlO6
IxlO6
IxlO6
IxlO6
Actual
Concentration'3'
(spores/mL)
0.85 x 106
0.71 x 106
0.69 x 106
0.41 x 106
0.78 x 106
0.71 x 106
0.76 x 106
0.71 x 106
0.83 x 106
0.91 x 106
0.93 x 106
0.92 x 106
PD (%)
15
29
31
59
22
29
24
29
17
9
7
8
(a) The value reported is the average of three plates developed for each enumeration. The uncertainty about the
  average is approximately 8%.
4.1.3 Interferent solutions

For the interferent samples, the concentration of calcium and magnesium was confirmed by EPA
Method 200.7.(6)
4.2 Quality Control of Drinking Water Samples

A method blank sample consisting of DI water was analyzed once for approximately every 12
water samples analyzed for anthrax and once for approximately every 11 samples analyzed for
ricin for a frequency of approximately 9% for both anthrax and ricin. A positive control sample
was also analyzed once for approximately every 12 water samples for anthrax and once for
approximately every 16 samples for ricin for frequencies of approximately 9% for anthrax and
6% for ricin. While performance limits were not placed on the results of the positive control
sample, the vendor informed Battelle that, if the positive control samples did not cause a
significantly higher millivolt (mV) reading than the method blank, it would indicate to the
operator that the system was  not functioning properly.
                                           13

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4.3 Technical Systems Audit

The Battelle Quality Manager conducted a TSA to ensure that the verification test was performed
in accordance with the test/QA plan(1) and the AMS Center QMP.(8) As part of the audit, the
Battelle Quality Manager reviewed the standards and methods used, compared actual test
procedures with those specified in the test/QA plan,(1) and reviewed data acquisition and
handling procedures. Observations and findings from this audit were documented and submitted
to the Battelle Verification Test Coordinator for response. No findings were documented that
required any significant action. The records concerning the TSA are permanently stored with the
Battelle Quality Manager.
 4.4  Audit of Data Quality

At least 10% of the data acquired during the verification test was audited. Battelle's Quality
Manager or designee traced the data from the initial acquisition, through reduction and statistical
analysis, to final reporting, to ensure the integrity of the reported results. All calculations
performed on the data undergoing the audit were checked.


4.5 QA/QC Reporting

Each internal assessment and audit was documented in accordance with Sections 3.3.4 and 3.3.5
of the QMP for the ETV AMS Center.(8) Once the assessment report was prepared, the Battelle
Verification Test Coordinator responded to each potential problem and implemented any
necessary follow-up corrective action. The Battelle Quality Manager ensured that follow-up
corrective action was taken. The results of the TSA were sent to the EPA.
4.6 Data Review

Records generated in the verification test were reviewed before they were used to calculate,
evaluate, or report verification results. Table 4-3 summarizes the types of data recorded. The
review was performed by a technical staff member involved in the verification test, but not the
staff member who originally generated the record. The person performing the review added
his/her initials and the date to a hard copy of the record being reviewed.
                                           14

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Table 4-3. Summary of Data Recording Process
Data to Be Recorded
Dates and times of
test events
Sample collection and
preparation
information,
including chain-of-
custody
Biosensor procedures
and sample results
Anthrax enumeration
data
Reference method
procedures and
sample results
Responsible
Party
Battelle
Battelle and
Water
Utilities
providing
DW samples
Battelle
Battelle
ATEL
Where
Recorded
ETV data
sheets
ETV data
sheets,
laboratory
record books
and/or chain-
of-custody
forms
ETV data
sheets
Enumeration
data forms
Data
acquisition
system, as
appropriate
How Often
Recorded
Start/end of
test, and at each
change of a test
parameter
At time of
sample
collection and
preparation
Throughout test
duration
With every
enumeration
Throughout
sample analysis
process
Disposition
of Data
Used to
organize/check test
results; manually
incorporated in data
spreadsheets as
necessary
Used to
organize/check test
results; manually
incorporated in data
spreadsheets as
necessary
Manually
incorporated in data
spreadsheets
Manually
incorporated in data
spreadsheets
Transferred to
spreadsheets and
reported to Battelle
                                       15

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                                      Chapter 5
                  Statistical Methods and Reported Parameters


The methods presented in this chapter were used to verify the performance parameters listed in
Chapter 3.  The QTL Biosensor produces qualitative results; i.e., the Biosensor indicates only the
presence or absence of the contaminant and does not measure the concentration present.
Therefore, the data evaluation methods were applied in that context.
5.1 Qualitative Contaminant Presence/Absence

Contaminant presence/absence was assessed by comparing the mV reading for the contaminant-
only PT solutions with the mV threshold determined during the Method Detection Limit (MDL)
evaluation described in Section 5.4. Note that both the contaminant-only samples and MDL
samples were analyzed at the same time and all data were interpreted at the conclusion of all
analyses. Samples generating results above the MDL mV threshold level (determined to be 65
mV for anthrax and 76 mV for ricin) were considered positive. The QTL Biosensor features a
red/green light that is set at a factory determined mV level to switch from green (no contaminant
present) to red (contaminant present).  The red/green light feature was not evaluated during
verification testing and instead the MDL determined mV threshold was used.
5.2 False Positive/Negative Responses

A result was considered a false positive when a DI water or DW sample was spiked with a
potential interferent, a cross-reactive compound, or not spiked at all and a response above the
MDL mV threshold level was obtained. A result was considered a false negative when any DW
or interferent sample was spiked with a contaminant at 10 times the vendor stated limit of
detection and produced a response below the MDL mV threshold level. Interferent PT samples,
cross-reactivity PT samples, and DW samples were included in the analysis. The number of false
positive and negative results is reported.
5.3 Consistency

The reproducibility of the results was assessed by calculating the percentage of individual test
samples within a set (i.e., within a single concentration level or type of interferent) that produced
positive or negative results without variation among replicates.
                                           16

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5.4 Method Detection Limit

The MDL for each contaminant was determined according to procedures described in 40CFR136
Appendix B(9) and was assessed from seven replicate analyses of a fortified sample with the
target contaminant concentration approximately five times the vendor-stated LOD. The MDL
was calculated using the following equation:

                                     MDL = txS

Where t is the Student's value of 3.143 for a 99% confidence level when the degrees of freedom
(N-l) equals six, and S is the standard deviation of the replicate samples.
5.5 Other Performance Factors

Aspects of the QTL Biosensor performance such as ease of use, field portability, and sample
throughput are discussed in Section 6. Also addressed are qualitative observations of the
verification staff pertaining to the performance of the QTL Biosensor.
                                          17

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                                       Chapter 6
                                     Test Results
6.1 Qualitative Contaminant Presence/Absence

The responses for the QTL Biosensor using the contaminant-only PT samples containing anthrax
and ricin are shown in Tables 6-la and 6-lb and are discussed in the following sections.

6.1.1  Anthrax

The results obtained for the PT samples containing anthrax are given in Table 6-la.  The QTL
Biosensor generated positive results (i.e., had a mV reading greater than the MDL mV threshold
of 65mV) for concentrations of anthrax in all three replicates of solutions containing five times
the vendor-stated LOD (5 x 105 spores/mL) and higher. At the vendor-stated LOD (1 x 105
spores/mL) only one of three replicates was considered positive. There were no positive results
at the LD concentration of 200 spores/mL, which was not unexpected given that the LD
concentration is considerably lower than the vendor-stated LOD.

6.1.2  Ricin

The results obtained for the PT samples containing ricin are given in Table 6-lb. The QTL
Biosensor generated positive results (i.e., had a mV reading greater than the MDL mV threshold
of 76  mV) for concentrations of ricin in all three replicates of solutions containing five times the
vendor-stated LOD (0.25 mg/L) and higher. These positive results included the LD level for
ricin of 15 mg/L. At the vendor-stated LOD (0.05 mg/L) none of the three replicates generated a
response greater than the 76 mV MDL threshold, although two readings approached that level at
64 mV and 66 mV.
6.2 False Positive/Negative Responses

Three types of samples were analyzed to evaluate the susceptibility of the QTL Biosensor to
false positive and negative results. These included interferent PT samples, consisting of DI water
fortified with Ca and Mg or with humic and fulvic acids, both with and without the addition of
target contaminants; cross-reactivity PT samples consisting of DI water fortified with a
contaminant similar biologically or chemically with the target contaminant; and DW samples
both concentrated and unconcentrated and both with and without the addition of target
contaminants. In addition, a preservative blank containing sodium azide, which is used as a
preservative in commercially available ricin, was analyzed with the ricin test cartridges to
evaluate the potential for interference from the preservative. A false positive result was defined

                                           18

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as a result above the MDL threshold mV reading (determined to be 65 mV for anthrax and 76
mV for ricin) in the absence of the target contaminant and a false negative result was defined as a
result below the MDL threshold mV reading from a DW or interferent sample containing the
target contaminant at levels ten times the vendor-stated LOD.

Table 6-la. Anthrax Contaminant-Only PT Sample  Results-Contaminant
Presence/Absence Evaluation
Testing
Level
LD
LOD
5xLOD
10 x LOD
50 x LOD
Concentration
(spores/mL)
200
IxlO5
5xl05
IxlO6
5xl06
Result
(my)
23
18
17
54
45
67
124
209
205
299
380
540
1546
1594
1512
No. of
Positive
Results(a)
0
1
3
3
3
LD = Lethal dose concentration.
LOD = Vendor-stated limit of detection.
(a) Number of positive results (as indicated by a mV reading >65) out of three replicates.
Shaded areas indicate positive results.
                                            19

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Table 6-lb. Ricin Contaminant-Only PT Sample Results-Contaminant Presence/Absence
Evaluation
Testing
Level
LOD
5xLOD
10 x LOD
50 x LOD
LD
Concentration
(mg/L)
0.05
0.25
0.5
2.5
15
Result
(my)
64
66
33
125
175
245
199
207
205
396
682
466
954
945
827
No. of
Positive
Results(a)
0
3
3
3
3
LD = Lethal dose concentration.
LOD= Vendor-stated limit of detection.
(a) Number of positive results (as indicated by a mV reading >76) out of three replicates.
Shaded areas indicate positive results.
                                             20

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6.2.1  Interfered PT Samples

The results from the interferent PT samples are given in Table 6-2.  The QTL Biosensor anthrax-
specific cartridges generated false positive results from the PT samples containing calcium and
magnesium, but no contaminant. The ricin-specific cartridges generated no false positive results
in the presence of possible interferences, but no contaminant. For the anthrax-specific
cartridges, there were no false negative results when contaminant was present in solutions
containing possible interferences; however, for the ricin-specific cartridges there were false
negative results for two out of three replicates in the presence of 250 mg/L each of calcium and
magnesium. A preservative blank (0.16 mg/L sodium azide) was also analyzed with the ricin-
specific test cartridge to assess possible interference from the preservative in the commercially
available ricin. The preservative did not cause any  false positive responses (average of three
readings equaled 22 ± 7 mV).
6.2.2  DW Samples

Table 6-3 shows the results of testing with drinking water obtained from four different
geographic locations, both unconcentrated and concentrated and both with and without
contaminant.

For the unspiked, unconcentrated water samples, the QTL Biosensor anthrax-specific cartridge
generated false positive results in at least one of three replicates in water from all locations
except Ohio, for which there were no false positive results. With the anthrax-specific  cartridge,
false positive results were generated in at least two of three replicates in unspiked, concentrated
water samples from each of the four locations. In the presence of anthrax, for the unconcentrated
water samples from each location, there was a false negative result in one of three replicates in
waters from California, Florida, and Ohio, but no false negative results in water from New York.
For concentrated waters in the presence of contaminant there were no false negative results using
the anthrax-specific cartridge.

The QTL Biosensor ricin-specific cartridge generated false positive results in one of three
replicates for both the unspiked, unconcentrated and unspiked, concentrated water samples from
Florida.  There were no other false positive results in either the unconcentrated or concentrated
waters from the other locations. There were no false negative results in the presence of ricin for
any of the waters, both concentrated and unconcentrated from all locations.

Because of the false positive results obtained in the presence of calcium and magnesium and in
various types of DW samples, the vendor has subsequently undertaken an evaluation of the effect
of calcium and magnesium on their technology performance.  The vendor has determined that
calcium interferes with the reagents used in their technology more than magnesium and is
working to incorporate a high affinity binder of cations (such as calcium and magnesium) into
their sample buffer lyophilates to mitigate this problem. Any improvement based on the addition
of a high affinity binder has not been verified through the ETV program.
                                            21

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Table 6-2.  Interferent PT Sample Results- False Positive/Negative Evaluation

Interferent
0.5 mg/L
each humic
and fulvic
acids
2.5 mg/L
each humic
and fulvic
acids
50 mg/L
each Ca and
Mg
250 mg/L
each Ca
and Mg
Anthrax
Anthrax
(spores/mL)
none
IxlO6
none
IxlO6
none
IxlO6
none
IxlO6
Result
(mV)
20
22
19
394
398
250
35
18
19
365
299
270
394
268
368
255
278
446
932
639
280
697
609
882
No. of
Positive
Results(a)
0
3
0
3
3
3
3
3
Ricin
Ricin
(mg/L)
none
0.5
none
0.5
none
0.5
none
0.5
Result
(mV)
25
18
20
474
294
305
35
47
27
424
586
365
27
22
23
246
406
515
40
40
20
35
59
88
No. of
Positive
Results(a)
0
3
0
3
0
3
0
1
(a) Number of positive results out of three replicates as indicated by a mV reading >65 for anthrax and >76 for ricin.
Shaded areas indicate positive results.
                                               22

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Table 6-3.  DW Sample Results-False Positive/Negative Evaluation

DW Sample
California
California-
Concentrated
Florida
Florida-
Concentrated
Anthrax
Anthrax
(spores/mL)
none
IxlO6
none
IxlO6
none
IxlO6
none
IxlO6
Result
(mV)
644
541
131
807
62
1186
620
1126
850
627
558
272
48
68
1088
62
350
924
269
227
397
1394
588
432
No. of
Positive
Results(a)
3
2
3
3
2
2
3
3
Ricin
Ricin
(mg/L)
none
0.5
none
0.5
none
0.5
none
0.5
Result
(mV)
16
16
15
317
229
271
21
20
14
313
253
196
117
53
25
384
281
174
86
18
19
406
218
404
No. of
Positive
Results(a)
0
3
0
3
1
3
1
3
(a) Number of positive results out of three replicates as indicated by a mV reading >65 for anthrax and >76 for ricin.
Shaded areas indicate positive results.
                                               23

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Table 6-3.  DW Sample Results- False Positive/Negative Evaluation (continued)

DW Sample
New York
New York-
Concentrated
Ohio
Ohio-
Concentrated
Anthrax
Anthrax
(spores/mL)
none
IxlO6
none
IxlO6
none
IxlO6
none
IxlO6
Result
(mV)
38
123
31
114
93
149
40
76
157
78
110
143
28
41
48
56
164
79
41
141
341
140
302
249
No. of
Positive
Results(a)
1
3
2
3
0
2
2
3
Ricin
Ricin
(mg/L)
none
0.5
none
0.5
none
0.5
none
0.5
Result
(mV)
27
26
24
261
163
194
24
20
30
162
216
242
17
20
17
218
223
285
18
22
25
232
291
309
No. of
Positive
Results(a)
0
3
0
3
0
3
0
3
(a) Number of positive results out of three replicates as indicated by a mV reading >65 for anthrax and >76 for ricin.
Shaded areas indicate positive results.
                                               24

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6.2.3  Cross-Reactivity PT Samples

The results from the cross-reactivity PT samples are given in Table 6-4. A PT sample fortified
with a spore or chemical similar to each target contaminant was analyzed in the absence of any
of the target contaminant. The mV readings obtained for each cross reactivity compound are
given for each sample.  No false positive results were obtained with the potentially cross-reactive
compounds tested.

Table 6-4. Potentially Cross-Reactive PT Sample Results
Cross-Reactivity Compound
Anthrax: Bacillus thuringiensis
(1 xlO6 spores/mL)
Ricin: Lectin from soybean
(0.5 mg/L)
Result (mV)
22
24
20
23
26
23
No. of Positive
Results(a)
0
0
  Number of positive results out of three replicates as indicated by a mV reading >65 for anthrax and >76 for ricin.
6.3 Consistency

Using the QTL Biosensor and the anthrax-specific cartridge, results were consistent (i.e.,
produced positive or negative results without variation among replicates) in 21 out of 29 sets of
replicates or 72%.  Using the ricin-specific cartridge, results were consistent in 26 out of 29 sets
of replicates or 90%. Replicates included in the consistency calculation are the contaminant-only
PT samples, the interferent PT samples and the DW samples.
6.4 Method Detection Limit

The MDL for each contaminant was determined as described in Section 5.4. Note that the
contaminant, interferent, DW and MDL samples were analyzed at the same time and MDL
threshold values were determined afterwards, then applied to the sample data to interpret results.
Results are presented in Table 6-5. A mV reading of 65 was determined as the MDL threshold
value for anthrax and a mV reading of 76 was determined as the MDL threshold value for ricin.
Based on the data presented in tables 6-la and 6-lb, these mV MDL levels correspond to
contaminant concentrations in between the LOD and 5 x LOD concentrations tested.
                                           25

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Table 6-5. Method Detection Limit
Analyte
Anthrax
Ricin
Concentration
5xl05
spores/mL
0.25 mg/L
Testing
Level
5xLOD
5xLOD
Result
(mV)
168
192
127
181
166
167
181
125
178
175
135
143
117
129
Average
Result
(mV)
169
143
Standard
Deviation
(mV)
21
24
MDL
(mV)
65
76
LOD = vendor-stated limit of detection
6.5 Other Performance Factors
6.5.1  Ease of Use

The QTL Biosensor came with clearly written and informative instructions as well as a
demonstration video disc. Contents of the test cartridge kits were clearly labeled and storage
requirements were readily available. Overall, all packaging was easy to open, even when the
verification testing staff were outfitted with double-gloves.

The test cartridges come in packages containing everything needed to prepare a sample for
analysis with the QTL Biosensor. The cartridges were required to be stored between 2-8° C for
long term storage, but could be held at room temperature for up to six months. The test
cartridges came ready-to-use and required no preparation. The QTL Biosensor software was easy
to use and included a software feature called the "Sample Wizard" which prompted the operator
through the sample analyses through on-screen instructions. Use of the Sample Wizard is
optional and samples could be processed without the display prompts.  The surfaces of the QTL
Biosensor were easily wiped clean. Two syringes, a sample prep tube,  and a test cartridge were
generated as waste with each analysis.

As tested in this study, mV readings were obtained and manually recorded; however, the QTL
Biosensor can be set with a mV threshold that trips an indicator light from green, which indicates
                                           26

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a response below the set mV threshold, to red, indicating a result exceeding the mV threshold.
The indicator light feature was not evaluated during this study.

No formal scientific education would be required for using the QTL Biosensor and cartridges,
but general good laboratory skills are helpful. Because the kit is  intended for evaluating
biological and chemical agents, users should know and understand the procedures for safely
working with or near these agents before using this product.  The verification testing staff were
able to conduct tests with the kit after a two hour training  session.  Additionally, a non-technical
operator who had not received training from the vendor, and who relied solely on the instruction
manual and training disc was able to successfully operate  the QTL Biosensor.  QTL technical
support contact information, including phone, e-mail, fax  and website addresses are included in
the instruction manual for easy access to the vendor's contact information.

5.5.2 Field Portability

Field portability testing took place in a non-laboratory location with analyses performed by both
a trained operator and a non-technical, untrained first-time user.  Field portability testing was
accomplished by transporting the QTL Biosensor to a well lit shipping/receiving area.  The
temperature and relative humidity were ambient (20 +/- 2  °C  and 40-50%, respectively). The
equipment was light-weight and easy for one person to transport. A cooler was used to transport
the test cartridges to keep them between 2-8°C prior to use. Once at the field testing location, the
equipment was set up within minutes.  All reagents are pre-packaged and ready-to-use as
provided in the kit allowing a sample to be prepared and analyzed within five minutes  of set-up.
In addition to the cooler used to transport test cartridges, testing  staff needed to provide
something on which to record data and  a waste container;  otherwise, all other supplies were
provided with the QTL Biosensor and the test cartridge kit.

At this non-laboratory location, the QTL Biosensor was operated using its self-contained
rechargeable nickel-metal hybrid battery pack.  For long-term field deployment, a power source
would be needed to keep the QTL Biosensor battery charged. Additionally, for short-term use a
cooler is sufficient for transporting the test cartridges to keep them between 2-8°C; however, for
long-term field deployment a refrigerator would be useful to keep the  test cartridges cold.

The technology was field tested with a method blank and the vendor-provided positive control
cartridge.  Results in the field were similar to results obtained in the laboratory with the method
blank result well below and the positive control result well above the MDL mV threshold.

6.5.3 Throughput

Approximately 12  sample analyses were completed per hour. Each test cartridge is single-use.
                                            27

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                                  Chapter 7
                           Performance Summary
Table 7-1.  Anthrax Summary Table
Parameter
Contaminant-
only PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Sample Information
DI water
0.5 mg/L humic and fulvic
2.5 mg/L humic and fulvic
50 mg/L Ca and Mg
250 mg/L Ca and Mg
Unconcentrated CA
Concentrated CA
Unconcentrated FL
Concentrated FL
Unconcentrated NY
Concentrated NY
Unconcentrated OH
Concentrated OH
1 x 106 spores/mL
Bacillus thuringiensis
Anthrax Concentration
(spore/mL)
200 (lethal dose)
1 x 105 (vendor-stated
limit of detection)
5xl05
IxlO6
5xl06
unspiked
unspiked
IxlO6
IxlO6
unspiked
Positive Results out
of 3 Replicates
0
1
3
3
3
0
0
3
3
3
3
2
3
1
2
0
2
3
3
3
3
2
3
2
3
3
3
2
3
0
False positive results occurred in Ca and Mg interferent samples as well as the
Unconcentrated water from CA, FL, and NY and all concentrated drinking
water samples.
False negative results occurred only in the Unconcentrated CA, FL, and OH
drinking water samples.
Shading indicates results for unspiked sample.
                                      28

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Table 7-1.  Anthrax Summary Table (Continued)
   Consistency
Results were consistent (i.e., produced positive or negative results without
variation among replicates) in 21 out of 29 sets of replicates or 72%.
     Method
  Detection Limit
The method detection limit was determined to be the concentration generating
a 65 mV response.  It was between IxlO5 spores/mL (vendor-stated limit of
detection) and 5xl05 spores/mL.
      Other
   Performance
     Factors
Long term storage of test cartridges should be at 2-8° C, but cartridges may be
kept at room temperature for up to six months. Analysis software was user-
friendly. The QTL Biosensor uses electricity or rechargeable batteries and
includes a rugged carrying case. Test cartridges and detector were used inside
and outside a laboratory by trained operator as well as non-technically trained
operator; sample throughput was 12 samples per hour.
                                                  29

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Table 7-2.  Ricin Summary Table
Parameter
Contaminant-
only PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Method
Detection Limit
Sample Information
DI water
0.5 mg/L humic and fulvic
2.5 mg/L humic and fulvic
50 mg/L Ca and Mg
250 mg/L Ca and Mg
Unconcentrated CA
Concentrated CA
Unconcentrated FL
Concentrated FL
Unconcentrated NY
Concentrated NY
Unconcentrated OH
Concentrated OH
0.5 mg/L
Lectin from soybean
Ricin Concentration
(mg/L)
0.05 (vendor-stated limit of
detection)
0.25
0.5
2.5
15 (lethal dose)
unspiked
unspiked
0.5
0.5
unspiked
Positive Results out
of 3 Replicates
0
3
3
3
3
0
0
0
0
0
0
1
1
0
0
0
0
3
3
3
1
3
3
3
3
3
3
3
3
0
False positive results occurred in the Unconcentrated and concentrated FL
drinking water samples.
False negative results occurred only in the 250 mg/L Ca and Mg interferent
sample.
Results were consistent (i.e., produced positive or negative results without
variation among replicates) in 26 out of 29 sets of replicates or 90%.
The method detection limit was determined to be the concentration generating a
76 mV response. It was between 0.05 mg/L (vendor stated limit of detection)
and 0.25 mg/L.
      Other
   Performance
     Factors
Long term storage of test cartridges should be at 2-8° C but cartridges may be
kept at room temperature for up to six months. Analysis software was user-
friendly. The QTL Biosensor uses electricity or rechargeable batteries and
includes a rugged carrying case. Test cartridges and detector were used inside
and outside a laboratory by trained operator as well as non-technically trained
operator; sample throughput was 12 samples per hour.
Shading indicates results for unspiked sample.
                                                   30

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                                     Chapter 8
                                    References
1.   Test/QA Plan for Verification of Immunoassay Test Kits, Battelle, Columbus, Ohio,
    January 2004.

2.   American Public Health Association, et al. Standard Methods for the Examination of Water
    and Waste-water. 19th Edition, Washington, D.C., 1997.

3.   U.S. EPA, Methods for Chemical Analysis of Water and Wastes,  EPA/600/4-79/020,
    March 1983.

4.   U.S. EPA Method 524.2, "Purgeable Organic Compounds by Capillary Column GC/Mass
    Spectrometry," Methods for the Determination of Organic Compounds in Drinking Water—
    Supplement III, EPA/600/R-95/131, August 1995.

5.   U.S. EPA Method 552.2, "Haloacetic Acids and Dalapon by Liquid-Liquid Extraction,
    Derivatization and GC with Electron Capture Detector," Methods for the Determination of
    Organic Compounds in Drinking Water—Supplement III, EPA/600/R-95/131, August 1995.

6.   U.S. EPA Method 200.7, "Trace Elements in Water, Solids, and Biosolids by Inductively
    Coupled Plasma—Atomic Emission Spectrometry," EPA-821-R-01-010, January 2001.

7.   American Public Health Association, et al. Standard Methods for the Examination of Water
    and Waste-water. 20th Edition, Washington, D.C., 1998.

8.  Quality Management Plan (QMP)for the ETV Advanced Monitoring Systems Center,
   Version 5.0,  U.S. EPA Environmental Technology Verification Program, Battelle,
   Columbus, Ohio, March 2004.

9.  Code of Federal Regulations, Title 40, Part 136, Appendix B, Definition and Procedure for
   the Determination of the Method Detection Limit-Revision 1.11.
                                         31

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