ASTM E3395-23

• Abstract:

  SIGNIFICANCE AND USE; 5.1 Prompt and accurate identification of harmful biological agents on-scene is crucial to decision making for taking action and responding to incidents involving biological agents. ; 5.2 The detection and identification of a biological agent will inform how responders prepare for on-site activity (for example, selection of PPE and necessary precautionary actions), treat exposures, secure and decontaminate the incident site, and inform follow up actions to be taken after the incident has occurred. ; 5.3 Inclusivity and exclusivity test panels are used to ensure that biological agents targeted by the FSD can be detected (inclusivity) and that biological agents not targeted by the FSD are not detected (exclusivity). The environmental test panel is used to determine if there are potential interferences that could result in a false negative result when spiked whole biological agent is present.; SCOPE; 1.1 General: ; 1.1.1 This test method provides a procedure for characterizing the performance of nucleic acid-based field screening devices (FSDs) for the detection and identification of biological agents, when utilizing the test samples and statistical considerations described in Specification E3394. ; 1.1.2 This test method describes sample preparation and analysis protocols to use when characterizing the performance of nucleic acid-based field screening devices for the detection and identification of biological agents. ; 1.1.3 The intent of this test method is to provide a methodology to analyze samples in a manner that is analogous to how they are to be analyzed in the field by federal and state/local/tribal/territorial (SLTT) law enforcement and first responders, but under more controlled and reproducible conditions than those generally achievable when conducting field testing. The analysis of testing results as described in this test method and in Specification E3394 allow for a systematic way of measuring the statistical performance of FSDs. ; 1.2 Units: ; 1.2.1 The values stated in SI units are to be regarded as standard in this document. ; 1.2.2 When creating test sample mixtures, all concentrations are stated as copies/mL or genome equivalents/mL (GE/mL). ; 1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. ; 1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

• Status: Published
• Publication Date: 31-Oct-2023
• Technical Committee: E54 - Homeland Security Applications
• Drafting Committee: E54.01 - CBRNE Detection and CBRN Protection

Overview

ASTM E3395-23 – Standard Test Method for Characterizing Performance of Field Screening Devices for the Identification of Biological Agents provides a comprehensive procedure for evaluating the effectiveness of nucleic acid-based field screening devices (FSDs). Accurate, on-scene identification of biological agents is critical for emergency responders and informs appropriate actions in incidents involving potential biothreats. This standard enables federal, state, local, tribal, and territorial (SLTT) organizations, as well as law enforcement and first responders, to reliably assess FSD performance under controlled and reproducible laboratory conditions prior to field deployment.

Key Topics

• Performance Characterization: Outlines a structured approach to test and validate FSDs for detecting and identifying biological agents, ensuring devices can effectively distinguish between targeted and non-targeted agents.
• Sample Preparation and Analysis: Includes protocols for preparing test and control samples, conducting analyses following manufacturer’s recommendations, and recording results to ensure traceability and consistency.
• Inclusivity and Exclusivity Panels: Utilizes standardized panels of biological agents. The inclusivity panel verifies that FSDs detect intended targets, while the exclusivity panel confirms non-detection of non-target agents to avoid false positives.
• Environmental Panel Testing: Examines the performance of FSDs using samples that mimic real environmental backgrounds, highlighting the device's resilience against potential interferences that may lead to false negatives.
• Documentation and Reporting: Specifies the importance of full documentation, including all materials, processes, and outcomes, to support transparency and repeatability.
• Calibration and Control: Emphasizes the need for regular calibration, control sample analysis, and instrument maintenance as per manufacturer and safety guidelines.
• Statistical Performance Measurement: Details how to measure performance metrics such as probability of detection (POD), confidence intervals (CI), and lower confidence bounds (LCB) as a systematic approach to device evaluation.

Applications

• Emergency Response: Supports responders in quickly identifying biological threats, which is crucial when selecting personal protective equipment (PPE), organizing decontamination, and making treatment decisions after potential exposures.
• Field Deployment Readiness: Ensures FSDs are evaluated for reliability in laboratory settings that simulate real field conditions, reducing operational risk during critical incidents.
• Law Enforcement and Homeland Security: Provides law enforcement and security personnel with a robust methodology to screen suspicious materials and secure incident sites.
• Public Health and Safety: Contributes to the safe management of outbreaks or bioterrorism threats by validating tools that can identify potentially hazardous biological agents.

Related Standards

• ASTM E3394: Specification for Field Screening Devices Used for Identification of Biological Agents – establishes requirements for FSDs, including sample types and statistical methods referenced by E3395.
• ASTM E2458: Practices for Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being Biological Agents and Toxins from Nonporous Surfaces.
• ASTM E3131: Specification for Nucleic Acid-Based Systems for Bacterial Pathogen Screening.
• ASTM E3289 and E3290: Guidelines and test methods for field detection of hazardous substances (e.g., fentanyl), complementing biothreat detection.
• ASTM E2771: Terminology for Homeland Security Applications, providing standard definitions relevant to this standard.

Practical Value

ASTM E3395-23 provides a rigorous, standardized method for characterizing field screening devices designed for the rapid and reliable identification of biological agents. Its systematic approach underpins decision-making in emergency management, supports regulatory compliance, and ensures the safety of personnel and the public in the event of biological incidents. Incorporating inclusivity, exclusivity, and environmental testing panels enhances testing reliability by mimicking real-world conditions and minimizing false responses. By following this internationally recognized standard, organizations can implement certified best practices and remain at the forefront of homeland security, biosafety, and emergency preparedness.

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3395 − 23
Standard Test Method for
Characterizing Performance of Field Screening Devices for
the Identification of Biological Agents
This standard is issued under the fixed designation E3395; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 General: 2.1 ASTM Standards:
1.1.1 This test method provides a procedure for character- E2458 Practices for Bulk Sample Collection and Swab
izing the performance of nucleic acid-based field screening Sample Collection of Visible Powders Suspected of Being
devices (FSDs) for the detection and identification of biologi- Biological Agents and Toxins from Nonporous Surfaces
cal agents, when utilizing the test samples and statistical E2771 Terminology for Homeland Security Applications
considerations described in Specification E3394. E3131 Specification for Nucleic Acid-Based Systems for
1.1.2 This test method describes sample preparation and Bacterial Pathogen Screening of Suspicious Visible Pow-
analysis protocols to use when characterizing the performance ders
of nucleic acid-based field screening devices for the detection E3289 Guide for Using Equipment and Assays for Field
and identification of biological agents. Detection of Fentanyl and Fentanyl-Related Compounds
1.1.3 The intent of this test method is to provide a method- E3290 Test Method for Establishing Performance of Equip-
ology to analyze samples in a manner that is analogous to how ment and Assays for Field Detection of Fentanyl and
they are to be analyzed in the field by federal and state/local/ Fentanyl-Related Compounds
tribal/territorial (SLTT) law enforcement and first responders, E3394 Specification for Field Screening Devices Used for
but under more controlled and reproducible conditions than Identification of Biological Agents
those generally achievable when conducting field testing. The
2.2 Federal Standards:
analysis of testing results as described in this test method and
18 US Code 178 Definitions
in Specification E3394 allow for a systematic way of measur-
ing the statistical performance of FSDs. 3. Terminology
1.2 Units: 3.1 Definitions:
1.2.1 The values stated in SI units are to be regarded as 3.1.1 accuracy, n—closeness of agreement between a test
standard in this document. result and the accepted reference value. E2771
1.2.2 When creating test sample mixtures, all concentrations
3.1.2 assay, n—quantitative or qualitative test used to deter-
are stated as copies/mL or genome equivalents/mL (GE/mL).
mine the presence or absence of a chemical or biological
1.3 This standard does not purport to address all of the material.
safety concerns, if any, associated with its use. It is the 3.1.2.1 Discussion—Amended the definition from Specifi-
responsibility of the user of this standard to establish appro- cation E3131 to include chemical as well as biological
priate safety, health, and environmental practices and deter- material. E3131
mine the applicability of regulatory limitations prior to use.
3.1.3 biological agent, n—any microorganism (including,
1.4 This international standard was developed in accor-
but not limited to bacteria, viruses, fungi, rickettsia, or proto-
dance with internationally recognized principles on standard-
zoa); infectious substance; or any naturally occurring,
ization established in the Decision on Principles for the
bioengineered, or synthesized component of any such micro-
Development of International Standards, Guides and Recom-
organism or infectious substance capable of causing: (1) death,
mendations issued by the World Trade Organization Technical
disease, or other biological malfunction in a human, an animal,
Barriers to Trade (TBT) Committee.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
This test method is under the jurisdiction of ASTM Committee E54 on contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Homeland Security Applications and is the direct responsibility of Subcommittee Standards volume information, refer to the standard’s Document Summary page on
E54.01 on CBRNE Detection and CBRN Protection. the ASTM website.
Current edition approved Nov. 1, 2023. Published December 2023. DOI: Available from U.S. Government Publishing Office (GPO), 732 N. Capitol St.,
10.1520/E3395-23. NW, Washington, DC 20401, http://www.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3395 − 23
a plant, or other living organism; (2) deterioration of food, 3.1.11 genome, n—the complete set of genetic material of a
water, equipment, supplies, or material of any kind; or (3) human, animal, plant, or other living thing.
deleterious alteration of the environment. 18 USC 178
3.1.12 genome equivalents, GE, n—number of genome
3.1.3.1 Discussion—Also termed biothreat agent.
copies present in a given mass of nucleic acid (deoxyribo-
3.1.4 calibration, n—set of operations that establish, under nucleic (DNA) or ribonucleic acid (RNA)) that can be calcu-
specified conditions, the relationship between the values of lated by converting the size of a genome in base pairs to
micrograms of DNA or RNA.
quantities indicated by a measurement instrument or measuring
system or values represented by a material measure or a
3.1.13 inclusivity panel, n—collection of closely related
reference material and the corresponding values realized by
biological agents, viruses, or nucleic acids used during testing.
standards.
E3131
3.1.5 confidence interval, CI, n—range of values created
3.1.13.1 Discussion—Tests involving members of the inclu-
using a procedure that, when repeated many times, on distinct
sivity panel, if carried out, shall follow the methodology
data sets, generated from the same underlying stochastic
presented in this test method and in Specification E3394.
process, will bracket the true measure of performance, such as
3.1.14 inhibition, n—undesirable effect that can result in a
probability of detection (POD), the proportion of times stated
false negative result and is typically caused by the presence of
in the confidence level. E3131
compounds that interfere with the assay or detection process.
3.1.5.1 Discussion—This definition differs slightly from the
E3131
one in Specification E3131 by explicitly linking the interval to
3.1.15 limit of detection, LOD, n—lowest amount of analyte
the confidence level.
in a sample that can be detected with stated probability. E3131
3.1.6 confidence level, CL, n—probability value association
3.1.16 lower confidence bound, LCB, n—lowest value of a
with a CI; the percentage of intervals that can be expected to
one-sided CI created using a procedure that, when repeated
include the true population parameter in the long run. E3131
many times on distinct data sets generated from the same
3.1.7 environmental panel, n—collection of materials and
underlying stochastic process, will include the true measure of
compounds that can be found in the environment (indoor or
performance a proportion of times equal to the stated
outdoor).
probability. E3131
3.1.7.1 Discussion—The environmental panel is determined
3.1.16.1 Discussion—The LCB ensures that the POD attains
by the entity directing testing and will include samples
a satisfactory value for the CL selected and determines the
representative of aerosol backgrounds in the locations of
minimum number of samples that shall be analyzed. Slightly
interest. Note that this test method does not include discussion
modified from Specification E3131.
of aerosol filter sample collection, sample preparation
3.1.17 measurement process, n—step, or series of system-
conditions, or methods that may be necessary to extract
atic steps, used to detect a material or determine if a system or
collected material into a suitable liquid for analysis using
instrument performs as intended. E3289
nucleic acid-based methods. The environmental panel is used
as a more “real-world” test of a detection system’s performance
3.1.18 multiplex assay, n—assay that is capable of measur-
when spiked with a whole organism. The environmental panel
ing multiple biological agents or multiple targets for a single
should not result in any false positive or negative results and
agent in a single test sample. E3131
should not cause failure of any control samples.
3.1.18.1 Discussion—This definition differs slightly from
3.1.8 exclusivity panel, n—collection of near-neighbor bio-
the one in Specification E3131 by including multiple targets.
logical agents, viruses, or nucleic acids used during testing.
3.1.19 near neighbor, n—organism, virus, or nucleic acid
E3131
that is similar to a desired target biological agent but should not
3.1.8.1 Discussion—Results from tests involving members
result in a positive detection result. E3131
of the exclusivity panel, if carried out, shall follow the
3.1.19.1 Discussion—Exclusivity panel members include
methodology presented in this test method and in Specification
near neighbors.
E3394.
3.1.19.2 Discussion—Testing involving near neighbors, if
3.1.9 false negative, n—failure to detect a compound within
carried out, shall follow the methodology presented in this test
a sample when it is present at a concentration well above the
method and in Specification E3394.
limit of detection. E3289
3.1.20 panel, n—collection of samples used during testing.
3.1.9.1 Discussion—This definition differs slightly from the
3.1.20.1 Discussion—Examples of panels include inclusiv-
one in Guide E3289 by emphasizing concentrations above the
ity panel, exclusivity panel, and environmental panel.
limit of detection.
3.1.21 pooling, v—act of creating a single test sample (the
3.1.10 false positive, n—detection of a compound within a
pooled sample) that contains strains from different biological
sample when it is not present. E3289
agents. E3131
Eurachem Selection, Use and Interpretation of Proficiency Testing (PT)
Schemes by Laboratories, 2nd edition, 2011. www.eurachem.org. From https://dictionary.cambridge.org/us/dictionary/english/genome.
E3395 − 23
3.1.22 probability of detection, POD, n—proportion of posi- 5. Significance and Use
tive analytical outcomes in a stated number of tests for a
5.1 Prompt and accurate identification of harmful biological
qualitative method for a given matrix at a given concentration.
agents on-scene is crucial to decision making for taking action
3.1.22.1 reference material, n—material, sufficiently homo- and responding to incidents involving biological agents.
geneous and stable with respect to one or more specified
5.2 The detection and identification of a biological agent
properties, that has been established to be fit for its intended
will inform how responders prepare for on-site activity (for
use in the measurement process; properties can be quantitative
example, selection of PPE and necessary precautionary
or qualitative. E3131
actions), treat exposures, secure and decontaminate the inci-
3.1.23 risk, n—the probability of suffering a loss or harm or dent site, and inform follow up actions to be taken after the
injury; peril. E2458
incident has occurred.
3.1.24 spiked sample, n—sample that is created by adding a
5.3 Inclusivity and exclusivity test panels are used to ensure
known quantity of biological agent to a known quantity of
that biological agents targeted by the FSD can be detected
other material, such as environmental matrix materials.
(inclusivity) and that biological agents not targeted by the FSD
are not detected (exclusivity). The environmental test panel is
3.1.24.1 Discussion—Examples of spiked samples include
pathogens added to environmental matrix materials that are used to determine if there are potential interferences that could
result in a false negative result when spiked whole biological
typically prepared in a liquid (buffer) suspension.
agent is present.
3.1.25 strain, n—isolates or variants of the target biological
agent(s) that the method can detect (inclusivity strains) or
6. Apparatus
should not detect (exclusivity strains). E3131
6.1 Dead air box, for preparation of nucleic acid samples
3.1.26 test sample, n—amount and identity of a particular
and biological safety cabinet suitable for working with risk
substance (target and non-target compounds) prepared for
group (RG) 2 or 3 organisms as necessary.
testing. E3290
6.2 Mixing apparatus and containers.
3.2 Acronyms:
6.3 Dispensing devices, capable of delivering aliquots be-
3.2.1 CI—confidence interval.
tween 10.00 mL and 1.00 μL, calibrated following manufac-
3.2.2 CL—confidence level.
turer recommendations.
3.2.3 DNA—deoxyribonucleic acid.
6.4 Instrument and assay kit, in operational readiness.
3.2.4 FSD—field screening device.
7. Reagents and Materials
3.2.5 GE—genome equivalents.
7.1 Nucleic acids or organisms, as required for a given
3.2.6 LCB—lower confidence bound.
evaluation of an FSD.
3.2.7 PCR—polymerase chain reaction.
7.2 Nuclease-free water, for dilutions of test panel materials
3.2.8 POD—probability of detection. as necessary.
3.2.9 PPE—personal protective equipment. 7.3 Consumables needed for a given field screening device,
including buffer, assay cartridges, or other reagents and
3.2.10 RG—risk group.
materials, per manufacturer’s guidance.
3.2.11 RNA—ribonucleic acid.
7.4 Laboratory equipment, such as pipettes, and disposable
3.2.12 SLTT—state, local, tribal, and territorial.
labware such as pipette tips, and microcentrifuge tubes.
8. Hazards
4. Summary of Test Method
8.1 Take all necessary precautions when working with
4.1 Test samples are prepared that contain a known
infectious biological materials. Implement best practices for
concentration/amount of target and non-target analyte(s), as
working with biological materials including use of proper PPE.
defined in Annex A1 of Specification E3394.
8.2 Safety data sheets for all chemicals and pathogen data
4.2 Instrument and assay start-up, preparation, system
sheets for all biological agents and materials shall be consulted
checks, and calibrations are performed per manufacturer’s
prior to use.
instructions and guidance.
8.3 The user of this test method shall be aware of the
4.3 Test samples and control samples are analyzed accord-
hazards associated with the operation of the chosen instruments
ing to the manufacturer’s instructions and guidance.
or assays. Consult the manufacturer-provided user manual and
4.4 Required documentation is collected.
training materials for hazards specific to the instrument or
assay being used.
4.5 Any additional information required for samples and
testing conditions is recorded.
9. Required Documentation
4.6 Results are evaluated and recorded.
9.1 Include, at a minimum, enough information in the
4.7 Results are reported. documentation of all chemicals and materials used in testing,
E3395 − 23
sample preparation and dilutions to trace the material(s) to its created artificially by adding known amounts of materials that
source, including manufacturer/supplier, catalog or product are present in the area of interest.
number, or both, lot number, purchase date, and any
10.5.3.3 This standard does not address issues associated
manufacturer-provided quality information/certificates. with sample preparation (for example, spore and cell
disruption, extraction/cleanup) prior to nucleic acid analysis,
9.2 Record all calculations (for example, for dilutions).
which is necessary for some types of samples and detection
9.3 Prepare any instrument or assay for testing in accor-
systems.
dance with manufacturer’s guidance, including any calibration,
10.5.3.4 The environmental panel may be obtained from an
tuning, or operational checks. Record all protocols used and
aerosol filter extract that is collected under relevant conditions
results obtained.
in appropriate locations. Concentrations of potentially interfer-
ing components in air can vary with time of year, location, and
9.4 Record control sample identity and control sample
many other factors, making it impractical to test even a reduced
results of all test panel samples associated with those controls
number of all possible combinations involving more than a few
(see 10.10).
of these factors. Therefore, multiple environmental panel test
9.5 Save all associated raw data and data analysis/reporting
samples of the most representative environmental conditions
outputs
...