Biological Safety Manual – Chapter 16: Working With Human, Non-Human Primate, and Other Mammalian Cells and Tissues

Biological Safety Manual

Title

Biological Safety Manual – Chapter 16: Working With Human, Non-Human Primate, and Other Mammalian Cells and Tissues

Introduction

Prior to beginning any work in a University of North Carolina at Chapel Hill (“UNC-Chapel Hill” or “University”) lab, one must perform a risk assessment for the materials that will be handled to identify the potential hazards associated with a project – cell and tissue culture work is no exception to this principle. Although the risk of infection is generally recognized to be low for work with most cell cultures, the potential for laboratory acquired infections (LAIs) increases significantly when considering cell lines or tissues derived from non-human primate (NHP), humans, or primary cells from other mammalian species.

The following chapter will review the potential laboratory hazards and recommended practices for cell and tissue culture work, human and NHP specimen handling, and the University’s bloodborne pathogen (BBP) Exposure Control Plan.

UNC-Chapel Hill’s policies for good laboratory practice are guided by the following:

  • the U.S. Occupational Safety and Health Administration’s (OSHA) Bloodborne Pathogen Standard (29 CFR 1910.1030)1;
  • proper aseptic technique 2,3; and
  • the recommended practices for work with human, NHP, and mammalian cells/tissues described in the current edition of the U.S. Centers for Disease Control and Prevention’s (CDC) Biosafety in Microbiological and Biomedical Laboratories (BMBL).

Please note that laboratory workers are expected to know the associated hazards and appropriate handling procedures for their work to preserve personal, communal, and environmental health, in addition to sample integrity. Principle Investigators (PIs) are responsible for communicating any work hazards to their staff, providing the necessary trainings, and ensuring their staff maintains competency with the techniques that will be used in the lab.

Table of Contents

  1. Potential Laboratory Hazards Associated with Cell and Tissue Culture Work
  2. Recommended Practices
    1. Risk Assessments Related to Material Source and Type
      1. Tissue Source & Origin Species
      2. Cell or Tissue Type
      3. Culture Type
      4. Additional Considerations
      5. Risk Mitigation
  3. References
  4. Bloodborne Pathogen Exposure Control Plan for Laboratories
    1. Regulation
    2. Definitions
    3. Exposure Determination
    4. Universal Precautions
    5. Epidemiology
    6. Facilities and Practices
    7. Engineering Controls
    8. Work Practice Controls
    9. Personal Protective Equipment
    10. Housekeeping
    11. Waste Disposal
    12. HIV and HBV Research Laboratories
    13. Hepatitis B Vaccination
    14. Post-Exposure Procedures
    15. Post-Exposure Evaluation and Follow-Up
    16. Training
    17. Inspection
    18. Exposure Control for UNC-Chapel Hill Employees Located in Other Counties
    19. Appendix A: Job Classifications
    20. Appendix B: Hepatitis B Declination Form
  5. Safety Practices for Work with Non-Human Primate Tissues and Body Fluids
    1. Purpose
    2. Biosafety Information
    3. Cercopithecinae Herpes Virus 1 or Herpes B Virus: Risk Group 4 Organism
    4. Simian Immunodeficiency Virus (SIV): Risk Group 3 Organism
    5. Training
    6. Laboratory Safety for Research Involving Non-Human Primate Specimens
    7. Routes of Exposure
    8. Post-Exposure
    9. References

I. Potential Laboratory Hazards Associated with Cell and Tissue Culture Work

As stated in the introduction, many cell cultures are considered to be low risk and can be safely handled at Biosafety Level (BSL)-1 or BSL-2. The main hazards associated with culture work include:

  1. Exposure to pathogens harbored in the tissue or cells (e.g., BBPs)
  2. Exposure to tumorigenic cells
  3. Exposure to recombinant or synthetic nucleic acids (e.g., viral vectors)

Project-specific variables (e.g., cells/tissue type, manipulations, reagents) will impact the level of risk associated with a project, thus a comprehensive risk assessment should be performed prior to project initiation to ensure the appropriate safety precautions will be implemented. Please see the next section, Recommended Practices, for additional guidance on the risk assessment process.

Please note: it is UNC-CH policy to treat all unfixed human tissue samples and cell lines, including characterized cell lines, as potentially infectious with regard to BBPs. Thus, human and other primate cells should be handled using BSL-2 practices and containment, meaning all work should be performed in a BSC, all work materials need to be decontaminated prior to disposal (via autoclaving or chemical disinfectants), and personal protective equipment should include, at minimum, a lab coat, gloves, and eye protection. 6,10,11,12 

II. Recommended Practices

Prior to project initiation, investigators should perform a risk assessment to identify the potential hazards associated with their work. A comprehensive risk assessment will facilitate the selection of appropriate containment, personal protective equipment (PPE), and work practices. The following 5 sections should be assessed prior to project initiation:

  1. Source (origin species)
  2. Cell/Tissue Type
  3. Cell/Tissue Characteristics
  4. Culture type
  5. Manipulations

See [fig 22.2 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122109/]

i. Tissue Source and Origin Species

The risk of handling a particular cell culture or tissue sample increases relative to the genetic similarity to humans. That is, the more closely related the origin species is to humans, the higher risk the specimen poses to lab personnel. Accordingly, cell lines or tissues derived from humans or NHPs pose the highest risk to human health due to the fact that pathogens often evolve to be species-specific. Improper safety procedures with these cell types have a higher probability of resulting in LAIs.

The most notable risk for handling human- and NHP-derived samples is the potential exposure to BBPs such as Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV). Working with human, NHP, and other mammalian cell lines may present a risk of exposure to BBPs. All work with human blood and Other Potentially Infectious Material (OPIM), including body fluids, tissues, and primary cell lines must follow the requirements described in the OSHA Bloodborne Pathogen Standard. Old World NHP specimens (i.e., macaques) may contain Macacine herpesvirus (Herpes B) and Simian Immunodeficiency Virus (SIV). This material should always be considered potentially infected and should be handled with strict barrier precautions and with swift occupational responses for potential exposures. Herpes B virus infection in macaques is usually symptom-free, or causes only mild oral lesions. However, Herpes B virus infection in humans can be fatal.6

Please be aware that while it is generally true that pathogens have species-specific adaptations that act as barriers to zoonotic infections (and thus LAIs), there are exceptions to this rule. Certain pathogens have been well documented to cause cross-species infections(e.g., influenzas, SARS Co-V, West Nile virus). Working with other (non-human and non-NHP) mammalian, avian, and invertebrate cell lines generally present lower risks.

ii. Cell or Tissue Type

Another important consideration is cell or tissue type and whether there is a hazard associated with the capability of the cell to form tumors (e.g., oncogene expressing). Hematopoietic cells and lymphoid tissues can have tumorigenic potential and therefore have an increased risk for handling. Neural tissues and endothelial cells may be considered to have less risk, but an assessment must determine the probability of whether such cells contain other adventitious agents and take into account the tissue or cell source(s) and parameters related to the history of that source. Epithelial cells and fibroblasts present the lowest risk in terms of cell type and tumorigenic potential.7

iii. Culture Type

When working with cell lines, the culture type is another important consideration. Primary cell lines are derived by sampling directly from in vivo organ and tissue samples and have a higher risk of containing undetected pathogens. Therefore, these culture types have shorter lifespans of unknown characterization and present a higher potential risk while culturing. Continuous cell lines (i.e., cells immortalized with viral agents such as EBV, SV-40, or other viral agents) have been modified to grow for extended passages, perhaps even indefinitely. Continuous cultures can usually be more characterized with PCR and cytometric analyses; however, cells carrying viral genomic material still can pose increased risks in the event of inadvertent exposures, particularly for immune-compromised individuals.8 There has been a report of tumor development from an accidental needlestick injury.9 Permissive cell lines that support viral replication may have a heightened risk of contamination with viral pathogens. Well-established, and possibly even tested, cell lines are generally considered safer, but the possibility of adventitious contamination by an unspecified pathogen during use must be considered during the risk assessment process, and measures must be taken to lower the risk of contamination.10

iv. Additional Considerations

When conducting a risk assessment, consider if endogenous pathogens are present in the specimen or if the pathogens have been added intentionally. Another key consideration is if agents may have been added as a result of passaging of the line in animal model systems. Experimentally infected cell lines should be handled following safety recommendations for both potential endogenous pathogens and known pathogens added in the course of research. Any cell line with known endogenous pathogens should be handled following the safety recommendations for those pathogens. Risk assessment should also consider if any recombinant materials are expressed by the cell line and whether the cell line is a type that supports viral replication. Consult with the Institutional Biosafety Committee, or equivalent resource, when working with recombinant or synthetic nucleic acids in cell lines.11 Helpful guidelines exist to increase awareness of the problems encountered when working with cells in biomedical research and how to address them effectively.12

v. Risk Mitigation

At a minimum, human and other primate cells should be treated as potentially infectious and handled using BSL-2 practices, engineering controls, and facilities.13 The use of a biological safety cabinet (BSC) for culturing activities is the universally accepted best practice. Higher containment must be considered for cell lines harboring Risk Group 3 and 4 pathogens as indicated by the risk assessment; higher containment must be considered if the agents present become airborne when energy is imparted on the biological sample. Personal protective equipment (PPE) such as laboratory coats, gloves, and eye protection should be worn in tissue culture laboratories and additional PPE should be added as indicated by risk assessment. All waste culture material must be decontaminated before disposal. All laboratory staff working with human and NHP cells and tissues should be enrolled in an occupational medical program specific for BBPs, and staff should work under the policies and guidelines established by their institution’s Exposure Control Plan (ECP).

III. References

  1. Occupational exposure to bloodborne pathogens. Final Rule. Standard interpretations: applicability of 1910.1030 to established human cell lines, 29 C.F.R. Sect. 1910.1030 (1991).
  2. McGarrity GJ, Coriell LL. Procedures to reduce contamination of cell cultures. In Vitro. 1971;6:257-65.
  3. McGarrity GJ. Spread and control of mycoplasmal infection of cell culture. In Vitro. 1976;12:643-8.
  4. US Public Health Service. Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis. MMWR Recomm Rep. 2001;50(RR-11):1–52.
  5. Bloodborne pathogens, 29 C.F.R. Part 1910.1030 (1992).
  6. NASPHV; Centers for Disease Control and Prevention; Council of State and Territorial Epidemiologists; American Veterinary Medical Association. Compendium of measures to prevent disease associated with animals in public settings, 2009: National Association of State Public Health Veterinarians, Inc. (NASPHV). MMWR Recomm Rep. 2009;58(RR-5):1–21.
  7. Pauwels K, Herman P, Van Vaerenbergh B, Dai Do Thi C, Berghmans L, Waeterloos G, et al. Animal Cell Cultures: Risk Assessment and Biosafety Recommendations. Apple Biosaf. 2007;12(1):26–38.
  8. Caputo JL. Safety Procedures. In: Freshney RI, Freshney MG, editors. Culture of Immortalized Cells. New York: Wiley-Liss; 1996. p. 25–51.
  9. Gugel EA, Sanders ME. Needle-stick transmission of human colonic adenocarcinoma [letter]. N Engl J Med. 1986;315(23):1487.
  10. McGarrity GJ. Spread and control of mycoplasmal infection of cell culture. In Vitro. 1976;12(9):643–8.
  11. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). Bethesda (MD): National Institutes of Health, Office of Science Policy; 2019.
  12. Geraghty RJ, Capes-Davis A, Davis JM, Downward J, Freshney RI, Knezevic I, et al. Guidelines for the use of cell lines in biomedical research. Br J Cancer. 2014;111(6):1021–46.
  13. United States Department of Labor [Internet]. Washington (DC): Occupational Safety and Health Administration; c1994 [cited 2019 April 10]. Applicability of 1910.1030 to establish human cell lines. Available from: https://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=INTERPRETATIONS&p_id=21519

IV. Bloodborne Pathogen Exposure Control Plan for Laboratories

A. Regulation

The Occupational Safety and Health Administration (OSHA) affirms that certain biological materials may harbor BBPs and occupational exposure to blood and other potentially infectious materials (OPIM) poses a significant health risk to workers. The OSHA Bloodborne Pathogen Standard (29 CFR 1910.1030) provides guidance on how to minimize or eliminate work hazards using a combination of engineering and work practice controls, personal protective clothing and equipment, training, medical surveillance, signs and labels, and other provisions.

In accordance with the OSHA Bloodborne Pathogen Standard, the University has developed an Exposure Control Plan designed to mitigate and minimize occupational exposure to BBP. Compliance with the Exposure Control Plan is a condition of employment for all employees with occupational exposures, and this document must be made available to every employee identified to be at risk for BBP exposure. Appendix A lists a variety of positions that may have exposure risks associated with their job duties.

B. Definitions

  1. Blood: Human blood, human blood components, products made from human blood.
  2. Bloodborne pathogens (BBPs): Pathogenic microorganisms that are present in human blood and can cause disease in humans. Include, but are not limited to, hepatitis B virus (HBV), human immunodeficiency virus (HIV).
  3. Occupational Exposure: Reasonably anticipated skin, eye, mucous membrane, or parenteral contact with blood or other potentially infectious materials (defined below) that may result from the performance of an employee’s duties.
  4. Other Potentially Infectious Materials (OPIM):
    1. Certain human body fluids
      1. Semen
      2. Vaginal secretions
      3. Cerebrospinal fluid
      4. Synovial fluid
      5. Pleural fluid
      6. Pericardial fluid
      7. Peritoneal fluid
      8. Amniotic fluid
      9. Saliva in dental procedures
      10. Any body fluid visibly contaminated with blood
      11. All body fluids when it is difficult to differentiate between body fluids
    2. Any unfixed tissue or organ from a human, living or dead.
    3. HIV-containing cell or tissue cultures, organ cultures, and HIV- or HBV-containing culture medium or other solutions; and blood, organs, or other tissues from experimental animals infected with HIV or HBV.
    4. Human cell lines.

C. Exposure Determination

PIs are responsible for determining which tasks, procedures, and positions may result in occupational exposure, and are required to notify employees about their potential for occupational exposure to blood or OPIM. The risk assessment for occupational exposures must be made independent of safety controls (e.g., PPE, BSCs). Laboratory workers who are at risk for occupational exposure to BBPs must indicate this on the Laboratory Worker Registration form by selecting “Bloodborne pathogens.” Researchers who work with HIV-positive samples. HIV viral culture, or who are involved with other research requiring BSL-2 with enhanced precautions (BSL-2+) will indicate this on their Laboratory Worker Registration form by checking “HIV Researcher.” The information provided on the registration form will be used to enroll employees into UNC-Chapel Hill’s medical surveillance program.

Some laboratory procedures that pose an exposure risk include:

  1. Handling contaminated sharps, glass, equipment, or waste
  2. Procedures with the potential to produce aerosols or spatters (e.g., centrifugation, pipetting, opening tubes)
  3. Phlebotomy
  4. Aliquoting blood or OPIM

It is important for workers to be informed about their occupational hazards and the safeguards selected to mitigate occupational exposures. Hazard awareness enables individuals to locate and utilize the appropriate safety controls in their workplace, helping to minimize or eliminate exposure to blood and OPIM.

D. Universal Precautions

All human blood and OPIM should be treated as infectious for HIV, HBV, and other BBPs. In general, under the OSHA Bloodborne Pathogens Standard, Universal Precautions are to be observed to prevent contact with blood or “other potentially infectious materials.” With Universal Precautions, individuals should wear PPE when they anticipate coming into contact with blood, blood products, certain body fluids, or any body fluids visibly contaminated with blood.

E. Epidemiology

Many diseases are linked to BBPs, but few BBPs are frequently responsible for infections in the workplace. Important diseases associated with occupational exposure to BBPs include hepatitis B, hepatitis C and AIDS. Historically, work-related exposure incidents have occurred more frequently in occupations involving direct patient contact, but some infections have been linked to cleaning up a spill of potentially infectious materials, too. For this reason, workers should only attempt to clean-up potentially infectious material if they have received documented training on BBPs. UNC-Chapel Hill provides training for all employees with potential for occupational exposures. Below are summary statements for the primary BBPs, Hepatitis B virus, Hepatitis C virus, and Human immunodeficiency virus, that cover basic concepts about their associated diseases to allow workers to monitor for symptoms and, if needed, discuss them with a supervisor, family members, and a physician.

Hepatitis B virus

Between two thirds and three fourths of all Hepatitis B infections result in either no symptoms of infection or a relatively mild flu-like illness. Between 25% and 33% of the infections, however, take a much more severe clinical course. The symptoms include jaundice, dark urine, extreme fatigue, anorexia, nausea, abdominal pain, and sometimes joint pain, rash, and fever. Hospitalization is required in about 20% of the more severe clinical cases.

A safe, immunogenic, and effective vaccine to prevent hepatitis B has been available since 1982 and is recommended for employees with the potential for occupational exposure to blood and other body fluids.

Hepatitis C virus

Hepatitis C virus is the most frequently occurring BBP infection. At least 85% of persons with Hepatitis C Virus (HCV) infection become chronically infected, and chronic liver disease develops in an average of 67%. HCV is most efficiently transmitted by large or repeated percutaneous exposures to blood, such as through the transfusion of blood or blood products from infected donors and sharing of contaminated needles among injection drug users. Other bloodborne viruses, such as HBV, are transmitted not only by percutaneous exposures, but also by mucous membrane and apparent parenteral exposures.

One case of transmission of HCV from a blood splash to the conjunctiva was reported for a health care worker.

Human immunodeficiency virus

HIV adversely affects the immune system rendering the infected individual vulnerable to a wide range of clinical disorders. These conditions, some of which tend to recur, can be aggressive, rapidly progressive, difficult to treat, and less responsive to traditional modes of treatment. Due to the damage HIV wreaks on the immune system, untreated HIV infection can result in AIDS (acquired immunodeficiency syndrome). A person with HIV is considered to have progressed to AIDs based on their CD4 count, or the development of opportunistic infections.

The CDC has divided HIV disease progression into four stages, grouped according to infections or symptoms reported.

  • Group I: Within a month after exposure, an individual may experience acute retroviral syndrome, the first clinical evidence of HIV infection. This is a mononucleosis-like syndrome with signs and symptoms that can include fever, lymphadenopathy, myalgia, arthralgia, diarrhea, fatigue, and rash. Acute retroviral syndrome is usually self-limiting and followed by the development of antibodies.
  • Group II: Although most persons infected with HIV develop antibodies to the virus with 6-12 weeks after exposure, most of these individuals are asymptomatic for months to years following infection. However, they can transmit the virus to others throughout this time.
  • Group III: Although no other signs or symptoms are experienced, some HIV-infected patients will develop a persistent, generalized lymphadenopathy that lasts more than 3 months.
  • Group IV: Epidemiologic data indicates that most persons who are infected with HIV will eventually develop AIDS. AIDS can result in severe opportunistic infections that an individual with a normal immune system would only rarely experience, as well as a wide range of neurologic and oncogenic or neoplastic processes. Some patients may experience “constitutional disease” also known as HIV “wasting syndrome,” which may be characterized by severe, involuntary weight loss, chronic diarrhea, constant or intermittent weakness, and fever for 30 days or longer. This syndrome may result in death. Individuals with AIDS may also develop HIV encephalopathy, dementia, myelopathy or peripheral neuropathy. In addition, the virus is capable of affecting the peripheral nervous system causing severe pain and weakness or numbness in the limbs. There are specific diseases considered indicators of AIDS. Among these are parasitic diseases such as Pneumocystis carinii pneumonia; fungal diseases such as candidiasis of esophagus, trachea, bronchi or lungs; viral diseases such as cytomegalovirus disease of an organ other than the liver, spleen or lymph nodes; cancer/neoplastic diseases such as Kaposi’s sarcoma; and bacterial infections such as Mycobacteriumaviumn complex.

HIV is a fragile virus. It cannot live for very long outside the body. Reports dealing with HIV infection indicate that the risk of bloodborne transmission from inadvertent exposure is considerably less for HIV than for HBV infection.

HIV can enter the bloodstream the same as HBV: if potentially infectious materials (like blood) come into contact with an unprotected break in your skin such as an open wound, acne, rash, etc. or if you experience a splash into your eyes and/or nose. The risk of getting an HIV infection like this is considerably less for HIV than for HBV infection. The occupational risk of acquiring HIV like this is 1 in 200 compared with 1 in 33 for HBV.

F. Facilities and Practices

All work with human blood or OPIM must be conducted at BSL-2 as described in the current edition of the BMBL. BSL-2 is for work involving agents of moderate potential hazard to personnel and the environment. BSL-2 requires that laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists. Access to the BSL-2 laboratory is limited when work is being conducted, extreme precautions are taken with contaminated sharp items and certain procedures in which infectious aerosols or splashes may be created are conducted in BSCs or other physical containment equipment. If you are working at BSL-2 you are required to take UNC-Chapel Hill’s online BSL-2 training.

G. Engineering Controls

Engineering Controls specifically isolate or remove a BBP hazard from the workplace. Engineering controls used at the University include sharps disposal containers, safer needle devices, and BSCs:

Sharps Disposal Containers

Sharps containers must be easily accessible and located near areas where sharps are used. Contaminated sharps receptacles must be red, puncture-resistant, leak-proof and display a BIOHAZARD label. UNC-CH research laboratories should use these sharps containers available from Fisher Scientific, or a similar alternative from another provider. Sharps containers must remain upright and be prepared for disposal once they are 2/3 full. Sharps containers must not be overfilled. Prior to removal from the laboratory, sharps containers must be closed and should never be opened, emptied, or cleaned manually. If removing the sharps container from a BSC, always decontaminate the exterior of the container. Contaminated sharps containers must be autoclaved prior to final disposal.

Prior to autoclaving, the sharps container should have indicator tape placed in an X across the biohazard symbol. Containers contaminated sharps should be placed into an orange, polypropylene autoclavable bag. The autoclave bag should be marked with an X over its biohazard symbol before placing the sharps container inside. After autoclaving, the bags with the containers of sharps can be disposed of with the regular trash.

Sharps Disposal Containers

 

Additional information regarding biohazardous waste disposal procedures can be found on the EHS website. The appropriate caution labels for biohazardous sharps and non-hazardous sharps can also be found on the EHS website.

Sharps Elimination, Safer Needle Devices, and Sharps Precautions

Federal OSHA regulations (CFR 1910.1030) require biohazard laboratories to minimize their use of sharps whenever possible. If there is no alternative to using a needle for a laboratory procedure (i.e. injection into animals), the use of safer needle devices should be considered. Protective devices include those that have a built-in safety feature or mechanism and “needleless systems” that effectively reduce the risk of an exposure incident. Some examples of safer devices (including animation of how they work) can be found on the OSHA website.

Sharps precautions are an example of work controls that are put in place to minimize exposure risk. These precautions apply to any contaminated object that can penetrate the skin including needles, scalpels and glass objects. Specific considerations for needles and broken glass are outlined below:

Contaminated Needles
  1. Contaminated needles are not to be bent, broken, recapped, or otherwise manipulated by hand (e.g., needle tip removal), unless it can be demonstrated that no alternative is feasible.
  2. If recapping or needle removal must be performed manually it must be accomplished through the use of a mechanical device or a one-handed technique.
  3. Immediately after use, contaminated reusable sharps must be placed in a puncture-resistant container. Contaminated disposable needles are to be placed in sharps containers described above and autoclaved before disposal.
  4. Sharps containers will be prepared for disposal once they are 2/3 full.
Broken Glassware 
  1. If broken glassware is possibly contaminated, workers should not handle it directly. Instead, workers must wear gloves and use mechanical means to pick it up (e.g., brush and dust pan, tongs, forceps).
  2. If the glassware is biohazardous, it should be handled as described in UNC-Chapel Hill’s Biological Waste Disposal Policy.

The use of sharps containers for glass Pasteur pipette disposal has caused safety concerns, particularly in BSCs designated at BSL-2. As previously stated, overfilling sharps containers increases the risk of sharps injuries and occupational exposures. An acceptable disposal alternative for glass pipettes in the BSC is a receptacle like the Whitney Products Pipet Keeper.

Whitney Products Pipet Keeper

 

Biological Safety Cabinets

BSCs must be used when conducting procedures with a potential for creating aerosols or splashes of blood or OPIM. These procedures include:

  1. Centrifuging.
    1. Note: If the centrifuge has sealed rotor heads, or if safety cups will be used, centrifuging can be conducted outside the BSC provided that the rotor heads or safety cups are opened in the BSC.
  2. Grinding, blending, vigorous shaking or mixing.
  3. Sonic disruption.
  4. Opening containers whose internal pressures differ from ambient.
  5. Inoculating animals intranasally.
  6. Harvesting infected tissues from animals or embryonate eggs.
  7. Conducting experiments with high concentrations or large volumes (greater than 10 liters).

Improper use of BSCs can lead to loss of containment, compromising sample integrity or resulting in potential exposure to infectious material. The following practices should be followed when using the BSC:

  1. Decontaminate work surfaces before and after use.
  2. Wear appropriate PPE for the biological agent you are working with at all times, even when using a BSC.
  3. Do not crowd the work surface. Only keep materials needed for the procedure inside the BSC.
  4. Segregate clean and contaminated items on the work surface and try to keep workflow going from “clean to dirty.”
  5. Limit movement in and out of the BSC as it disrupts the air barrier.
  6. Do not use bunsen burners or other flame sources inside the BSC.
  7. Minimize disruption of BSC airflow by placing it away from doors, air vents, and common walkways. If another lab member is working in the BSC, avoid walking near the BSC or performing tasks that may create air currents (e.g., sweeping).

Dispose of pipettes inside the BSC using either a horizontal tray filled wth disinfectant or a small biohazard bag as the waste receptacle. Do not use a container outside of the BSC. As stated in #5, moving in and out of the BSC disrupts the air barrier and can lead to accidental release or exposure depending on the procedure and biological agent. Engineering controls must be maintained on a regular schedule. While annual recertification is only required for research involving agents at Risk Group 2 or higher, it is highly recommended that BSCs be certified annually when used for human blood or OPIM. To have a BSC certified, contact Precision Air Technologies (919-962-5507).

H. Work Practice Controls

Work Practice Controls reduce likelihood of exposure by altering the method for a procedure or task. Sharps precautions, as discussed above in “Sharps Elimination, Safer Needle Devices, and Sharps Precautions,” is one example of this type of safety control. Additional work practice controls are discussed below:

Hand Hygiene

Hands and any other contaminated skin are to be washed with soap and water immediately or as soon as feasible following contact of such body areas with blood or other potentially infectious materials. Hand washing facilities must be readily accessible and should be located within the laboratory where the blood/OPIM is used. Hands are to be washed immediately or as soon as feasible after removal of gloves or other personal protective equipment.

Incidental Exposure Mitigation

  1. Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses are prohibited in work areas where there is reasonable likelihood of occupational exposure. For more information, refer to Chapter 3 of the UNC-Chapel Hill Laboratory Safety Manual.
  2. Storage of food and drink is prohibited in refrigerators, freezers, shelves, BSCs, or on countertops or bench tops where blood or other potentially infectious materials are present.

Mouth pipetting/suctioning of blood or other potentially infectious materials is prohibited. Mechanical pipetting devices must be used.

Minimize Splashing

  1. All procedures involving blood or other potentially infectious materials must be performed in such a manner as to minimize splashing, spraying, spattering, and generation of droplets of these substances.
  2. When working in a BSC, sample handling will be performed at least 6 inches interior to the front edge of the work surface.

Labels

BIOHAZARD warning labels are posted when there is a chance that germs, including BBPs, may be present. Common places these labels are found are on freezers, incubators, centrifuges, BSCs, waste containers, etc. which are used with blood or other potentially infectious material; and other containers used to store, transport or ship blood or other potentially infectious materials. According to OSHA, BIOHAZARD warning labels must include the following legend: Universal Biohazard Symbol, and be fluorescent orange or orange-red with lettering or symbols in a contrasting color. Labels are affixed as close as feasible to the container by string, wire, adhesive, or other method that prevents their loss or unintentional removal.

Biohazard Warning Label

 

Specimen Containers

Specimens of blood or other potentially infectious materials are to be placed in a closed container, which displays a BIOHAZARD warning label and prevents leakage during collection, handling, processing, storage, transport, or shipping. If the specimen could puncture the container or if outside contamination of the primary container occurs, the primary container is to be placed within a second closable, labeled, leak-proof container.

Contaminated Equipment

All equipment (i.e. freezers, refrigerators, centrifuges, etc.) potentially contaminated with blood or OPIM must be labeled with the biohazard warning symbol. Contaminated equipment must be decontaminated with an EPA registered tuberculocidal disinfectant or a solution of 5.25 percent sodium hypochlorite, (household bleach), diluted between 1:10 and 1:100 with water prior to servicing or shipping. Portions of the equipment not feasible for decontamination are to be designated with a BIOHAZARD warning label and the information must be communicated to service personnel.

I. Personal Protective Equipment

Personal protective equipment (PPE) is specialized clothing or equipment worn by an employee for protection against a hazard. General work clothes (including uniforms) are not PPE. Whenever there is the potential for occupational exposure, personal protective equipment such as gloves, gowns, laboratory coats, face shields or masks and eye protection must be available and utilized. Personal protective equipment in the appropriate sizes is to be readily accessible at the worksite or issued to employees. If a garment(s) is penetrated by blood or other potentially infectious materials, the garment(s) must be removed immediately or as soon as feasible.

All personal protective equipment must be removed and placed in a designated container (for storage, decontamination, or disposal) prior to leaving the work area. PPE must not be worn outside of the laboratory area. Gloves must be removed prior to leaving the laboratory. DO NOT wear gloves on elevators or use them to open doors or touch equipment (i.e. phones, computers) that others will be handling without gloves.

The minimum PPE required for handling blood/OPIM in laboratories is gloves, safety glasses (or goggles) and lab coats. Additional PPE such as surgical masks or face shields may be required for procedures with high probability for splashing.

Gloves

Gloves are to be worn when it can be reasonably anticipated that the employee may have hand contact with blood, other potentially infectious materials, mucous membranes, and non-intact skin; and when handling or touching contaminated items or surfaces. Disposable (single use) gloves such as surgical or examination gloves must be replaced as soon as practical when contaminated or as soon as feasible if they are torn, punctured, or when their ability to function as a barrier is compromised. Disposable (single use) gloves are not to be washed or decontaminated for re-use. Utility gloves may be decontaminated for re-use if the integrity of the glove is not compromised. However, they must be discarded if they are cracked, peeling, torn, punctured, or exhibit other signs of deterioration or when their ability to function as a barrier is compromised. Hypoallergenic gloves, glove liners, powder less gloves, or other similar alternatives are to be readily accessible to those employees who are allergic to the gloves normally provided.

Masks, Eye Protection, and Face Shields

Masks in combination with eye protection devices such as goggles or glasses with solid side shields, or chin-length face shields, are to be worn whenever splashes, spray, spatter, or droplets of blood or other potentially infectious materials may be generated and eye, nose, or mouth contamination can be reasonably anticipated. Regular prescription glasses are not considered eye protection and safety glasses, goggles, or face shields must be worn over these glasses.

Lab Coats, Gowns, Aprons, and Other Protective Body Clothing

Appropriate protective clothing such as, but not limited to, lab coats, gowns, aprons, or similar outer garments are to be worn in occupational exposure situations. The type and characteristics will depend upon the task and degree of exposure anticipated. Your Principal Investigator will determine which PPE is appropriate for the work that you perform.

J. Housekeeping

Laboratories are to be maintained in a clean and sanitary condition. An appropriate written schedule for cleaning and method of decontamination are to be determined and implemented, based upon the location within the facility, type of surface to be cleaned, and tasks or procedures being performed in the area.

All equipment and work surfaces are to be cleaned and decontaminated with an appropriate disinfectant after completion of procedures, or, immediately after spills. Suitable disinfectants include those that are tuberculocidal (e.g. Vesphene, OMNI II Disinfectant) or a solution of 5.25% sodium hypochlorite (household bleach) diluted to 1:10 to 1:100 with water. Fresh solutions of diluted household bleach must be made daily (every 24 hours). Please see OSHA Directive CPL 02-02-069 (“Enforcement Procedures for the Occupational Exposure to Bloodborne Pathogens”) for more information.

Protective coverings, such as plastic wrap, aluminum foil, or imperviously-backed absorbent paper used to cover equipment and environmental surfaces, are to be removed and replaced as soon as feasible when they become overtly contaminated or at the end of the work shift if they may have become contaminated.

All bins, pails, cans, and similar receptacles intended for reuse which have a reasonable likelihood for becoming contaminated with blood or other potentially infectious materials are to be inspected and decontaminated on a regularly scheduled basis and cleaned and decontaminated immediately or as soon as feasible upon visible contamination.

Employees should handle contaminated laundry as little as possible. Employees must bag or containerize contaminated laundry at the location where the laundry was used. Employees must not sort or rinse contaminated laundry in the location of use. Employees must place and transport contaminated laundry in bags or containers labeled or color-coded. Employees must place and transport the laundry in bags or containers which prevent soak-through and/or leakage of fluids to the exterior whenever contaminated laundry is wet and presents a reasonable likelihood of soak-through of or leakage from the bag or container. Employees must not take home contaminated clothing or other laundry such as lab coats for cleaning. Personal clothing contaminated with blood or other potentially infectious material will be cleaned by a laundry service with arrangements being made through the employee’s department. Information about laundry services can be found at the UNC-Chapel Hill Laundry website.

Clean-up of Blood Spills

Spills may occur when containers of blood or other potentially infectious materials (OPIM) are dropped in the laboratory or may occur when an injured person drips blood on the floor. Employees who are exposed to blood or OPIM are to be thoroughly familiar with emergency and decontamination procedures so that the contamination is contained and exposure of individuals is minimized. The following procedure is suggested for clean-up of blood spills in research laboratories at UNC-Chapel Hill:

  1. Evacuate the area and allow 30 minutes for aerosols to dissipate prior to spill cleanup.
  2. Protective clothing should be worn when entering the spill area. Gloves, safety glasses or goggles, and a lab coat or disposable coveralls should be worn. For spills on the floor, a gown that may trail the floor when bending down should not be worn.
  3. Pour disinfectant solution around the spill and allow the liquid to flow into the spill. Paper towels soaked in disinfectant may be used to cover the area. Suitable disinfectants include those that are tuberculocidal or a solution of 5.25% sodium hypochlorite (household bleach) diluted 1:100 with water. To minimize re-aerosolization, avoid pouring the disinfectant solution directly onto the spill. Allow at least 20 minutes of contact time before cleaning up spill.
  4. For small liquid spills, wipe up spill with paper towels.
  5. For large spills, a bucket with 1:100 dilution of sodium hypochlorite and mop should be used.
  6. For large spills or spills containing sharp materials (broken glass, plastic), use a dust pan and squeegee to transfer contaminated materials (paper towels, glass, liquid, etc.) into an orange biohazard bag, tape or tie the bag closed and place in a second orange biohazard bag. Place the dustpan and squeegee in a separate biohazard bag for autoclaving.
  7. All potentially contaminated waste and PPE should be placed in biohazard bags for autoclaving.

K. Waste Disposal

Regulations and Requirements

The OSHA Bloodborne Pathogens Standard regulates the containment and labeling of blood and certain waste which may be contaminated with blood, as well as needles and other sharps. The North Carolina medical waste rules (15A NCAC 13 B .1200), require that “Regulated Medical Waste,” defined as “blood and body fluids in individual containers greater than 20 ml, microbiological waste, and pathological waste,” be treated before disposal in order to render the waste nonhazardous. Primary methods for treating biological waste in UNC-Chapel Hill campus research laboratories differ from those at UNC Hospitals. All biohazard waste generated on UNC-Chapel Hill campus must be treated by the lab prior to being transferred by housekeeping staff to an outdoor dumpster, and then transported to the landfill. In contrast, UNC Hospitals incinerates the majority of their biological waste onsite, mostly due to confidentiality issues pertaining to patient samples.

Most UNC-Chapel Hill campus laboratory generated biohazard waste, as defined above, falls under the category of “microbiological waste” as defined in the North Carolina medical waste rules. Critical elements of UNC-Chapel Hill Campus Biological Waste Disposal Policy are described below. The most commonly used effective treatment method for research laboratories under this policy is steam disinfection (autoclaving). Only by following these requirements can the perception and the fact of biosafety be achieved, from the laboratory worker generating the waste to the UNC support staff treating and transferring the waste to the landfill employees hauling and burying the waste. Please see the UNC-Chapel Hill Biohazard Waste Management webpage for more information.

Definition of Laboratory Generated Biohazard Waste

Biohazard waste at UNC-CH includes potentially infectious material such as blood and body fluids, cell lines (primary and established) and waste contaminated or potentially contaminated from research and teaching activities requiring containment at BSL 1, 2, or 3, or animal or plant BSL 1, 2, or 3. Biohazard waste also includes materials contaminated or potentially contaminated during the manipulation or clean-up of material requiring BSL 1, 2, or 3 such as disposable culture dishes, devices used to transfer, inoculate, and mix cultures, liquid or solid media and collection flasks, gloves, pipettes, sharp items and animal carcasses and bedding. Biohazard waste originating from designated BSL-2 or greater containment areas must indicate type of material (agent, toxin, rDNA, etc.) on the Schedule F (Biological Hazards) section of the Laboratory Safety Plan. Laboratories with biohazard wastes not specifically addressed by this document (such as waste with multiple hazards, e.g. chemical or radioactive biohazard waste) should consult with EHS for alternative treatment and disposal methods.

Biohazard Waste Collection Methods

Refer to the chart below for a snapshot of the four most common biohazard waste collection methods. These methods are described in greater detail in the UNC-Chapel Hill Biohazard Waste Disposal Policy. For Contaminated animal carcasses, body parts, and bedding, refer to that particular section below for disposal guidance.

Biohazard Waste Disposal Chart

 

Contaminated Sharps

Sharps include items such as razor blades, scalpels, lancets, syringes with/without needles, slide covers, and specimen tubes. Puncture resistant plastic containers used to collect sharps contaminated under the definition of biohazard waste (above) must bear the biohazard symbol marked with an “x” using autoclave indicator tape. The OSHA Bloodborne Pathogens Standard requires laboratories to minimize their use of sharps whenever possible and that needles are not to be recapped, purposely bent, broken, or otherwise manipulated by hand. To avoid accidents related to overfilling the containers, remove the containers for treatment or disposal when they are 2/3 full. Containers of contaminated sharps are to be autoclaved. After autoclaving, ensure the containers are capped tightly prior to disposal for removal by housekeeping. New labels are available on the EHS website.

Contaminated Sharps Label

 

Research Lab/Clinic Pipetting

For large-scale collection of Glass (Pasteur) and plastic pipettes contaminated under the definition of biohazard waste (above), line a puncture resistant outer container (such as the package the pipettes came in) with an orange autoclave bag. To avoid handling a bag full of pipettes, place the indicator tape “x” over the bag’s biohazard symbol prior to loading the bag with pipettes. The hard walled outer container itself should bear a biohazard symbol marked with autoclave tape. For frequently removed small scale collection (such as sterile pipetting in a BSC), line a small orange autoclave bag inside a long, thin, hard-walled collection container. Plan to fill this container with appropriate disinfectant upon beginning (may require liquid disposal authorization) or, when finished, loosely close the bag, disinfect the outside of the bag, and transfer it to your larger scale pipette collection container located outside of the BSC.

Pipette tips are to be collected on the benchtop in a small autoclave bag lining a wire stand or other container bearing the biohazard symbol. Loosely close the bag to allow for steam penetration and place with other solid biohazard waste.

Contaminated Solids

Contaminated solids consist of culture dishes, flasks, Petri dishes, solid waste cultures/stocks from the testing and production of biologicals, gloves, gowns, masks, and other solid material potentially contaminated under the definition of biohazard waste (above). Line a biohazard waste collection container with an orange autoclave bag bearing an indicator tape “x” over the biohazard symbol. The outer waste collection container must red in color with a lid and a biohazard symbol so it will not be mistaken for regular trash by housekeeping.. Remove bags at 2/3 full. Never place glass in these containers.

Liquids

Biohazard liquids consist of human blood, animal blood, body products, body fluids, liquid growth media, etc. Autoclaved liquid wastes may be discharged directly to the sanitary sewer (in accordance with the University sewer disposal policy). If this is not feasible visit the EHS Chemical Treatment of Liquid Microbiological Waste webpage to evaluate if chemical treatment of your liquid biohazard waste requires an application for approval to the NC Medical Waste Division.

Remember, for disposal of any chemically treated liquid biohazard waste down the sanitary sewer, care is to be taken to avoid splash and the drains are to be flushed with generous amounts of water.

Contaminated Animal Carcasses, Body Parts, and Bedding

Animal carcasses are disposed of through the UNC-Chapel Hill Division of Comparative Medicine (DCM; 919-962-5335). Animal carcasses and animal body parts from uninfected animals, transgenic animals, animals inoculated with infectious agents, and animal contaminated with carcinogens/chemicals are disposed of by incineration. These materials are to be placed in incineration boxes provided by the DCM. (No needles or other type of metal and no PVC plastic are to be placed in the collection boxes. Use only non-PVC plastic bags.) Carcasses contaminated with radioisotopes are picked up by EHS.

L. HIV and HBV Research Laboratories

Research laboratories engaged in the culture, production, concentration, experimentation, and manipulation or HIV and HBV are to carry out their procedures at  BSL-2 with BSL-3 Practices. This does not apply to clinical or diagnostic laboratories engaged solely in the analysis of blood, tissues, or organs.

The Environment Health and Safety office conducts annual inspections of HIV research laboratories, to ensure adherence to applicable biosafety practices.

Training

Employees working in HIV or HBV research laboratories must be advised of the potential hazards of working with HIV. They are required to read all written procedures and follow all procedures. These employees receive annual training from EHS that includes the following:

  • OSHA Bloodborne Pathogen Standard,
  • BSL-3 practices,
  • emergency procedures, and
  • medical surveillance.

The Principal Investigator is required to ensure that:

  1. Employees demonstrate proficiency in standard microbiological practices and techniques and in the practices and operations specific to the facility before being allowed to work with HIV or HBV.
  2. Employees have prior experience in the handling of human pathogens or tissue cultures before working with HIV or HBV. A training program must be provided to employees who have no prior experience in handling human pathogens. Initial work activities must not include the handling of infectious agents. A progression of work activities is to be assigned as techniques are learned and proficiency is developed. Employees are to participate in work activities involving infectious agents only after proficiency has been demonstrated.

Work Practices

The following work practices are required for HIV in addition to the work practices described for working with blood and OPIM:

  1. Written policies and procedures must be established for access control so that only persons who have been advised of the potential biohazard, who meet any specific entry requirements, and who comply with all entry and exit procedures are allowed to enter the work areas and animal rooms. Access to the lab is restricted to persons authorized by the PI.
  2. All activities involving HIV must be conducted in a BSC and not on the open bench.
  3. Employees must wear the following when working with HIV:
    • a closed-front gown,
    • gloves,
    • safety glasses/goggles, and
    • a surgical mask.
  4. Vacuum lines (building or vacuum pump) must be protected with liquid disinfectant traps and HEPA filters as shown below. The filter should be dated and changed on a regular schedule (depending on use).

    Vacuum Line Protection

    An in-line HEPA filter should be in place at BSCs and wherever media from potentially infectious cell cultures is being drawn off.

  5. Centrifuge safety cups and/or sealed centrifuge rotors must be used to contain aerosols. Safety cups must only be opened inside of the BSC.
  6. Containment caging is required for animals.
  7. Use of needles and syringes must be used only when there is no other alternative, i.e. parenteral injection and aspiration of fluids from animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units can be used for these purposes. Needles cannot be bent, sheared, or recapped and must be placed immediately into sharps disposal containers as described above.
  8. Spills, accidents, and exposures must be reported to the Principal Investigator and EHS.

Medical Surveillance

Employees that work with HIV must be offered HIV testing every 6 months.

Laboratory Facilities

HIV laboratory facilities must meet, at a minimum, the following requirements:

  1. Laboratory doors must be self-closing and must be kept closed when work involving HIV or HBV is in progress. Keycard or combination locks are required to control access to the lab.
  2. Signs must be posted at the entrance to work areas in HIV and HBV Research Laboratory and Production Facilities. These signs are to be fluorescent orange-red or predominantly so, with lettering or symbols in a contrasting color. The signs must have the following information on them:
    • the Universal Biohazard Symbol,
    • the name of the infectious agent,
    • special requirements for entering the area, and
    • the name and telephone number of the laboratory director or other responsible person.
  3. The HVAC system must create directional airflow from “clean” areas into the laboratory. The exhaust air cannot be recirculated to other areas of the building.
  4. A hand washing facility must be available in the laboratory.
  5. An eyewash must be available in the laboratory. The eyewash must be tested weekly to ensure proper function and a log of the testing must be maintained.
  6. A BSC must be available in the laboratory. The BSC must be certified annually.
  7. An autoclave must be available for decontamination of infectious waste.

M. Hepatitis B Vaccination

Hepatitis B vaccination must be made available after the employee has received information and training regarding the vaccine and within 10 working days of initial assignment to all employees who have occupational exposure unless the employee has previously received the complete hepatitis B vaccination series (and can provide documentation), antibody testing has revealed that the employee is immune, or the vaccine is contraindicated for medical reasons. Employees who decline to accept hepatitis B vaccination must sign the declination statement in Appendix B. If you decline the vaccination, you can be vaccinated at a later date, free of charge

The HBV vaccination involves a series of three injections, the second administered one month following the first, and the third administered six months following the second injection. If an employee terminates their employment before finishing all three injections, the University is not responsible for providing the remaining injections.

Procedures for Requesting a Vaccination

Employees who have the potential for occupational exposures must obtain their vaccine through the UNC-Chapel Hill Employee Occupational Health Clinic (UEOHC) The employee must call the UEOHC (919-966-9119) to schedule the first appointment. The UEOHC will schedule subsequent appointments to complete the vaccination series.

N. Post-Exposure Procedures

An exposure incident is a specific

  • eye, mouth, or other mucous membrane;
  • non-intact skin; or
  • parenteral contact

with blood or OPIM that results from the performance of an employee’s duties. Employees must follow the procedures listed below if an exposure incident occurs.

Percutaneous exposure (needle sticks, cuts, animal bites or scratches)

Remove contaminated gloves and if possible, allow the wound to bleed freely for a minute. Wash the wound with soap and water for five (5) minutes and apply sterile gauze or a bandage, if necessary. Decontaminate and remove protective lab clothing and proceed immediately to the UEOHC or the Emergency Room (ER).

Mucous membrane exposure

Rinse tissue surface with copious amounts of water. Eyes will be irrigated for at least five (5) minutes using the emergency eye wash station. Decontaminate and remove protective lab clothing and proceed immediately to the UEOHC or the ER.

O. Post-Exposure Evaluation and Follow-Up

After any exposure event, employees must:

During daytime hours (8:30 a.m. 4:30 p.m., M-F)

Go to the UEOHC for treatment, consultation, assessment, and documentation of exposure.

After-hours

Call Healthlink (919-966-7890) to report the BBP exposure and request that the doctor on call for UEOHC after-hours BBP exposures be called. The on-call doctor will determine the need for immediate prophylaxis and if needed, direct the worker to meet the doctor in the ER or otherwise arrange for appropriate blood tests to be drawn and medications to be dispensed.

The post exposure medical evaluation will include documentation of routes of exposure and circumstances of incident, identification of source individual and testing (if possible), blood tests for HIV, HBV with consent from employee and post exposure prophylaxis and counseling.

The Principal Investigator and EHS must be notified of all exposures. An Employer’s Report of Injury to Employee form (Form 19) must be completed by the employee at UEOHC. OSHA regulations require that this form be filed with the Environment, Health and Safety Office within 48 hours of the incident. The Environment Health & Safety Office will investigate the circumstances of the exposure incident. A report will be made regarding the incident, and recommendations will be made to avoid further exposure incidents.

The University’s protocol for management of occupational exposures to HIV was developed by infectious disease specialists in the Department of Medicine and is identical to the protocol followed by the UNC Hospitals Employees Health Clinic. Current protocols for HIV post-exposure prophylaxis, necessitate immediate reporting of occupational exposures, so that administration of antiretroviral prophylaxis can be promptly initiated.

Billing

Charges for necessary services will be billed to the Environment, Health and Safety Office and paid from the University’s workers’ compensation account. Workers’ compensation will also pay for any necessary follow-up.

Medical Records

Medical records will be kept in confidentiality at the UEOHC. Records are not disclosed or reported without the employee’s express written consent to any person within or outside the workplace except as may be required by law. Employee medical records are kept for at least the duration of employment plus 30 years.

Student Exposures

Student Health Services (SHS) evaluates BBP exposures for UNC-Chapel Hill students including health affairs students. Students must go to the Student Health Clinic if the exposure occurs when SHS is open. The Acute Care Physician Extender or the on-call physician for SHS will evaluate the student. If the exposure occurs when SHS is closed, the student will call HealthLink at 919-966-7890. HealthLink will contact the SHS physician on-call who will then handle the initial evaluation and refer to the Infectious Disease Fellow if necessary.

P. Training

The OSHA Bloodborne Pathogen Standard requires that annual training be provided to all employees with the potential for occupational exposures. The required training is a condition for employment for all employees with the potential for occupational exposures. UNC-Chapel Hill provides online Bloodborne Pathogen training for laboratory employees.

This online training provides the following:

  • an overview of the requirements of the OSHA Bloodborne Pathogens Standard,
  • an explanation of the epidemiology and symptoms of bloodborne diseases, and
  • an outline of the University’s exposure control plan.

The Environment, Health and Safety Office will also conduct classroom sessions of this training at the request of any employee or department.

EHS keeps all training documentation.

Q. Inspection

EHS will inspect annually all facilities covered by the OSHA Bloodborne Pathogen Standard. The inspection documents that all employees covered by the standard have received training and been offered the hepatitis B vaccine. The supervisor must document in the lab safety plan that engineering controls and work practices to prevent occupational exposures are in place.

R. Exposure Control for UNC-Chapel Hill Employees Located in Other Counties

UNC-Chapel Hill employees working in other counties are to make arrangements to receive training, HBV vaccinations, and post-exposure evaluations and follow-up at other institutions or facilities located in their respective cities. They must send documentation for the required training and HBV vaccination records to their respective departments in Chapel Hill and to EHS. They must also receive a copy of the UNC-Chapel Hill Exposure Control Plan. UNC-Chapel Hill employees working at another institution must follow the Exposure Control Plan for that institution.

S. Appendix A: Job Classifications Identified as Potential Occupational Exposure

  • Access Control Shop Lead Technician
  • Access Installation Technician
  • Access Technician II
  • Access Technician III
  • Acting Director
  • Adjunct Assistant Professor
  • Adjunct Associate Professor
  • Adjunct Instructor
  • Adjunct Professor
  • Administrative Manager
  • Administrative Officer
  • Administrative Secretary
  • Administrative Services Assistant
  • Administrative Support Associate
  • Administrative Support Specialist
  • Animal Research Technician
  • Animal Transport Coordinator
  • Applications Analyst
  • Applications Specialist
  • Assistant Coach
  • Assistant Director
  • Assistant Professor
  • Assistant Teacher
  • Assistant Vice Chancellor
  • Associate Biological Safety Officer
  • Associate Director
  • Associate Professor
  • Associate Vice Chancellor
  • Audiologist
  • Biological Safety Specialist
  • Building Environmental Technician
  • Business Officer
  • Business Services Coordinator
  • Cage Processing Technician
  • Campus Hardware Lead Technician
  • Campus Maintenance Manager
  • Cell Culture Core Manager
  • Chemical Hygiene Officer
  • Clerical Support Associate
  • Clerical Support Supervisor
  • Clinic Assistant
  • Clinic Manager
  • Clinical Assistant Professor
  • Clinical Associate Professor
  • Clinical Dispensary Support
  • Clinical Fellow
  • Clinical Instructor
  • Clinical Nurse
  • Clinical Professor
  • Clinical Research Assistant
  • Clinical Research Associate
  • Clinical Research Coordinator
  • Clinical Research Nurse
  • Clinical Research Specialist
  • Clinical Support Associate
  • Clinical Trials Coordinator
  • Community Educator
  • Community Liaison Coordinator
  • Construction Manager
  • Day Care Teacher
  • Dean
  • Dental Assistant
  • Dental Assistant Supervisor
  • Dental Equipment Technician
  • Dental Hygienist
  • Dental Lab Case Coordinator
  • Dental Patient Records Supervisor
  • Dental School Receptionist
  • Dental Technician
  • Dentist
  • Department Manager
  • Detective
  • Direct Care Shift Leader
  • Direct Care Staff
  • Direct Care Supervisor
  • Director
  • Distinguished Associate Professor
  • Distinguished Professor
  • Division Administrator
  • Electrician
  • Electronics Specialist
  • EMCS Operator
  • Endocrine Assay Lab Director
  • Environmental Compliance Officer
  • Environmental Specialist
  • Ergonomist
  • Export Compliance Shipping Specialist
  • External Operations Supervisor
  • Facility Maintenance Supervisor
  • Facility Maintenance Technician
  • Facility Manager
  • Financial Counselor
  • Fire Safety Inspector
  • Fire Safety Professional
  • Fire Sprinkler Technician
  • Functional Assay Technician
  • General Utility Worker
  • Graduate Assistant
  • Hazardous Materials Manager
  • Hazardous Materials Specialist
  • Hazardous Materials Tech
  • Head Museum Guard
  • Health Physics Technician
  • Health Physics Technologist
  • Hemophilia Coordinator
  • Hemophilia Nurse
  • Housekeeper
  • Human Applications Lab Manager
  • Human Services Practitioner
  • Human Services Program Support Technician
  • HVAC Mechanic
  • HVAC Technician
  • Industrial Hygiene Manager
  • Industrial Hygienist
  • Insulation Project Manager
  • Insulator
  • Insurance Specialist
  • Internal Operations Supervisor
  • Job Coach
  • Lab Assistant
  • Lab Manager
  • Lab Processing Coordinator
  • Lab Research Specialist
  • Lab Technician
  • Laboratories Manager I
  • Laboratory Animal Technician
  • Laboratory Assistant
  • Laboratory Manager
  • Laboratory Research Specialist
  • Laboratory Technician
  • Lead Health Physics Technologist
  • Lead Technician
  • Lead Worker
  • Lecturer
  • Licensed Practical Nurse
  • Lieutenant
  • Lieutenant Of Investigations
  • Life Safety Technician
  • Mail Center Manager
  • Mail Clerk
  • Mail Sorter
  • Maintenance Foreman
  • Maintenance Mechanic
  • Maintenance Mechanic III
  • Maintenance Mechanic IV
  • Maintenance Mechanic V
  • Manager Of Patient Accounts
  • Materials Management Coordinator
  • Materials Manager
  • Medicaid Specialist
  • Medical Assistant
  • Medical Diagnostic Technician
  • Medical Lab Supervisor
  • Medical Lab Technician
  • Medical Lab Technician II
  • Medical Lab Technologist
  • Medical Laboratory Supervisor
  • Medical Laboratory Technician
  • Medical Laboratory Technologist
  • Medical Outreach Liaison
  • Medical Supply Technician
  • Medical Therapeutic Specialist
  • Networking Technician
  • Nurse Clinician
  • Nurse Clinician II
  • Nurse Consultant
  • Nurse Interviewer
  • Nurse Practitioner
  • Nurse Supervisor
  • Nursing Assistant
  • Nursing Assistant II
  • Nursing/Medical Assistant
  • Occupational Field Hygienist
  • Occupational Field Specialist
  • Office Assistant
  • Office Supervisor
  • Oncology Research Nurse Coordinator
  • Operations Supervisor
  • Oral Pathology Lab Supervisor
  • Os1 Crew Leader
  • Parking Enforcement Officer
  • Patient Records Technician
  • Patient Relations Rep
  • Patient Relations Supervisor
  • Patrol Squad Lieutenant
  • Pharmacist
  • Pharmacy Technician
  • Physician Assistant
  • Physician Extender
  • Physician Extender I
  • Physician Extender II
  • Plumber
  • Police Officer
  • Postage Meter Operator
  • Postdoctoral Research Associate
  • Postdoctoral Trainee
  • Pre-Doctoral Fellow
  • Preschool Assistant
  • Preschool Supervisor
  • Preventive Maintenance Inspect
  • Process Development Specialist
  • Processing Assistant
  • Professional Nurse
  • Professor
  • Program Assistant
  • Program Coordinator
  • Program Manager
  • Project Coordinator
  • Project Interviewer
  • Project Manager
  • Property Security Officer
  • Property Security Supervisor
  • Psycho Educational Therapist
  • Public Health Nursing
  • Public Safety Officer
  • Public Safety Telecommunicator
  • RCA Coordinator
  • Recruitment Coordinator
  • Regulatory Assistant
  • Research Specialist
  • Research Analyst
  • Research Analyst Il
  • Research Assistant
  • Research Assistant Professor
  • Research Assistant Technician
  • Research Associate
  • Research Associate Professor
  • Research Coordinator
  • Research Fellow
  • Research Instructor
  • Research Intern
  • Research Lab Assistant
  • Research Lab Specialist
  • Research Nurse
  • Research Nurse Consultant
  • Research Nurse Coordinator
  • Research Operations Manager
  • Research Professor
  • Research Specialist
  • Research Study Coordinator
  • Research Study Nurse
  • Research Technician
  • Research Technician I
  • Research Technician II
  • Reserve
  • Reserve Officer
  • Resident
  • Resident & Student Clinic Nurse
  • Respiratory & Research Associate
  • Roofer
  • Safety Officer
  • Safety Prevention Officer
  • Security
  • Security Guard
  • Senior Hazardous Materials Specialist
  • Senior Laboratory Technician
  • Senior Research Specialist
  • Sergeant
  • Sheet Metal Mechanic
  • Social Research Assistant
  • Social Research Associate
  • Social Research Specialist
  • Social Work Practitioner
  • Social Worker
  • Spanish Interpreter
  • Student Worker
  • Student Services Assistant
  • Study Coordinator
  • Substance Abuse Counselor
  • Support Services Associate
  • Support Services Captain
  • Supported Employment Supervisor
  • Surgical Assistant
  • Surgical Assistant Supervisor
  • Systems Analyst
  • Systems Specialist
  • Teaching Assistant
  • Team Leader
  • Tech Support Analyst Tech
  • Support Specialist Tissue
  • Culture Specialist University
  • Industrial Hygienist
  • Vascular Surgery Physicians Assistant
  • Vehicle/Equipment Operator
  • Veterinary Technician
  • Visual Arts Specialist
  • Vocational Supervisor
  • Workplace Safety Manager
  • X-Ray Technician
  • Zone Manager

T. Appendix B: Hepatitis B Vaccine Declination Form

DIRECTIONS: Please complete the following if you have previously received the Hepatitis B Vaccination series or if you are declining the Hepatitis B Vaccination series. After printing and completing the form, you can place it in campus mail to the UEOHC at CB #1649. It will be reviewed by a clinic provider and you will be contacted if any further information is required. If you have any questions please call the UEOHC at 919-966-9119.

The Hepatitis B Vaccine Declination form is attached to this webpage.

V. Safety Practices for Work with Non-Human Primate Tissues & Body Fluids

A. Purpose

To identify and mitigate risks associated with unfixed NHP specimens including cells, tissues, organs and bodily fluids.

B. Biosafety Information

NHP specimens, such as those from macaque monkeys, may contain herpes B virus and Simian Immunodeficiency virus (SIV). All macaques regardless of their origin should be considered potentially infected with herpes B as animals with no detectable antibody titer are not necessarily B virus-free1. NHP specimens should be handled with strict barrier precaution protocols and injuries should be tended to immediately according to the recommendations of the B Virus Working Group led by the NIH and CDC2.

C. Cercopithecinae Herpes Virus 1 or Herpes B Virus: Risk Group 4 Organism

Herpes B virus occurs as a natural infection of Asiatic macaque monkeys and some 10% of newly caught rhesus monkeys have antibodies against the virus. Herpes B virus is the only member of the family of simplex herpesviruses that can cause zoonotic infections. Human infections have been identified in at least 50 instances, with approximately 80% mortality when untreated. There remains an approximate 20% mortality in the absence of timely treatment with antiviral agents3. From 1987-2004, five additional fatal infections bring the number of lethal infections to 29 since the discovery of the virus in 1933.

Zoonoses have been reported following virus transmission through a bite, scratch, or splash to the eyes, nose, mouth, or broken skin. Cases of B virus have been reported after exposure to monkey cell cultures and to central nervous system tissue. There is no apparent evidence of B virus infection in the animals or their cells and tissues, making it imperative that all suspected exposures be treated according to recommended standards. In most documented cases of B virus zoonosis, virus was not recovered from potential sources. The loss of five lives in the past two decades underscores that B virus infections have a low probability of occurrence, but when they do occur it is with high consequences.

D. Simian Immunodeficiency Virus (SIV): Risk Group 3 Organism

Retroviruses such as SIV, are transmitted by sexual contact, parenteral inoculation, or exposure to mucus membranes such as those in the eyes nose and mouth. SIV has been isolated from blood, CSF, and a variety of tissues of infected nonhuman primates. Limited data exist on the concentration of virus in semen, saliva, cervical secretions, urine, breast milk, and amniotic fluid.

Workers have been reported to develop antibodies to simian immunodeficiency virus (SIV) following exposures. One case was associated with a needle-stick that occurred while the worker was manipulating a blood-contaminated needle after bleeding an SIV-infected macaque monkey. Another case involved a laboratory worker who handled macaque SIV-infected blood specimens without gloves. Though no specific incident was recalled, this worker had dermatitis on the forearms and hands while working with the infected blood specimens. A third worker was exposed to SIV-infected primate blood through a needle-stick and subsequently developed antibodies to SIV. To date there is no evidence of illness or immunological incompetence in any of these workers.

E. Training

Research involving NHP specimens must be conducted at BSL-2+). The laboratory director is responsible for ensuring that all personnel comply with the following training requirements prior to working at BSL-2+.

Prior experience and specific training programs required:

All personnel must:

  • Complete UNC-Chapel Hill Laboratory Environment training.
  • Complete UNC-Chapel Hill training for BSL-2 procedures.
  • Demonstrate proficiency in standard microbiological practices and techniques.
    • BSL-2 proficiency is certified by the PI and the associated documentation is maintained in the Biosafety Manual.
  • Complete annual BSL-2+/Herpes B training given by EHS.

F. Laboratory Safety for Research Involving Non-Human Primate Specimens

Research involving NHP specimens must be conducted at Biosafety Level 2+ (BSL-2+) involving BSL-2 facilities with BSL-3 practices summarized below.

  • Access to the laboratory is restricted to the fewest number of individuals necessary to conduct experimental procedures.
  • Conducted in a placarded laboratory with a sink, eye wash and negative air pressure.
  • All experimental manipulations are performed within a BSC.
  • Centrifugation performed in sealed tubes within a centrifuge equipped with either sealed rotors or safety cups.
  • Use of sharps and glassware is minimized or eliminated if at all possible.
  • Personal Protective Equipment (PPE) includes double gloves, a dedicated lab coat or closed front gown, as well as eye and face protection. Disposable lab coats or gowns may be utilized, but must be disposed after use.
  • Infectious materials transported out of the laboratory must be in a sealed, leak-proof container labeled with a biohazard sticker.
  • Work surfaces must be disinfected prior to and after use as well as after spills.
  • Waste must be chemically treated or autoclaved prior to disposal.

BSL-4 containment, which is not available at UNC-Chapel Hill, is required for culture and propagation of herpes B virus.

Heat treatment (Greater than 56° Celsius for 1 hour) or adjusted pH (pH <5 or pH >11 after 10 minutes at 25° Celsius) inactivate herpes viruses5 and can be utilized to downgrade containment to BSL-2.

Cryogenic preservation vials or “cryovials” stored in the liquid phase of liquid nitrogen can rupture upon warming if liquid nitrogen has infiltrated them, resulting in an explosion hazard. Pieces of the cryovial may be propelled towards personnel resulting in physical injury, particularly to the eyes, and exposure to the cryovial’s contents. For this reason, cryogenic storage of NHP specimens is strongly discouraged. Contact the Biological Safety Officer for guidance if an experimental design requires storage in liquid nitrogen.

G. Routes of Exposure

B virus infection in humans usually occurs as a result of bites or scratches from macaques – a genus of Old World monkeys that serve as the natural host – or from direct or indirect contact of broken skin or mucous membranes with infected monkey tissues or fluids. The virus can be present in the saliva, feces, urine, or nervous tissue of infected monkeys and may be harbored in cell cultures derived from infected monkeys.

Possible routes of transmission to humans include

  • Bite or scratch from an infected animal
  • Sharps, needle stick from contaminated syringe
  • Scratch or cut from contaminated cage or other sharp-edged surface
  • Exposure to nervous tissue or skull of infected animal (especially brain)

B virus may survive for hours on the surface of objects, particularly on surfaces that are moist.

Factors that contribute to the severity of an infection include:

  • Deep cuts – difficulty with cleaning poses increased risk of infection.
  • Exposure to human head or neck – Virus has shorter route to central nervous system (brain).

The injury need not be severe for infection to occur, although non-penetrating wounds are thought to carry a lower risk of transmission.

H. Post-Exposure

Herpes B

Post-exposure prophylaxis for Herpes B is only offered for exposures involving macaque monkey samples. Superficial wounds/scratches and appropriately cleaned wounds (i.e. cleaned within five (5) minutes of exposure and for 15 minutes) are considered low risk. Post-exposure prophylaxis is recommended for the following groups and should be started within 72 hours of exposure:

  • Deep wounds and punctures (such as bites);
  • Puncture with needles containing material from the CNS, eyelids, or mucosa;
  • Laceration of the head, neck, and torso;
  • Inadequately cleaned skin exposure;
  • Exposure to high-risk source (a macaque that is ill, immunosuppressed, known to be shedding virus or that has lesions compatible with B virus disease); and/or
  • Puncture or laceration after exposure to objects contaminated with fluid from oral or genital lesions or fluid known to contain B virus.

Valacyclovir 1g by mouth every eight (8) hours for 14 days is the recommended post-exposure prophylaxis for adults and non-pregnant women. Acyclovir 800mg by mouth five (5) times a day for 14 days is an alternative regiment. (Cohen et al. Recommendations for prevention of and therapy for exposure to B virus (Cercopithecine Herpesvirus 1). Clinical Infectious Disease 2002;35:1191-1203)

The NHP tissue specimen involved in the potential Herpes B exposure/injury should be secured at less than 60° Celsius (unless it is whole blood in which case it should be kept at 2-6° Celsius) so that it may be used for confirmation purposes. Virologic and serologic analysis will be used to evaluate exposure if an employee develops symptoms of infection within 1-2 weeks of a laboratory incident.

If the employee is being seen at the ER, they should take a copy of the laboratory Standard Operating Procedure with them.

I. References

  1. Centers for Disease Control and Prevention. (2020). Biosafety in Microbiological and Biomedical Laboratories (6th ed.). See Section VIII-E: Viral Agents. https://www.cdc.gov/labs/BMBL.html
  2. The B Virus Working Group. Guidelines for prevention of Herpesvirus simiae (B virus) infection in monkey handlers. J Med Primatol. 1988;17(2):77-83.
  3. Cohen JI, Davenport DS, Stewart JA, et al. Recommendations for prevention of and therapy for exposure to B virus (Cercopithecine herpesvirus 1). Clin Infect Dis. 2002;35(10):1191-1203.
  4. Centers for Disease Control and Prevention. Fatal cercopithecine herpesvirus 1 (B virus) infection following a muccutaneous exposure and Interim Recommendations for worker protection. MMWR Morb Mortal Wkly Rep. 1998;47:1073-6,1083.
  5. Croughan WS and Behbehani AM. Comparative Study of Inactivation of Herpes Simplex Virus Types 1 and 2 by Commonly Used Antiseptic Agents. J. Clin. Microbiol.
  6. Weigler BJ, Biology of B Virus in Macaque and Human Hosts: A Review. Clin Infect Dis. 1992; 14(2): 555-567.

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