What document provides the guidelines for working with infectious diseases in animal research?
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Recommendations of a CDC-convened, Biosafety Blue Ribbon PanelPlease note: An erratum has been published for this article. To view the erratum, please click here. J. Michael Miller, PhD1 Rex Astles, PhD2 Timothy Baszler, DVM, PhD3 Kimberle Chapin, MD4 Roberta Carey, PhD1 Lynne Garcia, MS5 Larry Gray, PhD6 Davise Larone, PhD7 Michael Pentella, PhD8 Anne Pollock, MT1 Daniel S. Shapiro, MD9 Elizabeth Weirich, MS1 Danny Wiedbrauk, PhD10 1National Center for Emerging and Zoonotic Infectious Diseases, CDC 2Laboratory Science, Policy and Practice Program Office, CDC 3College of Veterinary Medicine, Washington State University, Pullman, WA 4Lifespan Academic Medical Centers, Providence, RI 5LSG and Associates, Santa Monica, CA 6TriHealth Laboratories, Cincinnati, OH 7Weill Medical College of Cornell University, New York, NY 8University of Iowa Hygienic Laboratory, Iowa City, IA 9Lahey Clinic, Burlington, MA 10Warde Medical Laboratory, Ann Arbor, MI The material in this report originated in the National Center for Emerging and Zoonotic Infectious Diseases, Beth P. Bell, MD, MPH, Director. Corresponding preparer: J. Michael Miller, PhD, Microbiology Technical Services, LLC, Dunwoody, GA 30338. Telephone: 678-428-6319; Fax: 770-396-0955; E-mail: . Summary Prevention of injuries and occupational infections in U.S. laboratories has been a concern for many years. CDC and the National Institutes of Health addressed the topic in their publication Biosafety in Microbiological and Biomedical Laboratories, now in its 5th edition (BMBL-5). BMBL-5, however, was not designed to address the day-to-day operations of diagnostic laboratories in human and animal medicine. In 2008, CDC convened a Blue Ribbon Panel of laboratory representatives from a variety of agencies, laboratory organizations, and facilities to review laboratory biosafety in diagnostic laboratories. The members of this panel recommended that biosafety guidelines be developed to address the unique operational needs of the diagnostic laboratory community and that they be science based and made available broadly. These guidelines promote a culture of safety and include recommendations that supplement BMBL-5 by addressing the unique needs of the diagnostic laboratory. They are not requirements but recommendations that represent current science and sound judgment that can foster a safe working environment for all laboratorians. Throughout these guidelines, quality laboratory science is reinforced by a common-sense approach to biosafety in day-to-day activities. Because many of the same diagnostic techniques are used in human and animal diagnostic laboratories, the text is presented with this in mind. All functions of the human and animal diagnostic laboratory — microbiology, chemistry, hematology, and pathology with autopsy and necropsy guidance — are addressed. A specific section for veterinary diagnostic laboratories addresses the veterinary issues not shared by other human laboratory departments. Recommendations for all laboratories include use of Class IIA2 biological safety cabinets that are inspected annually; frequent hand washing; use of appropriate disinfectants, including 1:10 dilutions of household bleach; dependence on risk assessments for many activities; development of written safety protocols that address the risks of chemicals in the laboratory; the need for negative airflow into the laboratory; areas of the laboratory in which use of gloves is optional or is recommended; and the national need for a central site for surveillance and nonpunitive reporting of laboratory incidents/exposures, injuries, and infections. 1. Introduction: A Culture of Safety for Diagnostic LaboratoriesThis report offers guidance and recommends biosafety practices specifically for human and animal clinical diagnostic laboratories and is intended to supplement the 5th edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL-5), developed by CDC and the National Institutes of Health (1). This document was written not to replace existing biosafety guidelines, but to 1) improve the safety of activities in clinical diagnostic laboratories, 2) encourage laboratory workers to think about safety issues they might not previously have considered or addressed, and 3) encourage laboratorians to create and foster a culture of safety in their laboratories. Should any of the guidelines provided herein conflict with federal, state, or local laws or regulatory requirements, the laboratorian should defer to the federal, state, or local requirements. This culture of safety is also supported by the Clinical and Laboratory Standards Institute (2). Work in a diagnostic laboratory entails safety considerations beyond the biological component; therefore, these guidelines also address a few of the more important day-to-day safety issues that affect laboratorians in settings where biological safety is a major focus. According to the U.S. Bureau of Labor Statistics, in 2008, approximately 328,000 medical laboratory technicians and technologists worked in human diagnostic laboratories in the United States. An estimated 500,000 persons in all professions work in human and animal diagnostic laboratories. Any of these workers who have chronic medical conditions or receive immunosuppressive therapy would be at increased risk for a laboratory-acquired infection (LAI) after a laboratory exposure. Precise risk for infection after exposure is unknown because determining the source or the mode of transmission often is difficult. No national surveillance system is available. LAIs and exposures have been reported since early in the 20th century, but only in the 1970s were sufficient data available to attempt quantitative assessments of risk. Recent MMWR reports (3–11) have indicated that bacteria account for >40% of infections, with >37 species reported as etiologic agents in LAIs; however, other microbes are often implicated. Hepatitis B has been the most frequent laboratory-acquired viral infection, with a rate of 3.5–4.6 cases per 1000 workers, which is two to four times that of the general population. Any laboratorian who collects or handles tubes of blood is vulnerable (12). Early surveys of LAIs found that laboratory personnel were three to nine times more likely than the general population to become infected with Mycobacterium tuberculosis (13,14). In a 1986 survey of approximately 4000 workers in 54 public health and 165 hospital laboratories in the United States, 3.5/1000 employee infections occurred in hospital laboratories, and 1.4/1000 employee infections occurred in public health laboratories (15). In a 1994–1995 survey of 25,000 laboratory workers from 397 clinical laboratories in the United Kingdom, the overall rate of LAI was 18/100,000 employees (16). In a 2005 CDC study of bacterial meningitis in U.S. laboratorians, Neisseria meningitidis accounted for a substantial number of LAIs. The attack rate of this organism in the general population was 13/100,000 persons. The attack rate in the general population aged 30–59 years (the estimated age range of the average laboratorian) was 0.3 per 100,000. The attack rate for microbiologists (aged 30–59 years) was 20/100,000 (17). LAIs have also included fungal and parasitic infections. The most common agents of laboratory-acquired fungal infections are the dimorphic fungi Blastomyces, Histoplasma, and Coccidioides (18,19); most reported infections were caused by inhalation of conidia. Reported parasite-associated LAIs were caused primarily by Leishmania, Plasmodium, Toxoplasma, Chagas disease organism, and other trypanosomes (20). Of the 52 cases of laboratory-acquired malaria, 56% were vector borne (from mosquitoes used in research, not clinical laboratories). Most infected health-care workers acquired infection from needle sticks during preparation of blood smears or while drawing blood. In clinical chemistry laboratories, data from 17 New York hospitals listed needle puncture (103 cases), acid or alkali spills (46), glass cuts (44), splash in eye (19), and bruises and cuts (45) as the most frequent exposures (21). Needle puncture, glass cuts, splash in eye, and bruises and cuts have the highest potential for infection from microbes. In the hematology laboratory, the major causes of injuries are likely to be exposure to blood and body fluids; needle sticks, aerosols from centrifuge or removal of tube stoppers, tube breakage; or contaminated gloves (22). In non-microbiology sections of the diagnostic laboratory, the primary mistake may be assuming that a given specimen contains no infectious agents and then working with little attention to risk for infection. This scenario can be particularly problematic in laboratories developing new technologies, such as molecular and biochemical technologies, and in point-of-care diagnostics performed by staff unaccustomed to testing that requires biosafety considerations and use of barrier techniques such as personal protective equipment. 1.1. MethodsThe risks and causes of LAIs have been documented. However, there is a dearth of evidence-based research and publications focused on biosafety; particularly missing are studies documenting safe practices in the day-to-day operations of diagnostic laboratories. In 2008, CDC convened a Blue Ribbon Panel of laboratory representatives from a variety of agencies, laboratory organizations, and facilities to review laboratory biosafety in diagnostic laboratories. Members of the panel were either selected by the invited national laboratory organization they represented or were invited by CDC because of their roles in biosafety at the national level. The organizations participating in the panel represented the majority of laboratory technologists in the United States. In addition, some members of the panel were representatives of the biosafety community. The Blue Ribbon Panel recommended that biosafety guidelines be developed to address the unique operational needs of the diagnostic laboratory community and that they be science based and made available broadly. Panel members reviewed the guidelines that were developed and synthesized by the writing team. Official endorsements by the organizations they represented were not required, although each representative was required to submit written approval of the recommendations. Edits and comments from each participant were carefully considered and incorporated where appropriate. The guidelines provided herein are synthesized and supported from systematic reviews of peer-reviewed publications of evidence-based data from which recommendations could be made, justifying common-sense approaches that should be articulated, and where safe procedures have been described and proven. Because of the lack of evidence-based research in much of the current literature on biosafety practices, no attempt was made to weight the evidence and resulting recommendations (i.e., strong or weak). In the absence of supporting evidence-based research and documentation, some recommendations are based on expert opinion by international experts in the field of microbiology and must be appropriately applied until evidence-based research can substantiate their validity. The authors reviewed and approved their own sections and also evaluated how their topics accurately reflected and supported the goals of the entire document. Each section of recommendations was reviewed both within CDC and by the relevant national organizations whose members would embrace these guidelines. These included the College of American Pathologists, Greater New York Hospital Association Regional Laboratory Task Force, American Society for Microbiology, American Clinical Laboratory Association, Association of Public Health Laboratories, American Society for Clinical Laboratory Science, American Society for Clinical Pathology, American Biological Safety Association, American Association of Veterinary Laboratory Diagnosticians, and individual physicians and subject matter experts. Future research in biosafety practices in the laboratory will contribute to further recommendations and will substantiate others as well as provide opportunities to revise this document. 1.2. RiskPersons working in clinical diagnostic laboratories are exposed to many risks (1). Whether the patients are humans or animals and whether laboratorians work in microbiology or elsewhere in the laboratory, the human and animal diagnostic laboratory is a challenging environment. The more that laboratorians become aware of and adhere to recommended, science-based safety precautions, the lower the risk. The goal of a safety program is to lower the risk to as close as possible to zero, although zero risk is as yet unattainable as long as patient specimens and live organisms are manipulated. Protection of laboratorians, coworkers, patients, families, and the environment is the greatest safety concern. 1.3. Laboratory ExposuresLaboratory exposures occur more often than is generally suspected. Other laboratory incidents such as minor scrapes or cuts, insignificant spills, or unrecognized aerosols occur even more frequently and might not cause an exposure that results in an LAI. In this report, "laboratory exposures" refer to events that put employees at risk for an LAI and events that result in actual acquisition of LAIs. Except for reporting requirements imposed by CDC's Select Agent Program, which deals with handling of specific, potentially hazardous biological agents and toxins, no national surveillance system is in place to which medical laboratory exposures and subsequent work-related infections are reported. Increased attention has been focused on laboratory biosafety and biosecurity since 2001 but has been largely limited to precautions required for agents of bioterrorism. Other laboratory exposures and LAIs continue to occur, almost always because of a breakdown of established safety protocols. Because of the lack of an official surveillance mechanism for reporting LAIs and because of the fear of punitive action by an oversight agency if injuries are reported, the data needed to determine the extent and cause of LAIs are unavailable. In addition, there is a dearth of science-based insights on prevention of LAIs. The Blue Ribbon Panel recognizes the need for a voluntary, nonpunitive surveillance and reporting system with the potential for anonymity to be implemented in the United States. Such a system would allow for reporting and evaluation of all LAIs and would potentially lead to training and interventions to facilitate a negligible incidence rate. 1.4. Routes of Laboratory InfectionThe five most predominant routes of LAIs are
The first four routes are relatively easy to detect, but they account for <20% of all reported LAIs (23,24). No distinguishable exposure events were identified in approximately 80% of LAIs reported before 1978 (24–26). In many cases, the only association was that the infected person worked with a microbiological agent or was in the vicinity of a person handling a microbiological agent. The inability to identify a specific event was also reported in a more recent study (27), which found that the probable sources of LAIs were apparent in only 50% of cases. These data suggest that unsuspected infectious aerosols can play a large role in LAIs (1,23,24,28). 1.5. A Culture of SafetyThe concept of a "culture of safety," as described in this report, encourages all human and animal diagnostic laboratories to promote an organizational culture of systematic assessment of all work processes and procedures to identify associated risks and implement plans to mitigate those risks. In addition to the often unknown biohazard risk associated with handling diagnostic specimens, each section of the diagnostic laboratory has procedures and processes for handling known infectious agents that convey excessive risk for exposure and possible infection and/or occupational injury. These risks typically are associated with design flaws or lack of or inadequacy of safety procedures and training (1,2). In addition, the day-to-day operations of a human or animal diagnostic laboratory differ markedly from those of an academic or research laboratory and require different biosafety guidelines; these differences prompted the focus of this report on medical laboratory communities, their occupational risks, potential for exposure, and opportunities to mitigate those risks. Successful establishment of a culture of safety requires that laboratory safety become an integral and apparent priority to the organization, embraced first and foremost by top management and with the concomitant infrastructure support required to foster safe behaviors among its employees (29–31). As required by the Clinical Laboratory Improvement Amendments, the College of American Pathologists, and other accrediting agencies, a laboratory director needs to assume the responsibility for
1.6. Laboratory Design and Architectural Planning for MicrobiologyLaboratory design is fundamental to the safety of laboratory workers, hospital staff, and patients. The Clinical and Laboratory Standards Institute document, Laboratory Design; Approved Guideline (32), discusses laboratory design in detail. Because remediating poorly designed laboratory workspace is difficult, or even impossible, design warrants careful planning and consideration of safety issues. The following are suggestions to consider in the design or renovation of the diagnostic laboratory. Although there is no national standard requirement for an amount of space per person working in the laboratory, 300–350 sq. ft/person within a laboratory department is a reasonable figure to provide a safe work area. Ideally, allow a minimum 5-foot space between the worker (at a laboratory chair) and any object behind the worker to provide reasonable maneuverability.
2. Biological Risk Assessment and Biosafety Guidelines2.1. Risk AssessmentThe laboratory director is ultimately responsible for identifying potential hazards, assessing risks associated with those hazards, and establishing precautions and standard procedures to minimize employee exposure to those risks. Because the identity of an infectious agent is initially unknown in the clinical laboratory, the general recommendation is that the biosafety level (BSL)-2 standard and special practices in Biosafety in Microbiological and Biomedical Laboratories, 5th edition (1) be followed for all work in the clinical laboratory, and the Occupational Safety and Health Administration's (OSHA's) Standard Precautions (gloves, gowns, and protective eyewear) (33) and BSL-2 practices (2) be employed during handling of all blood and body fluids. Other comprehensive resources are available (34,35). Risk assessment, as outlined here and in Section 12, may determine that decreasing or increasing the BSL practices or facilities is warranted (Figure 1). Qualitative biological risk assessment is a subjective process that involves professional judgments. Because of uncertainties or insufficient scientific data, risk assessments often are based on incomplete knowledge or information. Inherent limitations of and assumptions made in the process also exist, and the perception of acceptable risk differs for everyone. The risk is never zero, and potential for human error always exists. Identifying potential hazards in the laboratory is the first step in performing a risk assessment. Many categories of microbiological hazards are encountered from the time a specimen is collected until it is disposed of permanently. A comprehensive approach for identifying hazards in the laboratory will include information from a variety of sources. Methods to ascertain hazard information can include benchmarking, walkabouts, interviews, detailed inspections, incident reviews, workflow and process analysis, and facility design. No one standard approach or correct method exists for conducting a risk assessment; However, several strategies are available, such as using a risk prioritization matrix, conducting a job hazard analysis; or listing potential scenarios of problems during a procedure, task, or activity. The process involves the following five steps:
Standardization of the risk assessment process at an institution can greatly improve the clarity and quality of this process. Training staff in risk assessment is critical to achieving these objectives. 2.1.1. Step 1. Identify the hazards associated with an infectious agent or material.
2.1.2. Step 2. Identify activities that might cause exposure to the agent or material.
2.1.3. Step 3. Consider the competencies and experience of laboratory personnel.
2.1.4. Step 4. Evaluate and prioritize risks. Risks are evaluated according to the likelihood of occurrence and severity of consequences (Table 2).
2.1.5. Step 5. Develop, implement, and evaluate controls to minimize the risk for exposure.
2.2. Principles of Biosafety (1)2.2.1. Containment "Containment" describes safe methods for managing infectious materials in the laboratory to reduce or eliminate exposure of laboratory workers, other persons, and the environment.
2.2.2. Biosafety Levels (Table 4)BSLs provide appropriate levels of containment needed for the operations performed, the documented or suspected routes of transmission of the infectious agent, and the laboratory function or activities. The four BSLs, designated 1–4, are based on combinations of laboratory practice and techniques, safety equipment (primary barriers), and laboratory facilities (secondary barriers). Each BSL builds on the previous level to provide additional containment. Laboratory directors are responsible for determining which BSL is appropriate for work in their specific laboratories.
2.3. Material Safety Data Sheets for Organisms and ChemicalsMaterial Safety Data Sheets (MSDS) for chemicals are available from the manufacturer, supplier, or an official Internet site. The Division of Occupational Health and Safety, National Institutes of Health, has promulgated guidelines for handling genetically manipulated organisms and has additional instructions for accessing MSDS (http://dohs.ors.od.nih.gov/material_safety_data_main.htm). 2.4. Biosafety Manual
3. Fundamental Safety Practices in Diagnostic LaboratoriesMany safety procedures, guidelines, and principles apply to all sections of the diagnostic laboratory. The recommendations presented in this section represent a broad view of safety throughout the laboratory. More detailed recommendations can be found in Biosafety in Microbiological and Biomedical Laboratories (BMBL-5) and in the World Health Organization's Laboratory Biosafety Manual (1,36). Hospitals, clinical laboratories, state and local health departments, CDC, and the American Society for Microbiology have established and/or published guidelines to follow when suspected agents of bioterrorism have been or could be released in the community. However, routine clinical laboratory testing may provide the first evidence of an unexpected bioterrorism event. Routine clinical specimens also may harbor unusual or exotic infectious agents that are dangerous to amplify in culture. These agents are often difficult to identify, and the routine bench technologist might continue work on the culture by passage, repeated staining, nucleic acid testing, neutralization, and other methods. This continued workup places the technologist and others in the laboratory at risk for infection. Ideally, these specimens are not to be processed or tested in the routine laboratory, and they can be removed from the testing stream if the suspected agent is known. Relationships with the state public health laboratory, and subsequently with the Laboratory Response Network, are critical in this effort. Once the testing process has begun, the bench technologist must have clear and concise instructions about when to seek assistance from the laboratory supervisor and/or director. 3.1. Specimen Receiving and Log-In/Setup Station
3.1.1. Leaking containers
3.1.2. Visible contamination of the outside of containers
3.1.3. Loose caps
3.1.4. Operational procedures
3.1.5. Manual removal of sealed caps; specimen aliquotting and pipetting
3.1.6. Pneumatic tube systems
3.2. Personal Precautions.If engineering controls are in place to prevent splashes or sprays, the requirement for PPE can be modified on the basis of a risk assessment and evidence of the effectiveness of the engineering control to prevent exposure from splashes or sprays. Examples of engineering controls include use of a BSC, having sealed safety cups or heads in centrifuges, and negative air flow into the laboratory. 3.2.1. Work at the open bench
CDC continues to recommend that sniffing culture plates should be prohibited. Isolates of small gram-negative or gram-variable rods (e.g., gram-negative coccobacilli) should be manipulated within a BSC.
3.2.2. Personal protective equipment Engineering controls (2.1.5. Step 5) should always be the first line of defense to minimize exposures. PPE includes a variety of items, such as gloves, laboratory coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, and goggles, that are designed to protect the laboratory worker from exposure to physical, biological, and chemical hazards. Distributing PPE to each employee as needed helps to ensure access to appropriate PPE. PPE is often used in combination with BSCs and other devices that contain the agents or materials being handled. In some situations where working in a BSC is impractical, PPE, including splash shields, may form the primary barrier between personnel and hazardous materials (1). (See Section 3.1). The Occupational Safety and Health Administration (OSHA) defines PPE as "appropriate" if it does not permit blood or other potentially infectious materials to pass through or reach the employee's street clothes, undergarments, skin, eyes, mouth, or other mucous membranes under normal conditions of use (33).
3.3. Biological Safety Cabinet
3.4. Disinfection3.4.1. Good work practices
3.4.2. Bleach solutions (sodium hypochlorite) (38)
3.5. Waste ManagementA clinical laboratory must establish a waste management plan.
3.5.1. Decontamination of medical waste before transport and disposal
3.5.2. Management of discarded cultures and stocks
3.5.3. Discarding a select agent
3.5.4. Autoclave safety
3.6. Dry Ice3.6.1. General information Under certain circumstances, dry ice can be an explosion hazard. Dry ice is solidified carbon dioxide (CO) and it is extremely cold (-109° F [-79° C]). Unlike water-ice, dry ice sublimates (changes directly from solid to gas) as it warms, releasing CO gas. CO vapor is considerably heavier than air; in confined, poorly ventilated spaces, it can displace air, causing asphyxiation.
3.6.2. Disposal of dry ice
3.7. Electrical Safety
3.8. Gases in the Laboratory: Compressed Gas CylindersCompressed CO cylinders are often used to provide gases for CO incubators; the risks associated with these incubators are minimal as long as the room is well ventilated. 3.8.1. Hazards
3.8.2. Minimizing hazards Many of these potential hazards can be minimized by adoption of safe handling practices.
3.9. Liquid Gases (Cryogens)Cryogenic liquids are liquefied gases that have a normal boiling point below -238°F (-150°C). Liquid nitrogen is used in the microbiology laboratory to freeze and preserve cells and virus stocks. The electron microscopy laboratory, frozen section suites, and grossing stations for surgical pathology frequently use liquid nitrogen; some laboratories also use liquid helium. The principal hazards associated with handling cryogenic fluids include cold contact burns and freezing, asphyxiation, explosion, and material embrittlement. 3.9.1. Cold contact burns and freezing
3.9.2. Asphyxiation hazards
3.9.3. Explosion hazards
3.9.4. Cryotube explosions
3.9.5. Embrittlement
3.9.6. Infectious disease hazards
3.10. Slip, Trip, and Fall HazardsSlips, trips, and falls can cause a laboratory worker to drop or spill vessels containing infectious agents or dangerous chemicals. They can also lead to skin punctures and abrasions that make laboratory workers more vulnerable to LAIs. Good housekeeping is the most fundamental means for reducing slips, trips, and falls. Without good housekeeping, any other preventive measures (e.g., installation of sophisticated flooring, specialty footwear, or training on techniques of walking and safe falling) will never be fully effective. 3.10.1. Slips
3.10.2. Trips
3.11. Ultralow-Temperature FreezersWear thermally resistant gloves and a laboratory coat when handling items stored at ultralow temperatures. Specimens stored at ultralow temperatures are extremely cold [-70°C to -85°C]), and paradoxically, direct contact with the skin can cause severe burns. 3.12. Ultraviolet light
3.13. Vacuum devicesVacuum-assisted filtration devices and side-arm suction flasks are used routinely in the general laboratory, whereas the electron microscopy laboratory uses vacuum-assisted evaporators, freeze-driers, freeze-fracture, and sputter coater units. Vacuum-assisted devices present implosion hazards and risk aerosol generation. 3.13.1. Implosion safety
3.13.2. Aerosol generation
3.13.3. Aerosol protection measures
3.13.4. Disposal of liquid wastes from vacuum-assisted aspiration traps
3.14. Biological Hazards3.14.1. Punctures and cuts Skin punctures and cuts can directly introduce an infectious agent into the body and can provide a route whereby a secondary agent can enter.
3.14.2. Ingestion and contact with infectious agents
3.14.3. Spills and splashes onto skin and mucous membranes
3.14.4. Aerosols and droplets Any procedure that imparts energy to a microbial suspension can produce infectious aerosols (1,23). Procedures and equipment frequently associated with aerosol production include pipetting, mixing with a pipette or a vortex mixer, and use of blenders, centrifugation, and ultrasonic devices (sonicators) (1,23,47). These procedures and equipment generate respirable particles that remain airborne for protracted periods. When inhaled, these tiny particles can be retained in the lungs. These procedures and equipment also generate larger droplets that can contain larger quantities of infectious agents. The larger droplets settle out of the air rapidly, contaminating, work surfaces as well as the gloved hands and possibly the mucous membranes of persons performing the procedure. Respirable particles are relatively small and do not vary widely in size distribution. In contrast, hand and surface contamination is substantial and varies widely (1,48). The potential risk from exposure to larger-size droplets requires as much attention in a risk assessment as the risk from respirable particles.
3.15. Ultrasonic Devices
3.16. Clean versus Dirty Areas of the LaboratoryIn the microbiology laboratory, all the technical work areas of the department are considered dirty. The same concepts of demarcation and separation of molecular testing areas that are described in this section can be used to establish clean and dirty areas in other parts of the diagnostic laboratory. 3.16.1. Clean areas
3.16.2. Offices Offices (e.g., of supervisors and laboratory director) that open into the clinical laboratory represent hybrid areas within the laboratory. These offices are not typically designed or maintained in a manner that allows for easy or efficient disinfection.
3.16.3. Dirty areas
3.17. InstrumentationWhether automated or manual, procedures with the potential for producing specimen aerosols and droplets (e.g., stopper removal, vortexing, opening or piercing evacuated tubes, using automatic sample dispensers) require PPE and engineering controls designed to prevent exposure to infectious agents. 3.17.1. Water baths and water (humidification) pans in CO incubators
3.17.2. Centrifuges and cytocentrifuges
3.17.3. Automated analyzers
3.17.4. Vacuum-assisted aspiration devices (See Section 3.13.) 3.17.5. ELISA plate washers in microbiology
3.17.6. Identification, blood culture, and PCR instruments Bacterial identification and antimicrobial susceptibility instruments, blood culture instruments, PCR instruments, and other laboratory instruments and devices are to be cleaned or disinfected according to the manufacturer's directions or recommendations. The routine and emergency cleaning procedure for each instrument must be a part of the safety component of the procedure manual. 3.18. Rapid Tests (Kits)
3.19. Unidirectional Work Flow and Separation of Work Areas
4. Tuberculosis LaboratoryTuberculosis (TB) resulting from exposure to infectious aerosols remains a major risk for laboratorians. There is no safe level of exposure since exposure to as few as 1–10 organisms can cause disease. An estimated 8%–30% of laboratorians may experience tuberculin conversions (52). To reduce exposures to Mycobacterium tuberculosis, a hierarchy of controls must be employed, including safe work practices, use of containment equipment, and specially designed laboratory facilities (1). Tuberculosis laboratories need to be separate and isolated from the main microbiology laboratory. Develop all policies and practices related to safety using a risk assessment process that is documented in the laboratory's biosafety manual.
4.1. Specimen Receiving and Log-In/Setup StationIn most clinical laboratories specimens are first received in the main microbiology laboratory (biosafety level [BSL]-2), where they are logged in and processed for other bacteriologic testing. The specimens submitted for TB analysis are moved to the TB laboratory for further processing specific for TB. 4.1.1. Specimen receiving in the main microbiology laboratory
4.1.2. Specimen receiving in other laboratory sections
4.1.3. Leaking containers
4.1.4. Visible contamination on the outside of container
4.2. Stains and DisposalPrepare smears in a BSC because aerosols, droplets and splatters can be generated. Unstained smears may contain viable tubercle bacilli and are to be handled with caution. 4.2.1. Gram stain Specimens submitted for routine cultures, especially sputum and other respiratory specimens, may contain tubercle bacilli and must be handled with care regardless of whether or not acid-fast bacillus (AFB) cultures were ordered. 4.2.2. Acid-fast stains — Kinyoun, Ziehl-Neelsen, auromine–rhodomine (fluorescent)
4.3. Culture Reading and Acceptable Activities at the Open Bench
4.4. Personal Precautions and Work PracticesPrecautions and work practices are selected with regard to the potential quantity of tubercule bacilli encountered in the procedure being performed. Hence, specimens have a lower concentration than a culture, in which the number of organisms is amplified. Because aerosols are generated whenever energy is imparted into the specimen, all protocols in the TB laboratory are evaluated through the risk assessment process for the potential to generate aerosols. Common aerosol-generating procedures are pouring liquid cultures and supernatant fluids, using fixed-volume automatic pipetters, and mixing liquid cultures with a pipette.
4.4.1. Personal protective equipment
4.4.2. Respiratory protection
4.5. Disinfection
4.6. Decontamination and Disposal of Laboratory Waste
4.7. Spill Cleanup
4.8. Clean versus Dirty Areas of the Laboratory
4.9. AFB Blood Cultures
4.10. Instrumentation
4.11. Testing4.11.1. Rapid testing (direct molecular test kits)
4.11.2. Molecular testing
5. Autopsy/Necropsy, Surgical Pathology
5.1. Autopsy/Necropsy–Associated InfectionsThe source of most laboratory-acquired infections and hazardous exposures that occur during autopsy/necropsy is unknown, and all autopsies and necropsies are to be considered risky (1,56).
5.1.1. Bloodborne pathogens Human-health–care workers involved in performance of autopsies are at high risk for occupationally acquired bloodborne pathogens because of both the injuries sustained and the population undergoing autopsy. Transmission risk is highest per exposure for hepatitis B virus, then hepatitis C virus and human immunodeficiency virus, respectively. These infections have been documented from autopsies as well as during embalming (1,2,56,60–62). 5.1.2. Other infections Specific data for other bloodborne pathogens, such as cytomegalovirus, are lacking, but infectious transmission is possible and risk may be higher especially for pregnant (serologically negative) or immunocompromised workers. Assess persons at higher risk for infection on a case-by-case basis and allow them to consent to participating in the autopsy only after being counseled (2,63). 5.1.3. Infectious aerosols Autopsies/necropsies of cadavers with suspected zoonotic agents generate potentially infectious aerosols. Although Mycobacterium tuberculosis is the prototypical pathogen most noted to be transmitted by aerosolization, persons who had meningococcemia, anthrax, rickettsiosis and legionellosis are other examples. Manipulation of infectious tissue can result in both airborne particles in a size (<5 µm) that floats on air currents for extended periods and can subsequently reach the pulmonary alveoli and small-droplet particles (>5 µm) that settle more quickly. Contamination may occur from fluid-aspirating hoses, from spraying the cadaver, and from oscillating saws. The aerosols created stay within the autopsy area and can result in subsequent contact with mouth and eyes, inhalation, or ingestion and can contaminate inanimate surfaces such as computers, telephones and camera equipment (56,57). 5.1.4. Organisms that require additional safety practices
5.1.5. Other biosafety exposures
5.1.6. Reporting to the mortician Report known bloodborne pathogens or other suspected aerosolization danger to the mortician and others potentially handling the body to limit subsequent transmissions that may occur during transport or embalming (69). 5.1.7. Necropsy remains of animals Dispose of animal cadavers with potential zoonotic infectious agents by appropriate decontamination (e.g., incineration, alkaline digestion or other methods), and do not return them to animal owners for private burial. 5.2. The Autopsy/Necropsy Suite5.2.1. Inspect the body/carcass
5.2.2. Safety guidelines for the suite
5.3. Chemicals (Formaldehyde)Formaldehyde (3.7%–4.0%) used for specimen preservation is the most common toxic chemical to which autopsy workers are exposed. The chemical is volatile and toxic and causes irritation to the eyes, mucous membranes, and skin and is associated with increased risk for all cancers. Occupational Safety and Health Administration (OSHA) regulations specify an exposure limit of 0.75 ppm as an 8-hour time-weighted average, and 2.0 ppm for short-term (15-minute) exposures (70). If formaldehyde can be detected by smell, it likely means exposure is occurring at a concentration beyond acceptable limits. Limit exposure to formaldehyde in the following manner.
5.4. Spills
5.5. Protective Equipment5.5.1. Safety equipment
5.5.2. PPE for autopsy/necropsy personnel
5.6. Disinfection and Cleaning Procedures for Equipment and Instruments5.6.1. Human autopsy
5.6.2. Human autopsy/animal necropsy The following guidelines for disinfection and cleaning following an autopsy or necropsy apply to both types of procedures.
5.7. Waste management5.7.1. Human tissue Either incinerate all pathological waste, since this is considered hazardous material and is regulated by the U.S. Department of Transportation (DOT), or transport pathological waste to on-site or off-site treatment facilities in clearly labeled, dedicated, leakproof containers or carts that meet DOT requirements. DOT sharps waste containers need to be puncture-proof in addition to meeting these requirements. State, local, and regional regulations may also apply and need to be addressed. 5.7.2. Animal tissue Dispose of all animal necropsy waste (tissues or postnecropsy cadaver) using an appropriate method as determined by the case-by-case risk analysis assessment (incineration, autoclaving and standard waste disposal, rendering, composting, cremation, private burial). 5.7.3. Other waste Shred autoclave red-bag waste if appropriate. State, local, and regional regulations may also apply and need to be addressed. (See Section 3.5, Waste Management.) 5.8. Clean versus Dirty AreasClean areas might include an administrative area and bathrooms with showers. Air from these areas should be exhausted differently than from the autopsy suite (56,76,77). All other areas are considered dirty, and appropriate PPE is required. 5.9. Surgical Pathology
5.9.1. Specimen receiving and log-in
5.9.2. Work at the open bench
5.9.3. Clean versus dirty areas of the laboratory All of the surgical pathology specialty areas (cytology, histology, grossing or frozen section rooms) are considered dirty areas if fresh specimens or body fluids are received or processed in an open room (not in a BSC or separately vented area). 5.9.4. Tissue stains Multiple staining procedures are performed in histology and cytology. The most common are included here. Some of these stains are prepared with ethanol and some with methanol, which can have an impact on management options for their waste. Provide material safety data sheets (MSDS) for each component in the laboratory.
5.9.5. Fixatives
5.10. Engineering Controls and Facility RenovationsWhen updating or renovating autopsy and other areas of the anatomic pathology laboratory that process fresh tissue and body fluids, the following should be considered.
5.11. Creutzfeldt-Jakob DiseaseSpecial precautions for autopsy and autopsy suite decontamination, brain-cutting, and histologic tissue preparation procedures are required when processing cases of possible CJD (1,56,65,67,86). 5.11.1. Autopsy Perform autopsies using BSL-2 precautions augmented by BSL-3 facility ventilation and respiratory precautions. Wear standard autopsy PPE. Limit the autopsy to brain removal. Restrict participants to only those who are necessary. Double-bag the brain and place it in a plastic container for freezing or fix it in 3.7%–4% formaldehyde after sectioning. Formaldehyde fixation occurs for 10–14 days before histologic sections are collected. 5.11.2. Histologic preparations
6. Parasitology LaboratoryExposure to infectious parasites during diagnostic procedures may result from handling specimens, drawing blood, performing various types of concentration procedures, culturing organisms, and conducting animal inoculation studies. Relevant parasites and their possible routes of infection are listed in Table 7 and Box 1. Table 8 contains information on resistance to antiseptics and disinfectants. 6.1. Specimen Receiving and Log-In/Setup Station
6.1.1. Leaking containers
6.1.2. Loose caps
6.2. Stains and Reagents (88,89)6.2.1. Trichrome stain
6.2.2. Hematoxylin stain
6.2.3. Iodine
6.2.4. Acid-fast stains (modified)
6.2.5. Giemsa stain
6.2.6. Wright stain
6.2.7. Formalin (HCHO)
6.2.8. Mercury-based fixatives
6.2.9. Zinc-based fixatives (containing formalin)
6.2.10. Copper-based fixatives (containing no formalin)
6.2.11. Xylene and alcohols
6.3. Working at the Bench
6.4. Personal Precautions6.4.1. Biological safety cabinet versus fume hood
6.4.2. Personal protective equipment
6.4.3. Immunization
6.4.4. Disinfection General recommendations for the microbiology laboratory are sufficient for use in the diagnostic parasitology section; these would include guidelines for disinfection of countertops, telephones, computers, equipment, and hands-free telephones. 6.5. Dirty versus Clean Areas of the LaboratoryGeneral guidelines for the microbiology laboratory also apply for the parasitology section of the laboratory. No special recommendations are necessary. 6.6. InstrumentationSafety requirements for the use of instruments are the same as those used for a general microbiology laboratory and are primarily involved with specimen handling. 6.7. Antibody and Antigen Parasitology TestingSafety requirements for antibody and antigen testing are the same as those used for a general microbiology or immunology laboratory and are primarily involved with specimen handling. 7. Mycology LaboratoryAlthough not a strict requirement, it is recommended that mycology laboratories that culture for filamentous fungi and manipulate those organisms be separate and isolated from the main microbiology laboratory with negative air pressure moving into the room from the main laboratory. Direct access to a Class II biological safety cabinet (BSC) is critical for this activity whether mycology work is conducted in a separate room or in an isolated section of the main laboratory. Most mycology diagnostic work can be conducted in the biosafety level (BSL)-2 laboratory. 7.1. Specimen Receiving and Log-In/SetUp Station7.1.1. Leaking containers Guidelines for the general microbiology laboratory apply also for the mycology laboratory. No special recommendations are necessary. 7.1.2. Visible contamination on outside of container Guidelines for the general microbiology laboratory apply also for the mycology laboratory. No special recommendations are necessary. 7.1.3. Loose caps Guidelines for the general microbiology laboratory apply also for the mycology laboratory. No special recommendations are necessary. 7.2. Stains and Disposal7.2.1. Gram stain The Gram stain is not the optimum stain for fungus, but if used particularly for yeast, the same guidelines that apply to bacteriology/clinical microbiology are followed for mycology. 7.2.2. Mycology stains
7.3. Culture Reading at the Bench (1,113)
7.4. Personal Precautions7.4.1. Biosafety cabinet
7.4.2. Personal protective equipment
7.4.3. Disinfection Recommendations for the general microbiology laboratory are sufficient for use in the mycology laboratory; these include guidelines for disinfection of countertops and items such as telephones, computers, equipment, and hands-free telephones. 7.4.4. Decontamination and disposal of laboratory waste
7.5. Clean versus Dirty Areas of the LaboratoryGuidelines for the general microbiology laboratory apply also for the mycology laboratory. 7.6. Select Agents and Pathogenic Moulds
7.7. Blood Culture Bench
7.8. InstrumentationInstruments used for mycology studies are most commonly those for continuously monitored blood culture and for yeast identification. Follow the same guidelines that apply to bacteriology/clinical microbiology. 7.9. Rapid Testing (Kits)
7.10. Molecular TestingFollow the clinical microbiology safety guidelines for mycology with the additional advisory that mold isolates must be handled in a BSC during extraction of nucleic acids. 8. Virology Laboratory8.1. Specimen Processing and Log-In Bench8.1.1. Biohazards associated with specimen receiving and log-in The clinical virology laboratory receives a wide variety of clinical specimens for virus detection. Because the infectious nature of this material is largely unknown, special care must be taken to prevent contamination of personnel, the environment, and other clinical specimens.
8.1.2. Leaking containers
8.1.3. Visible contamination on outside of container Specimens with a small amount of contamination (e.g., a dried blood spot) on the outside of the container are to be brought to the attention of the laboratory director. The director can examine the specimen and determine if it is suitable for testing and whether it constitutes a hazard to laboratory personnel. 8.1.4. Special precautions for suspicious specimens
8.2. Stains, Chemicals, and Disposal
8.2.1. Alcohols Ethanol, methanol, isopropyl alcohol, and alcohol blends are used in the virology laboratory to fix cells, for nucleic acid extraction and precipitation, and as a disinfectant.
8.2.2. Antibiotics Antibiotics in routine use include penicillin, streptomycin, gentamicin, ciprofloxacin, kanamycin, tetracycline, amphotericin B, and neomycin. These antibiotics can be found in culture media and viral transport media. Concentrated antibiotic mixtures are frequently used to increase the antibiotic concentrations in samples containing large numbers of bacteria or fungi. Concentrated antibiotic solutions can be purchased at 50 times (50×) and 100 times (100×) the working concentration. Although the risks associated with antibiotic preparation and use are relatively low in the virology laboratory, antibiotic preparation and handling has been associated with hypersensitivity reactions and contact dermatitus (115,116) and asthma (116–120) in hospital, pharmaceutical, and animal workers.
8.2.3. Bleach solutions (see 3.4.2) 8.2.4. Cycloheximide Cycloheximide is used as an antibiotic, protein synthesis inhibitor, and plant growth regulator. In the virology laboratory, cycloheximide is used in Chlamydia re-feed media.
Cycloheximide is inactivated by alkaline solutions (pH >7.0). Aspirating cycloheximide-containing culture fluids into vacuum traps containing a 1:10 bleach solution will inactivate the chemical. Most soaps and detergents are alkaline, and these agents will also inactivate cycloheximide. 8.2.5. Dimethyl sulfoxide Dimethyl sulfoxide (DMSO) is used as a cryoprotectant when freezing cell cultures. DMSO is a powerful solvent and can penetrate skin and latex gloves.
8.2.6. Electron microscopy stains, fixatives, and buffers
8.2.7. Electron microscopy embedding media (Meth)acrylates and epoxy-based materials are frequently used to embed biological samples for electron microscopy. Epoxy products include Epon, Araldite, Spurr resin, and Maraglas. Formvar (polyvinyl formal) is used as a support film for electron microscopy grids and for making replicas. Many of these compounds are toxic, carcinogenic or potentially carcinogenic and are known to cause skin irritation, dermatitis, and skin sensitization. Consult individual MSDS documents for more information.
8.2.8. Ethidium bromide Ethidium bromide (EtBr) is a DNA intercalating agent that is commonly used as a nonradioactive marker for visualizing nucleic acid bands in electrophoresis and other gel-based separations. EtBr is a potent mutagen, toxic after acute exposure, and is an irritant to the skin, eyes, mouth and the upper respiratory tract.
8.2.9. Evans blue Evans blue is used as a counterstain during fluorescence microscopy. Evans blue powders and solutions are skin irritants, but there is no known flammability, carcinogenicity, or teratogenicity warning associated with this compound.
8.2.10. Guanidinium solutions Guanidinium chloride, guanidinium thiocyanate, and guanidinium isothiocyanate are chaotropic agents used to disrupt cells and denature proteins (particularly RNases and DNases) during nucleic acid extraction procedures. These chemicals are strong irritants, and eye exposure can result in redness, irritation and pain. They are toxic if ingested and may cause neurologic disturbances. If inhaled, guanidinium compounds can cause respiratory tract irritation coughing, and shortness of breath.
8.2.11. Neutral red Neutral red is a pH indicator and a vital stain used in some plaque assays. It may be harmful if swallowed, inhaled, or absorbed through the skin and can cause irritation to the skin, eyes, and respiratory tract.
8.2.12. Merthiolate (thimerosal) Merthiolate, or thimerosal, is a mercury-containing antiseptic and antifungal agent used as a preservative in some laboratory solutions. Concentrated thimerosal is very toxic when inhaled, ingested, and in contact with skin.
8.2.13. Organic solvents
8.2.14. Sodium azide Sodium azide is a common preservative in many laboratory reagents, including monoclonal antibodies, buffers, and enzyme immunoassay reagents.
8.3. Handling Cell Cultures at the Bench
8.3.1. Cell lines
8.3.2. Cell culture practices Workers who handle or manipulate human or animal cells and tissues are at risk for possible exposure to potentially infectious latent and adventitious agents that may be present in those cells and tissues. CDC/National Institutes of Health recommended cell culture practices (1) include the following.
8.3.3. Biohazards associated with cell culture reading
8.3.4. Biohazards associated with liquid nitrogen use Liquid nitrogen can become contaminated when ampoules are broken in the dewar, and contaminants can be preserved in the nitrogen (23). These potentially infectious contaminants can contaminate other vials in the dewar and generate an infectious aerosol as the liquid nitrogen evaporates. Plastic cryotubes rated for liquid nitrogen temperatures are recommended for liquid nitrogen storage because they appear to be sturdier than glass ampoules and are less likely to break in the nitrogen. Glass ampoules are not recommended. Ampoules and cryotubes can explode when removed from liquid nitrogen creating infectious aerosols and droplets. See Section 3.9 for additional information. 8.4. Personal PrecautionsNo amount of safety engineering can reduce the physical, chemical, and biological risks in a laboratory environment if personal precautions are not employed consistently and rigorously. All laboratory workers and visitors are responsible for following established procedures regarding personal precautions. Directors and supervisors should periodically review their biosafety responsibilities (1). 8.4.1. Biological safety cabinet
8.4.2. Personal protective equipment
8.4.3. Disinfection Disinfection guidelines for the general microbiology laboratory are applicable to the virology laboratory (Section 3). 8.5. Decontamination and Disposal of Laboratory Waste
8.6. Clean versus Dirty Areas of the Laboratory
8.7. Early Recognition of High-Risk OrganismsRoutine clinical laboratory testing may provide the first evidence of an unexpected bioterrorism event, and routine clinical specimens may also harbor unusual or exotic infectious agents that are dangerous to amplify in culture. Early recognition of these possible high-risk organisms is critical, as is adherence to all fundamentals of laboratory safety. Events that require intervention by a supervisor or laboratory director are listed (Table 10). Although the majority of events are caused by inadvertent actions and pose no risk, laboratory technologists and directors should be aware that multiple high-risk causes are possible. How the laboratory responds to these trigger events will depend upon whether the laboratory has a BSL-3 facility and the capabilities of the state and local laboratory response network (LRN). 8.7.1. Fluorescent antibody testing bench
8.7.2. Suspicious or unusual results
8.7.3. Nucleic acid testing
8.8. Hazards Associated with the Electron Microscopy LaboratoryDiagnostic electron microscopy can be a relatively simple and rapid method for morphologic identification of agents in a specimen. Electron microscopy procedures can serve as a general screen to detect novel organisms or organisms that have altered genetic or immunologic properties that render them undetectable by nucleic acid or immunoassay protocols (192). Electron microscopy laboratories share many of the physical, chemical, and biological hazards described for the virology laboratory but also have some unique features. 8.8.1. Flammable and combustible liquids The electron microscopy laboratory uses a wide variety of flammable solvents, and the use of open flames is discouraged (see Section 8.2.13).
8.8.2. X-ray hazards The electron microscope will generate dangerous levels of X-rays within the microscope as high-energy electrons strike the metal components. Modern electron microscopes have sufficient shielding and lead-impregnated glass viewing ports that minimize dangers to the operator. However, modifications to the instrument, adding and removing accessories, and some maintenance procedures can compromise the shielding.
8.8.3. Electrical hazards See Section 3.7 for information regarding routine electrical safety in the electron microscopy laboratory.
8.8.4. Chemical hazards Several heavy metal stains and aggressive fixatives are used in the electron microscopy laboratory. See Section 8.2 and the MSDS materials provided by the manufacturers for guidelines for handling stains and fixatives. Embedding and filmmaking materials are chemical hazards, and many of these materials are dissolved in flammable organic solvents.
8.8.5. Cryogens and compressed gases The most commonly used cryogens used in the electron microscopy laboratory are liquid nitrogen and liquid helium. Compressed helium, CO and nitrogen are also used. Hazards and safety measures associated with these gases are summarized in Sections 3.8 and 3.9. 8.8.6. Specialized equipment The electron microscopy laboratory uses a number of specialized instruments whose use can be hazardous. For example, evaporators, freeze-driers, freeze-fracture, and sputter coater units use vacuum, and the vessels could implode. Implosion hazards are reviewed in Section 3.13.1.
8.8.7. Biological hazards The biological hazards of the electron microscopy laboratory are similar to those of the virology laboratory, and good laboratory practices must be followed.
8.9. Rapid Testing (Kits)Several FDA-approved, rapid immunodiagnostic tests for viral antigens and antibodies are available. Originally designed for point-of-care or near point-of-care testing, many of these tests are being used for testing in clinical virology laboratories. The following biosafety recommendations are based upon CDC biosafety guidance for handling clinical specimens or isolates containing 2009-H1N1 influenza A virus (194).
8.10. Molecular LaboratoryMolecular virology laboratories share many of the physical, chemical and biological hazards described for the virology laboratory, but they also present some unique hazards. 8.10.1. Electrical hazards See Section 3.7 for information regarding routine electrical safety in the molecular virology laboratory. Special high-voltage power sources are used in electrophoresis and nucleic acid sequencing equipment.
8.10.2. Ultraviolet light hazards
8.10.3. Chemical hazards The chemical hazards unique to the molecular virology laboratory include chloroform, ethidium bromide (Section 8.2.8) and guanidinium-based extraction reagents (Section 8.2.10). Avoid acute and long-term exposure to these. 8.10.4. Biological hazards The biological hazards in the molecular virology laboratory are similar to those of the virology laboratory, and good laboratory practices must be followed.
9. Chemistry LaboratoryAll specimens of human and animal origin tested by the chemistry, toxicology, or drug-testing laboratory may contain infectious agents. It is imperative to understand and minimize the risk of exposure to patient specimens through surface contact, aerosolization, or penetrating injury. Risk mitigation of laboratory-acquired infections is discussed in Sections 2 and 3. 9.1. Automated Analyzers (see also 3.17.3 and 10.6.3)Automated analyzers frequently have added features to help reduce operator exposures, but they do not totally eliminate the potential for exposure. A common feature in newer systems is closed system sampling.
9.2. Tissue Preparation for Chemical/Toxicological Analysis
9.3. Specific Analyzer RisksTo adequately assess the risk of active biohazards in analyzer effluents or processes, risk analysis should begin with assessment of procedures that occur prior to the use of specific analyzers. Sample preparation protocols may fully inactivate viruses and bacteria so that the risk of biohazardous aerosol generation in the analyzer effluent is essentially zero. One example is the use of protein-precipitation techniques or protein denaturing solvents in liquid chromatography, which would negate biohazard concerns in aerosols or effluents generated by the analyzer. 9.3.1. Graphite furnaces
9.3.2. Mass spectrometers
10. Hematology and Phlebotomy Laboratory10.1. Specimen Receiving and Log-In/Setup StationBiosafety guidelines for the hematology laboratory are the same as those for the microbiology laboratory and are described in Section 3.1. 10.2. Work at the Open BenchSee Section 3.2.1. 10.2.1. Standard operating procedures Standard operating procedures are described in Section 3.1.4. 10.2.2. Manual removal of sealed caps and specimen aliquotting/pipetting See Section 3.1.5. 10.2.3. Unfixed specimens
10.3. Personal PrecautionsGuidelines for personal precautions, including use of a BSC (Section 3.3), PPE (Section 3.2.1), and disinfection (Section 3.4) are described in Section 3. 10.4. Decontamination and Disposal of Laboratory Waste (39)See Section 3.5 for guidelines for decontamination and disposal of laboratory waste. 10.5. Dirty versus Clean Areas of the LaboratorySee Section 3.16. 10.6. InstrumentationWhether automated or manual, procedures with the potential for producing specimen aerosols and droplets (e.g., stopper removal, vortexing, opening or piercing evacuated tubes, automatic sample dispensers) require either PPE or engineering controls designed to prevent exposures to infectious agents. 10.6.1. Waterbaths See Section 3.17.1. 10.6.2. Centrifuges See Section 3.17.2. 10.6.3. Automated hematology/hemostasis analyzers Automated analyzers frequently have added features to help reduce operator exposures, but these do not totally eliminate potentials for exposure. A common feature in newer systems is closed system sampling. See Sections 3.17.3, 9.1, and 11.6.3 for additional information.
10.6.4 Flow cytometers (see Section 3.17.3) Occupational exposures in a routine flow cytometry (FCM) laboratory arise either from sample handling or, more specifically, from aerosols and droplets generated by the flow itself. Flow cytometric applications, e.g., phenotypic analysis, calcium flux evaluations, and apoptosis measurements of unfixed cells, when performed using jet-in-air flow cytometers with extremely high pressure settings can expose operators to potentially hazardous aerosols.
10.6.5. Automated slide stainers
10.6.6. Total or semiautomated hematology test systems
10.7. Rapid Testing (Kits) (Section 3.18)Consider used testing kits to be contaminated, and dispose of them appropriately in accordance with applicable local and state environmental regulations. 10.8. Molecular Testing (198)
10.9. Phlebotomy
10.9.1. General recommendations (200–203)
10.9.2. Dirty versus clean areas in the laboratory See Section 3.16. 10.9.3. Pneumatic tube systems See Section 3.1.6. 10.9.4. Personal precautions See Section 3.2. 10.9.5 Disinfection of work space See Section 3.4.1. Regardless of the method, the purpose of decontamination is to protect the phlebotomist, the patient and the environment, and anyone who enters a patient room/drawing station or who handles materials that have been carried into or out of the patient room/drawing station.
10.9.6. Disinfecting patient room work areas and drawing stations See Section 3.4.1.
10.9.7. Documentation of training and competency assessment in phlebotomy Assessment includes knowledge of, and adherence to, any applicable hospital infection control policies/procedures in patient settings and the concept of Standard Precautions. 11. Blood Bank11.1. Transfusion-Transmitted DiseasesMany infectious agents are transmitted through transfusion of infected blood; these include hepatitis B virus, hepatitis C virus, human immunodeficiency viruses 1 and 2, human T-cell lymphotropic viruses (HTLV-I and II), cytomegalovirus, parvovirus B19, West Nile virus , dengue virus, trypanosomiasis, malaria, and variant Creutzfeldt-Jakob disease. The AABB provides information on transfusion-transmitted diseases as well, available at http://www.aabb.org/Pages/Homepage.aspx. 11.2. Bloodborne Pathogen StandardThe Occupational Safety and Health Administration's (OSHA) Bloodborne Pathogen Standard, 29 CFR 1910.1030 must be adhered to in the blood bank laboratory (33). 11.3. Specimen Receiving and Log-In/Setup StationGuidelines for receiving and logging specimens and handling specimen containers are described in Section 3.1. 11.4. Work at the Open BenchWritten procedures for blood bank include specific work practices and work practice controls to mitigate potential exposures. Standard operational procedures (SOPs) and procedure manuals are described in Section 3.1.4. 11.4.1. Unfixed specimens
11.4.2. Biological safety cabinet A Class II biological safety cabinet (BSC; see Section 3.3) is required for all aerosol-generating processes. 11.4.3. Personal protective equipment See Section 3.2.2. 11.4.4. Disinfection See Section 3.4. 11.4.5. Decontamination and disposal of laboratory waste See Section 3.5 for discussion, including a waste management plan. 11.5. Clean versus Dirty Areas of the LaboratorySee Section 3.16. 11.6. InstrumentationSee Section 3.17. 11.6.1. Refrigerators and freezers For all refrigerators and freezers in the blood bank, establish a cleaning and maintenance protocol that will minimize contamination and extend the life of the equipment and also maintain the sophisticated cooling systems blood bank refrigerators require to provide uniform and quick temperature recovery when needed. (also see Section 3) Most newer blood bank laboratory refrigerators and freezers are stainless steel and have painted finishes and removable trays, which make cleaning and sanitizing an easier process.
11.6.2. Automated blood bank analyzers Automated or semi-automated instruments are now available that are adapted either to donor collection settings or patient transfusion settings. Although these instruments have the potential to replace much of the open bench testing in blood banks and donor collection settings, manual testing is still being used for some antibody detection and verification procedures and in smaller laboratories. All blood bank automated analyzers currently approved for use in the United States have added features to help reduce operator exposures, but they have not totally eliminated potential for exposure.
11.6.3. Total or semiautomated test systems See Section 10.6.6. 11.7. Test Kits and Reagent TraysSee Section 10.7. 11.8. Donor Blood Collection, Apheresis, and DispositionDonor collection and apheresis areas are considered patient care settings, and all applicable hospital patient care and infection control polices/procedures must be strictly adhered to.
12. Veterinary Diagnostic Laboratory12.1. IntroductionThis section provides practical guidelines for work practices that minimize biosafety hazards from veterinary diagnostic specimens. Many of the biosafety practice guidelines for human clinical microbiology laboratories are applicable in veterinary diagnostic laboratories. Similar to human clinical microbiology laboratories, the nature of the work performed in veterinary diagnostic laboratories puts these laboratorians, too, at risk for laboratory-acquired infections. Sixty percent of infectious diseases in humans are due to multihost pathogens that move across species lines (206,207), and during the past 30 years, 75% of the emerging human pathogen diseases (e.g., West Nile virus fever, highly pathogenic avian influenza, Lyme disease) have been zoonotic, i.e., transmitted between humans and animals (208). All nonhuman diagnostic specimens are potentially infectious to humans, although the degree of risk is less so than with handling and examination of human diagnostic specimens. Potential infectious agents in human diagnostic specimens are by definition human pathogens. Conversely, not all potential infectious agents in animal diagnostic specimens are human pathogens. The key to managing biosafety risk in veterinary diagnostic laboratories depends not only upon good general biosafety practices but, more importantly, on a practical risk assessment of the "unknown" diagnostic specimen. In general, veterinary diagnostic laboratories use biosafety level (BSL)-2 practices and facilities for general veterinary diagnostic work and do practical risk assessment of incoming accessions to determine whether decreased (BSL-1) or increased (BSL-3) biosafety practices or facilities are warranted. Where biosafety risk and practices differ between handling of human and animal diagnostic specimens, those differences are highlighted in this section. 12.2. Biological Risk Classification and Assessment12.2.1. Risk classification Two classifications of risk groups have been developed to facilitate the assessment of risk from various microbes and to recommend appropriate safety practices for the handling of those microbes (1). The World Organization for Animal Health (OIE) and World Health Organization (WHO) list four groups of biohazardous agents for humans and animals based upon level of risk and availability of effective treatment and prevention (Table 12) (209). CDC/National Institutes of Health (CDC/NIH) guidelines propose four biosafety levels and recommendations for appropriate containment practices for agents known to cause laboratory-acquired infections (Tables 12,13) (1). The two lists of risk groups are roughly equivalent, and neither makes allowance for persons who are particularly susceptible to infections by pre-existing conditions, such as a compromised immune system or pregnancy. In both risk group classification systems, increasing risk levels (numbers) imply increasing occupational risk from exposure to an agent and the need for additional containment for work with that agent.
12.2.2. Risk assessment See Section 2 for detailed risk assessment guidelines.
12.3. General Biosafety GuidelinesSee Section 3 for extensive and detailed biosafety guidelines generally applicable to all subdiscipline areas within a veterinary diagnostic laboratory.
12.3.1. Hand washing
12.3.2. Personal Protective Equipment
12.3.3. Staff training
12.3.4. Biological spill management
12.3.5. Immunization
12.4. Pathology (Necropsy and Surgical Pathology)See Section 5 for detailed biosafety guidelines applicable to necropsy, surgical pathology, and histology working areas in a veterinary diagnostic laboratory. 12.5. ParasitologySee Section 6. 12.6. MycologySee Section 7. 12.7. VirologySee Section 8. 12.8. ToxicologySee Section 9. 12.9. Hematology/SerologySee Section 10. 12.10. Molecular Diagnostics and Rapid TestsBiosafety guidelines to be followed when conducting molecular diagnostic testing (i.e., polymerase chain reaction [PCR]) or using rapid tests such as enzyme-linked immunosorbent assay (ELISA) can be specific to the particular testing being conducted. These are discussed in Section 3 and Sections 4, 5, 6, 7, 8 and 10, which deal with specific types of pathogens and testing. Section 8.10 provides the most thorough biosafety guidelines for molecular diagnostic testing. 12.11. Storage, Packaging, and ShippingSee Section 13 for detailed biosafety guidelines applicable to functions within a veterinary diagnostic laboratory regarding storage, packaging and shipping of infectious or diagnostic specimens. 12.12. Biosafety Education/TrainingSee Section 15 for practical guidelines regarding biosafety training within a veterinary diagnostic laboratory. 12.13. Biosafety Quality ImprovementSee Section 16 for guidelines regarding continual improvement of biosafety within a veterinary diagnostic laboratory. 13. Storing, Packaging, and Shipping Infectious Substances13.1. Storage of Infectious SubstancesInfectious substances in a clinical microbiology laboratory are encountered as fresh and processed patient specimens, cultures and subcultures, stored isolates, and serum or plasma. Invariably, all of these substances must occasionally be stored in some form and for some length of time, and many of these substances will be manipulated, relocated, and otherwise touched by laboratory workers. Therefore, storage of infectious substances is an important and integral component of worker safety in clinical microbiology laboratories. Handle all stored infectious substances using Standard Precautions and aseptic technique. Organisms responsible for external contamination of the storage vial will remain viable during storage and can be transmitted by manipulating the vial.
13.2. Packing and Shipping Infectious Substances
Note: The requirements and regulations governing the transport of infectious substances change frequently. Shippers are responsible for being aware of these changes, adhering to current regulations, obtaining permits in advance of shipping, and interpreting applicable regulations for themselves and their facilities. Persons shipping these substances are advised to check the web sites of the respective appropriate agencies. 13.2.1. Governing authorities and regulations
13.2.2. Importance of regulations The purpose of the regulations is to protect the public, emergency responders, laboratory workers, and personnel in the transportation industry from accidental exposure to the infectious contents of the packages. An important non–safety-related benefit of adherence to these regulations and requirements is minimizing the potential for damage to the contents of the package during transport and reducing the exposure of the shipper to criminal and civil liability associated with improper shipment of dangerous goods. 13.2.3. Exceptions
13.2.4. Specific regulations
13.2.5. U.S. Postal Service The U.S. Postal Service publishes its own regulations in the USPS Domestic Mail Manual (96). The USPS regulations for mailing hazardous materials generally adhere to DOT regulations; however, consult the USPS Domestic Mail Manual for specific needs and requirements. 13.3. Classification of Infectious Substances13.3.1. Classification All shipped goods must be classified using a three-step process to define dangerous goods that are shipped by commercial carriers. Classification allows the shipper to select the proper IATA packing instructions and directions to use, and provides information necessary to complete required documentation (a Shipper's Declaration for Dangerous Goods) if the substance is a Category A infectious substance. 13.3.2. Steps of classification
13.3.3. Category A infectious substances A Category A substance is "an infectious substance which is transported in a form that, when exposure to it occurs, is capable of causing permanent disability, or life-threatening or fatal disease to otherwise healthy humans or animals" (93).
13.3.4. Category B infectious substances A Category B substance is "an infectious substance that does not meet the criteria for inclusion in Category A" (93). Category B substances are not in a form generally capable of causing disability, life-threatening illness, or fatal disease. Category B substances must be assigned UN number UN3373 (Biological Substance, Category B). Following are examples of possible Category B substances:
13.3.5. Exempt human (or animal) specimens Exempt human or animal body site specimens are those for which there is "minimal likelihood there are pathogens present" (93). Examples of such specimens include urine or serum to be tested for glucose, cholesterol, hormone levels, prostate-specific antigen, and analytes used to evaluate heart and kidney function.
13.3.6. Exempt substances Many substances commonly encountered in clinical laboratories are exempt from strict infectious substance shipping requirements (Figure 2). Examples of such substances are
13.3.7. Patient specimens
13.3.8. Genetically modified organisms Genetically modified organisms usually meet either Category A or Category B criteria. If this is not the case, the organism must be classified as a "genetically modified microorganism" (Class 9, Miscellaneous Dangerous Goods) and packed and shipped as such. 13.3.9. Biological products Virtually all commercially available biological products are exempt from regulations for packing and shipping infectious substances. Examples of biological products include bacterial typing sera, vaccines, bacterial antigens, antimicrobial agents, reagents for identifying bacteria, and reagents used in antimicrobial susceptibility testing. 13.3.10. Infected animal
13.3.11. Medical waste
13.4. Naming Category A and Category B Substances
13.5 Packing Instructions and Packing Substances13.5.1. Packing instructions and directions
13.5.2. Marking and labeling outer packages
13.5.3. Specific markings and labels The following list cites the situations requiring a marker or label, and the specific markings or labels for that situation.
13.6. Documentation13.6.1. Shipper's Declaration for Dangerous Goods
13.6.2. Emergency response telephone number
13.6.3. Airbills IATA carriers are required to prepare airbills to describe air cargo and accompany shipments in transit. Some dangerous goods shipments, such as Biological Substances Category B shipments, require preparation of this document but not a Shipper's Declaration. Specific preparation instructions are detailed in each IATA package instruction and in the "Documentation" section of the Dangerous Goods Regulations. 13.7. RefrigerantsPackaging must be leakproof when wet ice is used. Dry ice is a Class 9 dangerous good; it must be packaged according to PI 954, and its use requires completion of a Shipper's Declaration if it is used to ship a Category A substance. Note: Dry ice is an explosion hazard and must never be placed into a tightly sealed container. Dry ice must be placed outside the secondary container, and the outer packaging must permit the release of CO. 13.8. Training and Certification
14. Emergency Procedures and ResponsibilitiesThe risk of acquiring a laboratory-associated infection (LAI) after physically contacting a microorganism (an "exposure") in the workplace is real, always present, and an integral part of working in a diagnostic laboratory, and in particular the clinical microbiology laboratory. The potential for an exposure exists whenever a laboratorian manipulates and transports microorganisms, processes and stores patient specimens, and operates instruments used in the process. Diagnostic laboratories can be safe places to work if standard and appropriate safe work practices and procedures are easily accessible, understood by employees, enforced, and followed. These procedures are to be properly outlined in an exposure control plan and laboratory manuals. These plans are composed of essential elements related to preventing an exposure, and, equally important, they describe employer and employee involvement and responsibilities before and after an exposure. Appropriate actions taken after an exposure can greatly reduce or even eliminate the chance that an exposure will result in an LAI. Well-designed plans with the full support of the director and higher management can reduce workers' chances of exposures to microorganisms and can help ensure a culture of safety in diagnostic laboratories. 14.1. Responsibilities of Employers Before an Exposure14.1.1. Exposure control plan
14.1.2. Documentation of potential exposures
14.1.3. Emergency response equipment and facilities
14.1.4. Immunizations The Advisory Committee on Immunization Practices, in addition to recommending immunization of health-care personnel with vaccines recommended for all adults (influenza, measles/mumps/rubella, varicella, and tetanus/diphtheria/pertussis), recommends meningococcal or hepatitis B vaccination for those at risk for occupational exposure (211,217,218).
14.1.5. Education of employees
14.2. Responsibilities of Employees Before an ExposureIt is the responsibility of laboratory employees to do the following:
14.3. Responsibilities of Employers After an Exposure14.3.1. Determination of the extent of exposure
14.3.2. Documentation of exposures
14.3.3. Consultation with employee health clinicians The employee and the supervisor of an employee who has experienced a potential exposure are to contact the employee health physician or nurse and discuss the exposure. These clinicians are the persons most likely to provide advice regarding timely chemoprophylaxis and to able to administer appropriate antimicrobial agents. 14.3.4. Counseling exposed employees
14.3.5. Exposure to Mycobacterium tuberculosis
14.3.6. Exposure to Neisseria meningitidis
14.3.7. Exposure to bloodborne pathogens
14.4. Responsibilities of Employees After an Exposure
15. Biosafety EducationBiosafety education efforts begin even before an employee begins working in the laboratory. The employer must develop an accurate job description so that the employee understands the job responsibilities. Knowledge, skills, and abilities needed for the job are to be defined. Evaluate incoming employees to see if they meet these criteria. Develop a mentoring plan and fill any training gaps before employees are placed in a position that would put them at risk for exposure. Evaluate and document the employees' competency before they are allowed to work independently. Educational opportunities to reinforce safe behaviors must be ongoing and supported by all levels of management and staff. In accordance with Occupational Safety and Health Administration (OSHA) requirements, education about the risks of exposure to infectious agents begins with a new employee's first orientation to the laboratory or assignment to technical work and is to be specific to the tasks the employee performs. Training must include an explanation of the use and limitations of methods that will reduce or prevent exposure to infectious materials. These include engineering controls, work practices, and personal protective equipment. Annual retraining for these employees must be provided within 1 year of their original training and should emphasize information on new engineering controls and practices. Annual safety training offers a chance to review key biosafety measures that may be forgotten during everyday work pressures. The responsibility for overseeing the safety education of laboratory personnel must be clearly assigned. This responsibility may be delegated to the biosafety officer or other staff member who has been given additional training through specialized courses or work experience and whose competency to perform the training has been verified. Because laboratory tests might be performed outside a traditional laboratory setting (e.g., doctor's office, outpatient clinic, community setting), these recommendations for training and education must be adapted to suit the employees performing the tests and the person who is overseeing them. 15.1. Biosafety Training/ExercisesEmployee training can be accomplished by any of several methods, and nearly all of these can be adapted or combined to fit the needs of employees in a particular laboratory.
15.2. Educational Reinforcement
15.3. Annual Checklist of Critical Safety Items and ProceduresThere is no one "official" set of questions for an annual safety checklist. Although many common activities might be performed by all personnel, customize the list to reflect the actual job duties. Analyze each work station for the type of biosafety risks associated with it, and target the checklist to each of these risks. If practical, ask individual laboratorians to draft their own checklists for the duties they perform, and have their list reviewed by their supervisor and safety officer. 15.4. Assessment and DocumentationEmployee training and competency assessment should be documented for the following:
15.5. Monitoring Compliance with Safety Procedures
16. Continuous Quality ImprovementIntegrate continuous quality improvement for biosafety with the continuous quality improvement for the entire laboratory. The 12 quality system essentials, as defined by the Clinical Laboratory Standards Institute, provide a comprehensive basis and reference for continuous quality improvement (222). More detailed and specific biosafety considerations have been listed for each of these elements (Table 17). AcknowledgmentsWe acknowledge the assistance of Tanya Graham, DVM, South Dakota State University, Brookings, SD; Larry Thompson, DVM, PhD, Nestle Purina Pet Care, St. Louis, MO; R. Ross Graham, DVM, PhD, Merrick and Company; Corrine Fantz, PhD, Emory University, Atlanta, GA; Thomas Burgess, PhD, and Quest Diagnostics, Tucker, GA. We appreciate the review and input into the document provided by the Office of Health and Safety, CDC; American Association of Veterinary Laboratory Diagnosticians; American Biological Safety Association; College of American Pathologists; American Society for Microbiology; Association of Public Health Laboratories; and subject matter experts at CDC. References
FIGURE 1. Risk assessment process for biologic hazards Alternate Text: The figure is a flow chart that presents the risk assessment process for a biologic hazard
FIGURE 2. Algorithm for classifying infectious substance for shipment Alternate Text: The figure is a flow chart that presents the process for classifying an infectious substance for shipment.
FIGURE 3. A completely labeled outer package. The primary container inside the package contains a Biological Substance, Category B infectious substance and is packed according to PI 650 Abbreviation: PI = packing instructions. Alternate Text: The figure is a diagram of a shipping package with the appropriate labeling for a Category B infectious substance. FIGURE 4. A completely labeled outer package. The primary container inside contains a liquid Category A infectious substance and is packed according to PI 620 Abbreviation: PI = packing instructions. Alternate Text: The figure is a diagram of a shipping package with the appropriate labeling for a liquid Category A infectious substance.
Blue Ribbon Panel for Issues of Clinical Laboratory Safety Kathleen G. Beavis, MD, College of American Pathologists, Chicago, Illinois; Ellen Jo Baron, PhD, Stanford, California; William R. Dunn, MS, Greater New York Hospital Association Regional Laboratory Task Force, New York, New York; Larry Gray, PhD, American Society for Microbiology, Cincinnati, Ohio; Bill Homovec, MPH, American Clinical Laboratory Association, Burlington, North Carolina; Michael Pentella, PhD, Association of Public Health Laboratories, Iowa City, Iowa; Bruce Ribner, MD, Atlanta, Georgia; William A. Rutala, PhD, Chapel Hill, North Carolina; Daniel S. Shapiro, MD, Burlington, Massachusetts; Lisa A. Skodack-Jones, MT, Salt Lake City, Utah; Christine Snyder, American Society for Clinical Laboratory Science, Helena, Montana; Robert L. Sunheimer, MS, American Society for Clinical Pathology, Syracuse, New York; Christina Z. Thompson, MS, American Biological Safety Association, Greenfield, Indiana. CDC Staff: Nancy L. Anderson, MMSc; Rex Astles, PhD; D. Joe Boone, PhD; David S. Bressler, MS; Roberta Carey, PhD; Casey Chosewood, MD; Mitchell L. Cohen, MD; Judy Delaney, MS; Thomas L. Hearn, DrPH; Kathleen F. Keyes, MS; Davis Lupo, PhD; Robert Martin, DrPH; Alison C. Mawle, PhD; Terra McConnel; J. Michael Miller, PhD; Shana Nesby, DVM; Janet K. Nicholson, PhD; John P. O'Connor, MS; Anne Pollock; John C. Ridderhof, DrPH; Pamela Robinson; Elizabeth G. Weirich, MS; Ae S. Youngpairoj. What are the guidelines for animal research?Ethical Guidelines for the Use of Animals in Research. Respect for animals' dignity.. Responsibility for considering options (Replace). The principle of proportionality: responsibility for considering and balancing suffering and benefit.. Responsibility for considering reducing the number of animals (Reduce). What are the two principal animal research regulatory documents used by the Office of laboratory Animal Welfare OLAW )?The PHS Policy and the U.S. Department of Agriculture's (USDA) Animal Welfare Regulations 2, are the two principal federal documents that set forth requirements for animal care and use by institutions using animals in research, testing, and education.
What is the regulation that provides guidelines for laboratory experimentation on microorganisms plants and animals?The biosafety regulation for laboratories involving microbiologic pathogens (State Council 2004; State Administration of Environmental Protection 2006; National Standards Committee 2008) should be followed, as appropriate.
Which guideline is used for the maintenance of laboratory animals?3. GOAL The goal of these guidelines is to promote the human care of animal used in biomedical and behavioural research and testing.
Which of the following is a function of the IACUC?Responsibilities. The IACUC is responsible for oversight of the animal care and use program and its components as described in the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (Policy) and the Guide for the Care and Use of Laboratory Animals (Guide ).
What are the three Rs in the legal standards for animal research?The 3 Rs stand for Replacement, Reduction and Refinement. Replacement alternatives refer to methods which avoid or replace the use of animals.
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