D.L. DeVincenzi, J. Bagby, M. Race, and J.D. Rummel, NASA CP-1999-208772,
NASA Ames Research Center, Moffett Field, CA (1999).
In 1996, several NASA-sponsored studies were underway to look at various aspects of a Mars Sample Return (MSR) mission. One of these studies by the Mars Exploration Long Term Science Working Group (MELTSWG) determined the need for additional study of five specific areas related to Planetary Protection (PP). One of the priority areas identified was the need to develop guidelines for return sample containment and quarantine analysis. In response to this need, the Mars Sample Quarantine Protocol Workshop was convened in June, 1997 to deal with three specific aspects of the initial handling of a returned Mars sample: 1) biocontainment, to prevent uncontrolled release of sample material into the terrestrial environment; 2) life detection, to examine the sample for evidence of live organisms; and 3) biohazard testing, to determine if the sample poses any threat to terrestrial life forms and the Earth’s biosphere.
Background: In order to constrain the scope of the Workshop, several starting assumptions were given: 1) The Mars Sample Return mission (MSR) will be launched in the 2005 opportunity; 2) the mission will return samples from biologically interesting sites based on data returned from missions in 1996, ’98, ’01, and ’03; 3) in a nominal mission, the sample will not be sterilized prior to return to Earth; 4) the amount of sample available for quarantine tests will be a small fraction of the total amount returned; and 5) biocontainment of the unsterilized sample will be maintained until quarantine testing for biohazards is accomplished.
FindingsThe Containment Subgroup discussed the development of recommendations that might be adopted by NASA for the safely controlled management of a Mars sample while a quarantine protocol is executed. Containment was defined as: “a system of protection of: 1) the Earth’s biosphere from release of ‘biological entities’ of martian origin, and 2) the integrity of the sample.”
Sample Return Canister: The entire system of containment – from Mars to Earth – must prevent the escape of potentially hazardous material. This means special design considerations for the canister and planning for Earth return procedures. Specific recommendations include:
- Decontamination of the exterior of the canister that contacts the martian surface;
- Contingencies for non-nominal events (ex. initial trajectory of Earth return vehicle biased to miss Earth; indicator system to monitor for breach of containment en route; on board system for sterilization in case of an in flight breach in containment; provisions to determine if a breach occurs during a hard impact at the landing site, and suitable sterilization for that event.)
Upon recovery of the canister and reconfirmation of proper containment, the canister must be transported to a quarantine facility in a container meeting regulatory requirements for safe transport of potentially hazardous biological material. Precautions for handling the sample return canister should include provisions for protective garments for the recovery crew and coordination with appropriate regulatory agencies such as USDA-APHIS and EPA.
Mars Receiving Laboratory (MRL): The unknown nature of any possible hazardous material in the sample warrants the use of the most stringent containment presently afforded to the most hazardous biological entities known on Earth; that is, a Biosafety Level 4 (BSL-4) operation. Appropriate containment is attained through the application of primary and secondary containment principles:
- Primary containment will be provided by utilizing Class III biosafety cabinets: comprised of glove boxes connected in sequence with sealable doors between cabinets and maintained under negative pressure.
- Secondary containment will be provided by the building: a ‘high-end’ BLS-3 structure which is sealed and maintained under negative pressure, with high efficiency particulate air (HEPA) filtered exhaust air, sterilized waste water, and with provision for personnel showers and appropriate use of disinfectants. While biological safety and physical security must be the prime considerations in the design of a Mars receiving facility, there could be alternative approaches to accomplish the needed containment besides a dedicated new facility. One such alternative includes providing a small MRL facility beside an existing approved BSL-4 laboratory (e.g. USAMRIID at Fort Detrick, Maryland, or CDC in Atlanta, Georgia) This would offer flexibility, availability of trained professionals and support staff, and possible simplification of the permit and approval process. Disadvantages include the possible reduction in control of samples by NASA while in the hands of another agency, and background organic residues and contamination that could interfere with sample interpretation. Existing policy for the transport and receipt of potentially hazardous agents requires CDC review of the facility, thereby providing an additional check on safety. Whatever alternative is selected, at least five years must be allowed for the construction and certification of such a highly technical facility and for the training of professional and support staff. Training periods are required for qualified personnel to become familiar with the new facility so their operations are safe, efficient, and accurate.
- Glove Box System: Glove boxes can be flexibly designed to include any laboratory equipment required by the protocols. Operational parts of equipment can be housed within the primary containment glove boxes, with electronics, control panels, etc. located outside the primary containment barrier. The report provides preliminary details on the need for careful planning of the sequence of steps for handling and opening the sample canister in the cabinet lines to avoid contamination on contained samples.
Containment Research and Technology Needs: Specific research areas recommended to accomplish successful containment both in transit and in the laboratory include:
- Challenge tests of HEPA filtration system should be undertaken using carbon-bearing particles from 10 nm to 100 nm in size.
- Research should be conducted to choose appropriate isotopes and particle sizes for use in flight verification and testing of canister seals (e.g., carbon compounds, radioactive-tagged particles).
- Select an appropriate indicator for canister seal integrity upon recovery
- Design effective processes to clean containment area of terrestrial biological entities and organics to avoid confusion during observations of the Mars samples.
- Systems must be developed and tested to maintain sample integrity when obtaining aliquots of material for quarantine testing
- Design research to provide a system for needle puncture of the ‘head space’ through a vacuum-sealed line; HEPA filters could be incorporated.
- Determine the suitable sterilization methods for the Mars sample.
The Life Detection Subgroup was assigned the task to develop a series of tests (a protocol) to detect the presence of live organisms, or of materials that have been derived from live organisms, in samples of material returned from Mars. The group first considered the likely aspects of viable organisms that might be detected and then determined the philosophy that should guide the life detection protocol, which in turn would dictate the sequence, techniques and handling requirements for the protocol. The subgroup also made recommendations on research needed to refine the eventual protocols.
The philosophy espoused by the subgroup aimed not only at detecting life, but distinguishing between potential martian life forms and terrestrial contamination. In particular: 1) there must be multiple lines of evidence to support an hypothesis that detected life is of martian origin, and 2) it is essential to understand the geological and potential ecological context of a sample in order to understand the nature of life that might be detected in the samples. A strong quality assurance and quality control (QA/QC) program was deemed essential, involving the use of chemical tracers in order to correlate the ‘detected’ material/organism(s) with the phase of the mission in which material was obtained.
In order to establish the appropriate context for life detection in a sample, a preliminary analysis of the sample was recommended to: 1) characterize the bulk mineralogy of the sample, 2) establish its elemental composition, 3) inventory the volatile and organic materials it may contain, 4) measure the redox couples present in the sample material, and 5) obtain a microscopic characterization of the sample surface and interior. As long as an adequate sterilization method could be defined which would not affect the results of the analysis, the Subgroup felt most of these analyses would not require the sample to be held in biological containment.
The Life Detection Subgroup prioritized three basic methods for accomplishing life detection:
- Organic chemical analysis and detection including search for functional groups containing reduced carbon, sulfur of nitrogen; analysis of possible kerogen materials for stable isotope abundances; detection of amino acids or possible proteins; analysis for amphiphiles in the form of fatty acids, hopanes, etc; a search for carbohydrates, nucleic acid bases, and related compounds (e.g., DNA, RNA, PNA, etc.); and potential detection of integrated cell walls or cell wall components such as lipopolysaccharides. Assuming current improvements in available technologies, it was felt that cellular life could be detected routinely at the level of 10-100 cells in a sample and as little as one cell in a 100 g sample.
- Light and/or electron microscopy to detect morphological indications of life, along with the trace mineralogy of the sample. Coupled with staining methods to reveal chemical evidence of life in conjunction with morphological methods, light microscopy was seen as having advantages over electron microscopy in terms of sample preparation, handling and real-time testing. Electron microscopy, particularly ion-probe techniques, can provide critical composition information about samples. The issue of what constitutes a ‘representative’ sample will need to be defined.
- Culturing of martian materials and/or living organisms: Although it will be difficult to generalize for putative martian organisms, cultivation as a life detection approach was recommend because of the potential to amplify the presence of life in a sample, to discriminate between a viable organism and materials that were once associated with biology (but not now alive), and to provide a natural link to hazard detection analyses. Attempted cultivation techniques should include not only conditions commensurate with the environment from which samples were obtained, but also the use of multiple media and carbon sources under both aerobic and anaerobic conditions, using both intact samples and processed sample materials. Given the low culturability of environmental microbes from Earth (~1%), culturability is of secondary or tertiary priority for life detection.
The Life Detection Protocol should be an integrated facet of the comprehensive analysis of samples for atmospheric, geophysical, and exobiological purposes. A comprehensive process for sample analysis and life detection was outlined which includes detailed comments about particular steps in the process such as the sample container, sample receiving , sample separation, microscopic/mineralogical/geochemical survey, life detection microscopy, and chemical analyses for signs of life. The Life Detection Subgroup recommended that the following considerations form the basic concept of chemical analysis techniques in life detection:
- Seek functional groups important for energy transfer rather than live biomass
- Seek to identify accumulated biomass-type molecules and cellular components rather than cells or single living entities
- Use more sensitive and less selective detectors for the first sample screening procedure. Rather than employing the selectivity of GC-MS or KC-MS as the first step, use highly sensitive infrared micro-calorimetric or lab-on-a-chip technology to provide high sensitivity detection of functional groups.
- Integrate remnant parts as a preliminary indication of possible extant life (the amount of functional groups remaining from remnant parts often exceeds the live biomass in samples on Earth.)
- It may not be possible to rely on DNR, RNA, proteins or even carbon-based molecular backbones as indicators because extraterrestrial life may be markedly different in detail from life on Earth. Focus initial screening efforts on amine and carboxyl functional groups to detect signs of life based on any backbone, C, N, P, S or Si. Comparison of stable isotopic signatures of non-life-like compounds (e.g., PAHs) and life-like compounds may provide additional information on the potential existence of life on Mars.
Life Detection Research and Technology Needs: NASA musts begin to incorporate life detection technologies into planning and anticipated sample receiving activities for MSR. In particular, a plan must be developed for the acquisition and operation of appropriate instrumentation within the sample handling facility, and appropriate sterilization protocols and methods must be developed to prepare samples for distribution to the wider scientific community.
The Biohazard Testing Subgroup was assigned the task of developing an up-to-date methodology to determine if returned martian sample materials are hazardous, regardless of whether life or biological entities are detected. The Subgroup proposed a tiered or stepwise approach to testing based heavily on protocols used by research and agencies for a wide range of biological agents. These tests would: 1) focus on a broad range of biohazards, 2) screen for indication of biological activity or disruption thereof, and 3) incorporate systematic feedback as data are gathered from the life detection studies , chemical analyses, and biohazard tests themselves. Emphasis was placed on hazards posed by organisms that replicate because of their potential for large scale negative impacts on Earth’s ecosystems.
Two priority biohazard concerns were addressed: 1) pathogenicity, and 2) ecological disruption. (Chemical toxicity was not considered a significant biohazard or global threat since toxic materials will not replicate and spread, and since proper laboratory protocols will protect those who work with the samples). Detailed information and discussion about various tests are provided in the appendix of the report. In general, the subgroup recommended the following:
Pathogenicity: Regardless of the outcome of preliminary life detection tests or chemical analyses, it will be prudent to screen samples for two types of pathogenicity – toxic and infectious – using tests specifically designed to detect biological activity or disruptions. In vitro methods are considered superior to whole organism tests for preliminary biohazard screening because of their sensitivity, simplicity and speed, as well as their widespread use, acceptance and interpretation. By selecting a suitably diverse range of in vitro tests and conditions, it will be possible to screen for biologically important outcomes that might be indicative of biohazards in a wide range of representative species and taxonomic groups. It would be advisable to include a range of in vitro tests that are routinely used by agencies and researchers when scanning for pathogenesis. In addition, the inclusion of two addition types of tests – a series of laboratory mice injection studies (because of their extensive use for pathogenicity and biohazard testing) and a series of tests using Tetrahymena (as a model for metazoan biochemistry) – were discussed. A recommended battery of tests for detection indication of potential pathogenicity in the sample might include:
- diverse microbial media that use varied laboratory initial conditions
- selected tissue cultures and cell lines from mammalian organ systems, fish and insects
- embryonating chicken eggs
- mouse injection studies
- Tetrahymena (protozoans)
- Plant tissue cultures (wheat, rice, potato).
Ecological Disruption: In the event of inadvertent introduction to the Earth’s biosphere of putative martian microbes, there would be little threat of widespread ecological disruption based on our comparative knowledge of martian and Earth conditions and our knowledge about microbial potential on Earth. Nevertheless, since the risk of potentially harmful effects is not zero, it will be prudent to screen for the ability of the returned sample to disrupt microbial ecosystems. Although such tests are not routinely done, it would be advisable to design and conduct suitable microcosm tests to screen for potential ecosystem effects or disruption in biogeochemical cycles. Two types of microcosm tests are recommend, the first designed to assay for disruptions of important representative microbial systems upon addition of martian material, and the second to determine if any undetected biological entities can grow or propagate in selected sterilized microcosm of representative terrestrial ecosystems
Criteria for Distribution of martian Samples: The Biohazard Testing Subgroup considered the many possible interpretations of data for the proposed battery of life detection and biohazard tests and developed a table providing an overview of various combinations of findings (Table 1 in report). In general, if any life forms are detected, even if preliminary test suggest they do not pose a biohazard, the Subgroup advised continued strict containment, rather than controlled distribution, at least initially. Strict containment should be maintained in light of any positive test results until findings are verified and/or a scientific panel provides further guidance on subsequent handling. All verification testing should use only in vitro tests under BSL-4 containment. No consensus was reached on what containment/ release recommendations should be made if all life detection and biohazard tests are negative. Additional discussion will be needed to translate the various test outcomes into specific recommendations for release of unsterilized materials from containment.
Biohazard Research and Technology Needs: Specific recommendations for R&D related to biohazard testing were identified in the following areas:
- Validation of methodological approach (cell and tissue test rather than whole organisms studies; pre-testing of efficacy; techniques for characterizing any isolated or suspected life forms etc.)
- Microcosm Research (development, effectiveness; predictive value; non-destructive, long-term observation and sampling, etc.)
- Representative samples, controls and replicates
- Other operational issues (training and monitoring programs for lab personnel; management of lab operations and facilities; issues related to limited quantities of material, sample allocation, research access, and evaluation of research proposals).
Five separate interim reports provide the archival record of discussions
and deliberations associated with the Mars Sample Handling Protocol Workshop
Series, which took place between March 2000 and June 2001. The stated
objective for the Workshop Series was: “For returned Mars samples, develop
a recommended list of comprehensive tests, and their sequential order,
that will be performed to fulfill the NRC recommendations that ‘rigorous
analyses determine that the materials do not contain any biological hazards.’”
The workshop series ultimately led to the development of a Working Draft Protocol, which was later reviewed and revised to become the Draft Test Protocol for Detecting Possible Biohazards in martian Samples Returned to Earth (NASA/CP-2002-211842). Each workshop began with a set of background tutorials, followed by breakout sub-groups that deliberated on various assigned topics, and finally by plenary presentation and discussion of findings.
NOTE: Sub-group findings from the workshop series do not represent stand-alone recommendations, but rather the incremental information used as input towards the development of the overall Draft Protocol. An overview of the workshop documents may be gleaned from the subgroup topics discussed at each of the five workshops.
Workshop 1 Subgroup Topics:
- Preliminary Sample Characterization Requirements
- Representative Sub Samples: Physical-Chemical Analyses (2 groups)
- Sequence And Types Of Tests; Range Of Results And Release Criteria
- Candidate Life Detection Tests-Qualifiers, Contraindications, Controls And Characterization
- Candidate Biohazard Tests : Qualifiers, Contraindications, Controls And Characterization
Workshop 2 Subgroup Topics:
- Life Detection
- Biohazard Testing (2 groups)
- Physical and Chemical Tests
- Molecular Biological Tests
- Organism- and Cellular-Based Tests
Workshop 2a (Sterilization) Subgroup Topics:
- Survival Mechanisms of Terrestrial Extremophiles As Models For Resistance To Sterilization
- Worst Case Scenario of Sterilizing martian Samples
- Sterilization Methods And Procedures To Preserve Sample Integrity For Future Scientific Research
- If No Carbon Or Polymers Found, Is Sterilization Necessary Prior To Sample Distribution?
- Can martian Samples Be Sterilized Effectively and Safely Distributed Outside Containment Facility Prior To Completion Of Life Detection and Biohazard Tests and Other Chemical Analyses? (2 groups)
- Can Sub-Samples Of Preserved martian Meteorites Serve As Models To Test Sterilization Methods, Procedures, And Effectiveness?
- What Are Effective Sterilization Methods For Samples Returned From Mars? (2 Groups)
Workshop 3 Subgroup Topics:
- Unifying Properties of Life
- Morphological Organization and Chemical Properties of Life
- Geochemical and Geophysical Properties of Life
- Chemical Methods for Life Detection
- Cell Biology Methods for Life Detection
- Additional Topic: What if Life is Detected?
Workshop 4 Subgroup Topics:
- Review And Assess The Draft Protocol For Physical/Chemical Testing
- Review And Assess The Draft Protocol For Life Detection Testing
- Review And Assess The Draft Protocol For Biohazard Testing
- Environmental And Health/Monitoring And Safety Issues
- Requirements Of The Draft Protocol For Facilities And Equipment
- Contingency Planning For Different Outcomes Of The Draft Protocol
- Personnel Considerations In Implementation Of The Draft Protocol
- Draft Protocol Implementation Process And Update Concepts
- Plenary Discussion on R&D needs for implementing the protocol
The Workshop 4 report also includes the Working Draft Protocol in Appendix A, the first consensus compilation of a proof-of-concept protocol developed during the workshop series.