By design, stream-in–air cell sorters produce aerosols. Therefore, the use of these instruments with certain biological agents constitutes a potential procedure hazard. This chapter establishes requirements for the design of laboratories housing cell sorters, the creation of laboratory or instrument-specific Standard Operating Procedures (SOPs), and the procedures for the safe operation of cell sorters and validation of their aerosol containment systems. 

The objectives and responsibilities set forth in this manual chapter are applicable to all NIH employees in all Institutes and Centers. NIH employees will comply with this policy and perform their duties in the safest possible manner.

Flow cytometric cell sorting has become an important technology in basic and clinical research laboratories. However, samples that are sorted may contain infectious biological agents, and standard procedures must be implemented to minimize risk of exposure to these potentially hazardous agents. In addition, these standard procedures must be consistent with NIH Manual Chapter 3035.

Laboratory procedures that generate aerosols are classified as the most important operational risk factor supporting the need for containment equipment and facility safeguards. The likelihood of aerosol production by cell sorters is high due to the possibility of fluid exiting a small orifice (usually 70µm) µm at high pressure (up to 70psi) impacting a hard surface.  Aerosol production is highest in the event of a partial obstruction of the nozzle orifice and subsequent stream deviation.  In recognition of this risk of aerosol exposure when sorting potentially infectious material, the NIH Biosafety Policy for Cell Sorters was approved in a memo to the Director, Division of Occupational Health and Safety (DOHS), ORS, in 2012. 

The fundamental objectives of any laboratory biosafety program should be containment of hazardous materials, and the development and implementation of procedures designed to reduce exposure based upon a thorough risk assessment.  This policy is meant to reduce or eliminate exposure of the outside environment and laboratory personnel to potentially hazardous agents during the operation of cell sorters.

This policy is derived from established biosafety principles as outlined in the Biosafety in Microbiological and Biomedical Laboratories (BMBL), current edition , and the current International Society for the Advancement of Cytometry (ISAC) Cell Sorter Biosafety Standards.

This policy supplements other safety policies at the National Institutes of Health (NIH), and does not reduce or alter the requirements of any other policies.

All laboratories in the NIH Intramural Research Program utilizing Cell Sorters follow procedures defined in this NIH Manual Chapter.  These procedures (outlined in Section H) consist of six sections, 1) Risk Assessment; 2) SOP Development; 3) Room Design; 4) Cell Sorter-Specific Safety Equipment and Practices; 5) User-Specific Safety Equipment; and 6) Disinfection.

References and copies of registration forms are available from the DOHS website. Occupational Safety and Health Administration (OSHA) references are available on the OSHA website. Additional references include:

1. Holmes KL. Characterization of aerosols produced by cell sorters and evaluation of containment. Cytometry A 2011;79:1000-1008.
2. NIH Policy Manual, Chapter 3035: Working Safely with Potentially Hazardous Biological Materials
3. Holmes KL, Fontes B, Hogarth P, Konz R, Monard S, Pletcher CH Jr, Wadley RB, Schmid I, Perfetto SP. International Society for the Advancement of Cytometry cell sorter biosafety standards. Cytometry A 2014;85:434-453.
4. Oberyszyn AS. Method for visualizing aerosol contamination in flow sorters. Curr Protoc Cytom 2002;Chapter 3:Unit 3.5.
5. Perfetto, S. P., et al. "Novel Impactor and Microsphere-Based Assay Used to Measure Containment of Aerosols Generated in a Flow Cytometer Cell Sorter." Cytometry A. 2018 Dec 18. doi: 10.1002/cyto.a.23680.
6. Rutala WA, Weber DJ, HICPAC. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008. 2011.
7. Biosafety in Microbiological and Biomedical Laboratories - Centers for Disease Control and Prevention/National Institutes of Health. The current edition is available at the DOHS Publications page.
8. NIH Bloodborne Pathogen Exposure Control Plan for Non-Hospital Personnel. Prepared in compliance with 29 CFR 1910.1030.
9. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). Federal Register, July 5, 1994 (59 FR 34496). Amendment - Federal Register, FR, January 5, 2001 (66 FR 1146) and all current amendments.
10. Occupational Exposure to Bloodborne Pathogens. Occupational Safety and Health Administration Standard 29 CFR 1910.1030, 66 FR 5325 January 18. 2001.
11. NIH Policy Manual, Chapter 1743: Keeping and Destroying Records

1. ​​​​​​Flow Cytometric Analyzer - Scientific instrument used to characterize cells or particles in a fluid stream, based upon their measured fluorescence and light scatter characteristics, but are incapable of sorting the cells.
2. Fluorescence - activated Sorter (FACS) - FACS is a trademarked name for BD Biosciences instruments. 

Note: The scope of this document is for Cell Sorters, not analyzers.  The SOP’s used as examples in this document are for BD FACS Aria cell sorters, hence the use of the name FACS. It is important to distinguish a Cell Sorter from a Flow Cytometric Analyzer, since the risk of aerosol production from analyzers is much lower.

3. Cell Sorter - Scientific instrument used to isolate cells or particles based upon their measured fluorescence and light scatter characteristics.  There are two classes of Cell Sorters: electrostatic droplet-based (also known as jet-in-air or stream-in-air) and mechanical cell sorters.   Electrostatic droplet-based sorters employ a liquid stream at high (up to 70 psi) pressure, carrying cells through a nozzle.  The stream is not confined and therefore is open to the air.  Mechanical sorters utilize fluid streams that are confined within tubing or microfluidic channels.  The term cell sorters used in this Chapter refer to electrostatic droplet-based sorters.
4. Biosafety Levels - A biosafety level is the level of the biocontainment precautions required to work with dangerous biological agents in an enclosed facility. The levels of containment range from the lowest biosafety level 1 (BSL-1) to the highest at level 4 (BSL-4). In the United States, the Centers for Disease Control and Prevention (CDC) have specified the requirements for each of these levels.
5. Infectious Biological Agent - A microorganism (including, but not limited to, bacteria (including rickettsiae), viruses, fungi, or protozoa) or prion, whether naturally occurring, bioengineered, artificial, or a component of such microorganism or prion that is capable of causing communicable disease in a human, animal, or plant.
6. Personal Protective Equipment (PPE) - Items of clothing (i.e. lab coats, shoe covers, face masks, gloves, etc.) or equipment (i.e. face shields, eye goggles, etc.) designed to prevent or limit exposure to potentially harmful agents.
7. Standard Operating Procedure (SOP) -Written procedures that describe, in detail, how to perform a particular task or overall duty/responsibility.
8. Mucous membrane protection - A device or combination of devices, such as a full face shield, surgical face mask combined with form fitting goggles or approved protective glasses, etc., which protect the mouth, nose and eyes from splash or droplet contamination.
9. NIH Facility - A facility owned, operated or leased by the NIH.
10. Cell Sorter in certified Biological Safety Cabinets (BSC) -  Class II BSC: manufactured to meet functional certification criteria for personnel and product protection as defined by NSF 49; Class I BSC: manufactured to meet functional certification criteria for personnel protection as defined by the BMBL and have an inward airflow velocity of 100 linear feet per minute.  High Efficiency Particulate Air (HEPA) filters are to be tested for leakage annually.
11. Select Agents – Select Agents are bio-agents which have been declared by the U.S. Department of Health and Human Services (HHS) or by the U.S. Department of Agriculture (USDA) to have the "potential to pose a severe threat to public health and safety". These bio-agents are divided into three broad categories: 1) HHS select agents and toxins (affecting humans); 2) USDA select agents and toxins (affecting agriculture); and 3) Overlap select agents and toxins (affecting both).
12. NIH Institutional Biosafety Committee (IBC) - The NIH Institutional Biosafety Committee reports to the Director, NIH or his or her designee on matters pertaining to the control of biological hazards.  The NIH IBC is the primary reviewing and biosafety approval body for all proposed research associated with the intramural use of microbiological agents and recombinant or synthetic research subject to the NIH Guidelines. The IBC also serves as an advisory body to the Division of Occupational Health and Safety (DOHS), ORS.
13. Human Pathogens - Human Pathogens are agents (such as viruses, bacteria, prions, or fungi) that cause disease in humans.
14. High Efficiency Particulate Air (HEPA) filter -A throwaway, extended-media, dry type filter with a rigid casing enclosing the full depth of the pleats. The filter shall exhibit a minimum efficiency of 99.97% when tested at an aerosol of 0.3 μm diameter.
15. Agent Risk Group-The classification of infectious 2microorganisms according to its capability to infect and cause disease in a susceptible human or animal host, its virulence as measured by the severity of disease, and the availability of preventative measures and effective treatment for the disease.  Four Risk Groups are defined in the BMBL, ranging from least likely to cause human disease (Risk Group 1) to highly likely to cause serious or lethal disease (Risk Group 4).

1. Director, NIH: Through DOHS and the Deputy Director for Intramural Research (DDIR), provides executive leadership in the development and implementation of biological safety policies, standards and procedures applicable to the NIH.
2. Deputy Director for Intramural Research (DDIR): The DDIR is the principal liaison with the NIH intramural research community regarding safety and health matters. The DDIR receives safety policies approved by ORS and communicates them to the IC Scientific Directors. Further, the DDIR raises safety concerns to ORS as they are brought to the DDIR’s attention from the intramural research community.
3. Designated Agency Safety and Health Official (DASHO): The Institutional Official responsible for management and administration of the NIH occupational safety and health program. This authority is delegated by the Director, NIH.
4. The Division of Occupational Health and Safety (DOHS): The DOHS provides the necessary staff   to effectively administer a comprehensive occupational safety and health program and it is responsible for:
   1. Providing general guidance on the selection and use of personal protective equipment (PPE) for laboratory personnel utilizing cell sorters. Specific PPE guidance shall also be provided for work involving infectious diseases, hazardous chemicals, etc., that may require additional PPE. If respirators are occupationally required, DOHS will select and approve them. Supervisors are not authorized to select or recommend the use of respiratory protection. 
   2. Providing guidance in performing risk assessment to PI's and employees operating cell sorters, including the determination of appropriate biosafety level appropriate for the type of samples and/or agents that will be sorted. Contact the Safety and Health Specialist from the DOHS home page.
5. Institute and Center (IC) Scientific Directors: Scientific Directors are responsible for ensuring full compliance with this policy within the IC.
6.  Principal Investigator (PI) or Immediate Supervisor (IS) is responsible for:
   1. Ensuring compliance from all personnel that work with Cell Sorters with all provisions of this Manual Chapter and any other special requirements or procedures specific to the facility within which they are working.
   2. The development and annual review of the Standard Operating Procedures (SOPs) for the Cell Sorting laboratory in compliance with this Manual Chapter. The SOPs should be reevaluated at least on an annual basis or whenever there is a change in instrument configuration that may affect biosafety.
7. Employees:
   1. Employees must comply with the provisions of this Manual Chapter and the established Standard Operating Procedures of the Cell Sorting Laboratory.  Employees are responsible for using prescribed personal protective equipment during performance of work.  

I. Risk Assessment

A risk assessment should be conducted for all samples/agents prior to sorting, and the appropriate biosafety level determined in collaboration with DOHS Safety and Health Specialists, subject matter experts, and the NIH Institutional Biosafety Committee (IBC).  For additional information, consult DOHS Publications page. The purpose of a risk assessment is to recognize and identify hazards and measure the risk or probability that something will happen because of that hazard.  The results of a comprehensive risk assessment determine the appropriate procedures and practices for cell sorting.  The designation of safety measures is dependent upon the risk and the severity of the consequences if exposure occurs.  Risk analysis takes into account the Risk Group of the agent and the procedures performed with the agent. 

Risk Assessment consists of five steps:

1. Identify and evaluate agent hazards
2. Identify laboratory procedure hazards
3. Make final determination of biosafety level (BSL) (See Appendix I)
4. Evaluate proficiencies of staff and integrity of safety equipment
5. Review risk assessment with DOHS Safety and Health Specialist, or NIH Institutional Biosafety Committee.

The Risk Group of a given agent can be determined from a variety of sources, most notably the current edition of the Biosafety in Microbiological and Biomedical Laboratories, (BMBL).  Cell sorting is considered a laboratory procedure hazard because of the potential for aerosol and/or slash exposure.  Agents that may be worked with at BSL-2 under normal laboratory procedures and practices, may require greater precautions as defined in this document as BSL-2 with enhanced precautions. Due to the risk of aerosol exposure in cell sorting, an aerosol management system is required at all biosafety levels and usually consists of a sort chamber evacuation pump equipped with a High Efficiency Particulate Air (HEPA) filter.  All aerosol management systems require validation (as indicated below), although the frequency of testing increases with increased biosafety levels. 

II. Standard Operating Procedure (SOP) Development for Cell Sorter Laboratories

An important outcome of any risk assessment process is the creation of standard operating procedures (SOPs).  An SOP must take into account hazards (agents and laboratory procedures) and specify practices and procedures designed to minimize or eliminate exposures to those hazards.  For cell sorters, the design of the instrument, especially containment or aerosol evacuation components, must be considered in the development of the SOP.  Each instrument must be evaluated for deficiencies in containment or aerosol evacuation design and appropriate procedures adopted to minimize risk.  An important example of this is that most cell sorters do not possess an interlock designed to prevent the operator from opening the sort chamber after a nozzle obstruction with subsequent stream deviation.  Therefore, the SOP should clearly address the procedures for evacuating the sort chamber of aerosols prior to opening the sort chamber, including a stated time period to wait after a clog induced stream deviation.

The general considerations for SOP development are outlined below:

1. Preparation before the sort
   1. Check fluids, empty waste
   2. Cover control surfaces with plastic wrap, including keyboards and mouse (or use washable keyboards).
   3. Perform containment testing
   4. Verify any automated decontamination functions
   5. Preparation of disinfectant solutions
   6. Sample preparation, i.e. staining, centrifugation, pipetting or manipulations that may generate aerosols should be performed in a manner to maximize containment and protect the worker
2. Procedures in the event of a nozzle obstruction
   1. Turn off stream
   2. Evacuate sort chamber prior to opening; increase Aerosol Management System (AMS) evacuation  rate
   3. Attempt to clear nozzle clog by stream flush routines, with sort chamber door closed.  If clog is not cleared,  remove the nozzle and dependent upon sample risk assessment, decontaminate nozzle before sonication
3. Decontamination procedures
   1. Decontaminate and clean sample lines, sort chamber and collection chamber.
   2. Decontaminate and clean surfaces around cytometer, especially near the sort chamber

Development of the SOP should also include consultation with DOHS Safety and Health Specialist who can provide guidance on general biosafety procedures as well as information on NIH policy.  Examples of SOPs for cell sorters are included in Appendix III to serve as templates for development of individual laboratory SOPs.  Finally, the SOP should be reevaluated at least on an annual basis or whenever there is a change in instrument configuration that may affect biosafety. 

III. Specific Requirements for Operation of Cell Sorters in NIH Laboratories Housing for Cell Sorters

Biosafety Level 2 (BSL-2) Laboratory – General:

1. The laboratory must meet all criteria for BSL-2 containment and be surveyed and posted by DOHS.
2. Air flow in the room is balanced to create negative airflow into the room.
3. Laboratories must have a sink for hand washing. The sink may be operated manually, hands-free, or automatically. It should be located near the exit door.
4. The laboratory should be designed so that it can be easily cleaned and decontaminated. Carpets and rugs in laboratories are not permitted.
5. Vacuum lines should be protected with HEPA filters, or their equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required.
6. An eyewash station must be readily available.

Biosafety Level 2 (BSL-2) - With Enhanced Precautions:

1. Ideally, the cell sorter is located in a separate, lockable room where no other laboratory activity is performed.  If the sorter is located in shared laboratory space, all Personal Protective Equipment (PPE) requirements (see G. Procedures IV - 4 User Specific Safety Equipment and Appendix I) should be followed by all personnel during sorting procedures.  The cell sorter should be placed in a location in the lab so that directional air flow is toward the cell sorter and away from other areas of the lab.  If the cell sorter is enclosed within a certified BSC (Class I or Class II), the requirement for placement of the cell sorter in a separate room may be abrogated dependent upon the overall risk assessment.
2. Air flow in the room is balanced to create negative airflow into the room.
3. The sorting room is locked to restrict access to allow the operator to concentrate on the sort and to maintain regular air flow and negative air pressure in the room.
4. During sorting procedures, a sign should be placed on the outside of the door to indicate that a potentially biohazardous sorting process is in progress. This sign also should contain all necessary information for entering the room safely, including warning for Class IIIb or IV lasers, if applicable.

Biosafety Level 3 (BSL-3) – Laboratory:

1. The laboratory must meet all criteria for BSL-3 containment and be surveyed and posted by DOHS.
2. The cell sorter must be located within a Class II certified BSC (can be recirculated).

Biosafety Level 4 (BSL-4) Laboratory:

1. The laboratory must meet all criteria for BSL-4 containment and be surveyed and posted by DOHS.
2. The cell sorter must be located within a Class II or Class III certified BSC.

IV. Cell Sorter-Specific Safety Equipment and Practices

1. Aerosol Containment: 

Aerosol management System (AMS): All cell sorters must be equipped with an aerosol management or evacuation system that is designed to evacuate the sort chamber and sort collection area of the cytometer.  It consists of an evacuator that creates negative pressure within those chambers, and transports aerosols through a HEPA or an ultra-low penetration air (ULPA) filter before exhausting to the room.  The AMS should be operated under all biosafety levels, BSL-2, BSL-2 with enhanced precautions, BSL-3, and BSL-4.

2. Validation of Aerosol Management Systems:  

Currently, the most widely accepted method of containment testing utilizes fluorescent plastic beads that are run on the instrument as a sample (See references 3, 4 and 5 above). The AMS must be tested under simulated worse-case “failure mode.” In this mode, the instrument is set to high pressure (usually 70psi), and fluorescent particles are concentrated to approach speeds of approximately 20,000-50,000 particles/second. The stream is forced to glance off of the waste catcher shield to create a large plume of aerosols and aerosols concentrated on a slide for subsequent analysis on a microscope.  Tolerance of aerosol escape is zero particles when the AMS is active and sort chamber door is closed.  This test (or other validated test for containment) is performed periodically (monthly or only when filters are exchanged) for BSL-2 labs and labs performing sorts under BSL-2 with enhanced precautions. The test is performed prior to every sort for BSL-3 labs.    Frequency of testing will be dependent upon the risk assessment and consultation with biosafety professionals and/or the IBC.  However, containment testing must be performed in the following circumstances:

1. Following instrument service or maintenance involving the sort chamber and/or AMS hose connections.
2. Following initial instrument installation or relocation.
3. Following change out of the standalone AMS filter.
4. For BSL-3 or 4 labs:
   1. Prior to every sort if the frequency of sorting is once/week or less
   2. Weekly, if the frequency of sorting is multiple sorts/week

3. Cell sorters in biological safety cabinets: 

Class II BSC’s enclosing cell sorters must be manufactured to meet functional certification criteria for personnel and product protection as defined by NSF 49.  Class I BSC’s enclosing cell sorters must be manufactured to meet functional certification criteria for personnel protection as defined by the BMBL, although it is recommended that the inward airflow velocity be 100 linear feet per minute or greater; HEPA filters must be tested for leakage annually. Cell sorters placed in BSC’s must have an AMS in which aerosol containment validation can be performed independent of the BSC blowers, i.e. with the BSC directional air current system turned off.  This is done to provide greater sensitivity when performing the cell sorter AMS containment tests.  The BSC must be validated initially at installation.  Frequent retesting and moni­toring proper functioning of the cabinet is mandatory, as per NSF 49 requirements.

4. User Specific Safety Equipment:
   1. Personal Protective Equipment (PPE) for Biosafety Level 2 (BSL-2) Laboratory:
      1. Front closure lab coat
      2. Gloves
      3. Eye Protection: Safety glasses
   2. Personal Protective Equipment (PPE) for Biosafety Level 2 (BSL-2) with enhanced precautions:
      1. Isolation-style solid-front or wrap-around gown
      2. Gloves
      3. Eye protection: Safety goggles face shield, splatter guard or integral respirator/face shield that provide mucous membrane protection as required for anticipated splashes or sprays of infectious agents or other hazardous materials.
      4. Respirator: National Institute for Occupational Safety and Health (NIOSH)-approved respirators must be worn during operation of the cell sorter under BSL-2 with enhanced precautions conditions.  Approved respirators include N-95, N-99, or N-100 filtering face-piece respirators or powered air-purifying respirators (PAPR) with integral face shield.  Respirators must remain on during all procedures associated with sample manipulation, including sample tube cap removal and loading of sample on instrument, or when removing collection tubes or other procedures where the sort or collection chamber is opened.  For non-primate samples containing agents that do not pose respiratory risk, mucous membrane protection may be substituted for respirators.  For example, the human pathogens leishmania and mouse models of toxoplasma infection are included in this category.
      5. Cell Sorters enclosed in a certified BSC: use of respirators as outlined above is recommended during instrument/sample manipulation within the BSC but can otherwise be removed during sorting procedures providing the BSC is operational, aerosol management system is active and all sort chamber and collection chamber doors are closed.  If the BSC-enclosed Cell Sorter is in a shared laboratory, respirators are not required for other laboratory personnel. 
      6. All individuals using respirators must be enrolled in the NIH Respiratory Protection Program. Supervisors are not authorized to select respiratory protective devices. Questions should be directed to the DOHS Technical Assistance Branch (TAB) at 301-496-2960
   3. Personal Protective Equipment (PPE) for Biosafety Level 3 (BSL-3) Laboratory:
      1. Liquid-resistant scrub suits or coveralls
      2. Gloves (double pair).  Change gloves when contaminated, integrity has been compromised, or when otherwise necessary. Gloves and protective clothing must not be worn outside the laboratory and must be disposed of with other contaminated waste.
      3. Eye Protection/Respirator: NIOSH-approved powered air purifying respirators (PAPR) with integral face shield must be worn at all times when in the laboratory.  If you have any questions, contact the DOHS Technical Assistance Branch at 301-496-2960.  Eye and face protection must be disposed of with other contaminated waste or decontaminated before reuse. 
   4. Disinfection:

The choice of disinfectant is dependent upon a variety of factors (4) including the agent in use, the chemical resistance of the cell sorter components, and potential of exposure of lab personnel to the chemical disinfectant.  Broad-spectrum disinfectants are desirable in a facility in which agent use is varied.  Sodium hypochlorite solutions (1:10 dilution of household bleach in H₂0; final concentration of 5,250-6,150 ppm of chlorine) offer several advantages over alcohols and other disinfectants; bleach has broad-spectrum antimicrobial activity, does not leave toxic residues, is unaffected by water hardness and is inexpensive and fast acting.  However, because of the corrosive nature to metals, exposure to instrumentation should be limited to times determined to be maximally efficacious to microbial killing.  In addition, bleach solutions must be prepared fresh due to loss of free available chlorine.  However, there are commercially available sprayers that mix the bleach and water when sprayed, eliminating the need to make fresh solutions daily.

All records pertaining to this chapter must be retained and disposed of under the authority of NIH Manual 1743,"Keeping and Destroying Records," Appendix 1, "NIH Records Control Schedules" (as amended). These records must be maintained in accordance with current NIH Records Management and Federal guidelines. Contact your IC Records Liaison or the NIH Records Officer for additional information.