Biosafety Precautions for Research with Human Clinical Specimens that May Contain Infectious Agents, including SARS-CoV-2

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Work with all unfixed human tissues must be conducted according to Biosafety Level 2 containment and work practices. Given the potential for exposure to SARS-CoV-2, additional precautions may be required.  

Upper and lower respiratory system specimens, such as nasopharyngeal and oropharyngeal swabs, sputum, mucus, and lung tissues may present increased risk of exposure to SARS-CoV-2. Stool samples may also contain the virus.  Blood and serum pose lower risk than the upper and lower respiratory system specimens. 

When handling specimens that pose unknown or higher risk of exposure to SARS-CoV-2, use the following enhanced biosafety precautions: 

  1. Work with biohazards inside a certified Class II Biosafety Cabinet (BSC). Waste generated must be collected within the BSC.
  2. Take precautions to achieve minimal skin contact with biohazards.
  3. All potentially contaminated items must be decontaminated before removal from the biosafety cabinet. 

The enhanced precautions described here are based upon recommendations from Centers for Disease Control and Prevention, World Health Organization and the Public Health Agency of Canada.   These guidelines will be updated as new information becomes available. 

Virus isolation in cell culture and initial characterization of viral agents recovered in cultures of SARS-CoV-2 specimens may only be conducted in a Biosafety Level 3 (BSL-3) laboratory using BSL-3 practices. 

Routine diagnostic testing of specimens, such as the following activities, can be handled in a BSL-2 laboratory using Standard Precautions:

  • Staining and microscopic analysis of fixed smears
  • Pathologic examination and processing of formalin-fixed or otherwise inactivated tissues
  • Molecular analysis of extracted nucleic acid preparations
  • Final packaging of specimens for transport to diagnostic laboratories for additional testing (specimens should already be in a sealed, decontaminated primary container)
  • Using inactivated specimens, such as specimens in nucleic acid extraction buffer
  • Performing electron microscopic studies with glutaraldehyde-fixed grids

Biosafety level 2 with enhancements described in this document should be used for

  • Procedures with a high likelihood to generate aerosols or droplets
  • concentration of samples prior to inactivation (e.g., centrifugation of a bronchoalveolar lavage sample);
  • sample preparation for nucleic acid extraction, molecular testing of nucleic acids, and antigen and antibody assays
  • Processing or preparation of specimens
  • Nucleic acid extractions of specimens
  • Performing diagnostic test (not involving virus propagation)
  • heat/chemical inactivation of specimens

Risk Assessment

All laboratories should perform a specific risk assessment to identify and mitigate risks. 

The primary factors to consider in risk assessment and selection of precautions fall into two broad categories:

1. Agent hazards

a. Are known pathogens present in the tissues?
b. Potential for exposure based on information available from provider of tissue
c. Its capability to infect and cause disease in a susceptible human or animal host
d. Route of transmission (percutaneous exposure, inhalation, mucous membrane, fomites, ingestion)
e. Its virulence as measured by the severity of disease
f. The availability of preventive measures and effective treatments for the disease
g. Infective dose
h. Stability in the environment
i. Host range
j. Endemic nature

2. Laboratory procedure hazards

a. Use of sharps
b. Large volumes of culture
c. Administration to animals
d. Potential for activities to create aerosols:

  • Pipetting
  • Blenders
  • Non-self-contained centrifuges
  • Sonicators
  • Vortex Mixers
  • Spills

3. Laboratory and Staff

a. Experience of laboratory staff handling specimens
b. Immune status of laboratory staff

Note:  Consult with University Health Service by calling 8-5035 if you have concerns about your immune status due to pregnancy, underlying health conditions or use of prescribed medications prior to beginning research with potentially infectious materials. 

 

Personal Protective Equipment (PPE)

Minimum PPE

Dedicate PPE within your laboratory for your experiments. Do not wear PPE to other non-lab areas and remove prior to leaving the BSL 2 laboratory. Depending on the number of labs you have and the nature of the work, researchers may need multiple lab coats for each area to avoid potential cross contamination

 

  1. Lab coat, preferably disposable with cuffed sleeves
  2. Sleeve covers should be worn to minimize contamination of wrists and lab coat sleeves.
  3. Consider double gloving, particularly for all work within a biological safety cabinet (BSC).  The outer pair can be removed before exiting the biosafety cabinet and a new pair put on when re-entering the biosafety cabinet.
  4. Wear safety glasses and disinfect after each use with 70% ethanol and air dry. Do not share with others in the lab.
  5. Wear a chin-length face shield or safety glasses and a surgical mask if working outside of the biosafety cabinet with biohazards on the bench. This will protect the researcher’s facial mucous membranes from exposure in the event of a spill outside the biosafety cabinet during transfer of material to and from the incubator.

Removal of PPE/Hand Washing

  1. In general, the most contaminated item is removed first. Usually, these are the outer pair of gloves if two pairs of gloves are worn. Discard gloves into the regulated medical waste receptacle.
  2. If only a single pair of gloves have been worn, disinfect your gloves with your lab’s disinfectant (such as 70% ethanol) when you have finished your work and allow at least a 30 second contact time.
  3. Wet some paper towels with your lab’s disinfectant and set aside. Disinfectant wipes can also be placed in the PPE removal location for the decontamination of reusable items after removal.
  4. After this 30 second decontamination step, remove your lab coat or gown.
  5. After removing your lab coat or gown, check for any visible contamination on your lab coat if reusable. Spray these areas with your lab’s disinfectant and allow to air dry. If wearing a disposable gown or lab coat, place in the biomedical waste container after removal.
  6. Spray your gloves with your lab’s disinfectant and “wash” your gloved hands together for 30 seconds to disinfect them once again.
  7. Remove your face shield or safety glasses and mask if worn. Place disposable items in the regulated medical waste receptacle. Use disinfectant-wet paper towels or disinfectant wipes to wipe down the face shield and safety glasses with disinfectant and allow to air dry.
  8. Remove your inner gloves aseptically, or by avoiding contact with the exterior of each glove and in a manner, that prevents the exterior of either glove from contacting your skin. Discard gloves in the regulated medical waste container.
  9. Wash your hands with soap and water for 30 seconds. Close the sink faucet off with paper towels after use. Do not touch the faucet handles with your hands after washing to avoid potential re-contamination of your hands

Removal of PPE Graphic »

 

Biosafety Cabinet (BSC)

  1. Perform all work with specimens within a certified Class II BSC.
  2. Turn on fan and allow it to operate for at least 10 minutes before beginning work.
  3. Place all items required for the experiment within the biosafety cabinet before starting work.
  4. Wipe items down with disinfectant or spray with 70% ethanol prior to placement within the biosafety cabinet.
  5. Keep the front and rear grilles clear when working within the biosafety cabinet. Avoid blocking the rear grille.
  6. Remind lab mates to minimize traffic and work behind the operator, as this may interfere with cabinet airflow.
  7. Wipe items down with disinfectant prior to removal from the biosafety cabinet.
  8. Wipe down biosafety cabinet with disinfectant after use (work surface, grilles, sides, back and inside front view screen).
  9. Decontaminate liquid waste with household bleach diluted 10% against the volume of the waste. Allow at least a 30-minute contact time for full decontamination.
  10. Collect dry waste (e.g. gloves, pipet wrappers, paper towels) in a small biohazard bag inside the biosafety cabinet. Seal bag prior to removal from biosafety cabinet.
  11. Transport waste to autoclave in a leakproof container.

 

Collection of Specimens

  1. University students and staff responsible for collecting human specimens must receive training from Environmental Health and Safety.  Request training at ehs@princeton.edu.
  2. Following minimum personal protective equipment must be available: full face protection, such as a face-shield, or safety glasses and a surgical mask, gloves and either a lab coat or disposable gown.  Depending upon the sample type and collection method, EHS may also recommend an N-95 respirator.

Sample Packaging

  1. Ensure that the exterior of the primary tube, bag or other container is disinfected after collection or prior to transfer to another laboratory. See disinfection section for information on appropriate disinfectants.
  2. After a sufficient contact time, at least 3 minutes, label the container and place inside a clean secondary container.
  3. Both primary and secondary containers must be leak-proof.
  4. Place paper towels in the bottom of the secondary container to absorb any liquids if there was a leak in the primary container during transport.
  5. Place a biohazard label on the outside of the exterior transport container with the lab’s contact information.

Domestic/International Shipment of Samples

  1. Consult with EHS to determine proper shipment method. Suspected and confirmed SARS-CoV-2 patient specimens, cultures, or isolates must be shipped as UN 3373 Biological Substance, Category B by a trained person.
  2. Dried blood spots can be shipped by mail or other carrier with no reasonable expectations of occupational exposure to blood or other potentially infectious material. Dried blood spots must be triple packaged:

a. The primary container is the filter paper matrix that contains the absorbed and dried blood.
b. A secondary container must enclose the primary (filter paper) container. The secondary container should have a fold-over flap or an inner envelope to secure the contents.
c. The third level of containment is an outer envelope of sturdy, high-quality paper. These levels of containment provide reasonable safety from occupational exposure and maintain optimal specimen integrity
d. You must affix or print the international biohazard symbol on the either the primary or secondary container to meet U.S. Occupational Safety and Health Administration requirements.  Use of plastic, foil bags, or other airtight, leak-proof sealed containers may result in heat buildup and moisture accumulation.
e. The outer shipping container must have a complete return address and delivery address. No content markings are required on the outer shipping container.

Initial Processing/Lysing of Specimens

If you are handling specimens from COVID-19 patients or suspect patients for testing or genetic analysis:

1. Wear minimum PPE*
2. Disinfect and open specimen containers in a biosafety cabinet.
3. Follow the lysing product instructions and allow the required contact time prior to considering the specimens to be “inactivated.”

Note: +ssRNA viruses, like SARS-CoV-2, are considered infectious as full-length RNA, as it could still establish an infection if it can gain entry to a human cell. CDC recommends handling full-length RNA using BSL 2 containment.

4. Disinfect the exterior of items prior to removal from the biosafety cabinet with the disinfectant you have selected for your research.
5. Continue to utilize PPE for working with RNA from this virus outside of the biosafety cabinet. A lab coat, safety glasses and mask or chin-length face shield, and gloves.
6. Follow the PPE removal*and hand washing guidance*

 

High-Risk Procedures

Flow Cytometry

  1. High speed sorting of unfixed human cells can generate a large quantity of aerosols in the event of a clog or deflection.  If you are using the Molecular Biology Flow Cytometry/Cell Sorter Core laboratory, samples must be fixed prior to the sort.  You must have documentation that the fixation method is effective against SARS-Cov-2.
  2. If you have your own high-speed cell sorter, notify EHS to request an evaluation of your process and for testing the containment of your cell sorter for the containment of biohazards.

Centrifugation

centrifugation

  1. Use sealed rotors or safety buckets as secondary containment for centrifugation.
  2. Load and unload the rotor or safety buckets within the biosafety cabinet.
  3. Do not overfill primary containers, limit to < ¾ full. Wipe exterior of all centrifuge tubes with disinfectant before loading.
  4. Seal rotors or buckets and wipe down with disinfectant, remove outer gloves inside the biosafety cabinet before transport to the centrifuge.
  5. Wait 2-5 minutes after the run to allow aerosols to settle in the event of a spill.
  6. Transport sealed rotor or safety bucket to biosafety cabinet to complete your experiment. Don new pair of outer gloves before continuing your work inside the biosafety cabinet.
  7. Decontaminate the rotor or safety bucket by spraying with 70% ethanol and allowing to air dry.
  8. In the event of a release of specimen during centrifugation, close lid of centrifuge, leave laboratory and contact EHS at 609-258-5294.

Sharps Elimination/ Precautions:

  1. Avoid the use of glass Pasteur pipettes or needles and syringes. Substitute plastic for glass whenever feasible.
  2. Alternatives to glass Pasteur pipettes include: plastic pipettes, plastic transfer pipettes, plastic gel loading pipette tips and pipette tip extenders, aspirators, and flexible plastic aspiration pipettes.
  3. Some researchers will either score and break the end off a 1 ml or 5 ml plastic pipette or remove the wool plug and use for aspirating cultures.
  4. If sharps cannot be avoided, maintain a sharps container in the immediate vicinity of use and discard intact needles and syringes immediately after use. Use a one- handed disposal method (keep a hand behind your back or by your side, and do not place your other hand on or near the opening of the sharps container).
  5. Never ever recap.
  6. If you must remove the needle from the syringe, use forceps, tweezers, a Kelly clamp or small pliers for this purpose.

Other Equipment

  1. HEPA-filtered incubators are recommended for tissue culture growth of biological specimens

Decontamination and Disinfection

  1. All surfaces and equipment must be disinfected after use. This includes all surfaces within the biosafety cabinet, used research materials, equipment, bench tops and other work surfaces, transport and transfer containers.
  2. SARS-CoV-2 and bloodborne pathogens such as HIV, HBV and HCV are inactivated with disinfectants that contain 62 – 90% Ethanol, 1-5% bleach in water solutions (made fresh daily) and >0.5% Hydrogen Peroxide. If you wish to use other disinfectants, please select from this list: https://www.epa.gov/pesticide-registration/list-n-disinfectants-use-against-sars-cov-2

Best Practices for Disinfection

  1. Ensure that the area is cleaned prior to initiating the disinfection process where applicable.   Use a “double” disinfection process, where the first application ensures that the surfaces are cleaned and the 2nd application is the disinfection.
  2. Ensure that the chemical disinfectant that you select for decontamination has proven efficacy against the biohazards in use or anticipated.
  3. The amount of the chemical matters significantly. Follow the manufacturer’s recommendations for dilution if purchasing a commercial disinfectant.
  4. No disinfectant works immediately. Disinfectants must be left on the surfaces or items to be decontaminated for a specified time and this varies by individual biohazard. Contact times of 1, 3, 5 or 10 minutes or even longer may be needed. Apply disinfectant until surfaces are glistening wet and allow surface to air dry. If your disinfectant has a higher evaporation rate (e.g. alcohols), and a longer contact time is needed, you may need more than one application.
  5. Ensure that all surfaces are completely covered with the disinfectant. Merely spraying the disinfectant on a surface, especially if only applied quickly or lightly, can leave spaces in between the disinfectant drops.

Biomedical waste disposal

  1. Follow the University’s regulated medical waste procedures, found here.
  2. Wastes that contain human pathogens or higher risk recombinant DNA research wastes, must be autoclaved prior to disposal inside the regulated medical waste containers.
  3. Liquid biohazard waste can either be autoclaved on slow-exhaust or chemically inactivated using household bleach at a final concentration of 10% against the volume of the waste to be treated for a contact time of 30 minutes. Dispose of liquid waste via lab sink drain, flushed with cool water.
  4. Urine and blood can be disposed of via the sanitary sewer system. Wear a chin-length face shield safety glasses and a surgical mask.  After disposal, flush the drain with cool water and then disinfectant.

 

Spills and Exposures

Follow normal procedure for spills of potentially infectious materials, found here.

Exposures:

An exposure is defined as contact with broken skin, eyes, nose, mouth, other mucous membranes, a percutaneous injury with a contaminated sharp, or contact with an infectious agent over a large area of apparently intact skin.

DO NOT DELAY!

CLEAN IT.                                

SKIN Exposures: Immediately remove contaminated clothing and wash the contaminated area with soap and water for 15 minutes.

EYE Exposures: Immediately flush the eye with water for at least 15 minutes at an eyewash or faucet. Remove contact lenses while flushing the eye.

GET TREATED.

  • If an injury is life-threatening or you need transport assistance, call 911.
  • Monday through Friday, 8 a.m. to 4 p.m., seek treatment at University Health Services. Ask a co-worker to call ahead (609 258 5035).
  • For exposures that occur during evening and weekend hours, contact the on-call Global Health Physician at 609-258-7971to seek advice on next steps. 

REPORT IT.

  • Report all exposures to your immediate supervisor and Principal Investigator.
  • Principal Investigators are responsible for reporting exposure incidents to EHS Biosafety.

 

References:

Centers for Disease Control and Prevention: Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19)) https://www.cdc.gov/coronavirus/2019-nCoV/lab/lab-biosafety-guidelines.html

Chang L, Zhao L, Gong H, Wang Lunan, Wang L. Severe acute respiratory syndrome coronavirus 2 RNA detected in blood donations. Emerg Infect Dis. 2020 Jul [April 16, 2020]. https://doi.org/10.3201/eid2607.200839

Public Health Agency of Canada: SARS-CoV-2 (Severe acute respiratory syndrome-related coronavirus 2) https://www.canada.ca/en/public-health/services/laboratory-biosafety-bio...

Wang W, Xu Y, Gao R, et al. Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA. Published online March 11, 2020. doi:10.1001/jama.2020.3786

World Health OrganizationLaboratory biosafety guidance related to coronavirus disease 2019 (COVID-19)

https://www.who.int/publications-detail/laboratory-biosafety-guidance-re...(covid-19)