Biosafety Level 2 plus (BSL-2+) Safety Manual - UPMC Manual Name: Biosafety Level 2 plus (BSL-2+) Safety Manual Date Adopted: November 2, 2004 Date Revised: December 11, 2009 Revision

C:\Documents and Settings\neilsonl\Desktop\UPCI Website\2012 redesign\Shared Resources\Cytometry 2012\Biosafety Level 2

plus (BSL-2+) Safety Manual R4.docx

1

Biosafety Level 2 plus (BSL-2+) Safety Manual

UPCI Cytometry Facility Room 1.45, Hillman Cancer Center

Revision 4, December 11, 2009



2

Manual Name: Biosafety Level 2 plus (BSL-2+) Safety Manual

Date Adopted: November 2, 2004

Date Revised: December 11, 2009 Revision number: 4

Authors: Hongmei Shen, Albert D. Donnenberg, E. Michael Meyer

Supersedes Policy: n/a

Distribution: Cytometry Facility Staff, Investigators and staff using the MoFLo cell sorter or present during cell sorting.

Director Date Albert D. Donnenberg, Ph.D.

Manager Date E. Michael Meyer

University Biosafety Officer Date

Technologist and User Review By signing I indicate that I have read and understand this manual and agree to abide by the policies set forth in this document. I also affirm that I have successfully completed and am current in the required modules on Bloodborne Pathogens and Chemical Hygiene.

Date Signature



3



4

TABLE OF CONTENTS

PAGE

1. Overview of BSL-2+ facility and procedures .................................................... 6 Introduction ............................................................................................................ 6 BSL-2+ personnel requirements ........................................................................... 6 BSL-2+ facility, layout, and air handling .............................................................. 7 Medical requirements, hygiene, and good laboratory practices ....................... 7

2. Locations of storage and use of agents ........................................................... 8

Agents used in the BSL-2+ lab ............................................................................. 8 Location of storage of agents ............................................................................... 8

3. Standard operating procedures for BSL-2+ facility ....................................... 10

General BSL-2+ laboratory practices ................................................................. 10 Equipment Maintenance ...................................................................................... 13 Spill response in the BSL-2+ lab ........................................................................ 13 References ........................................................................................................... 14

4. Emergency response guidelines ..................................................................... 15

Emergency contact information .......................................................................... 15 Medical emergencies ........................................................................................... 15 Injuries .................................................................................................................. 15 Facility emergencies ............................................................................................ 16

5. Revision History ............................................................................................... 16 6. Appendix ........................................................................................................... 17



5

1. Overview of Biosafety Level 2+ (BSL-2+) Facility & Procedures Date: December 11, 2009 Purpose: To provide a general overview of the facility and procedures for all personnel working in the BSL2+ laboratory of the Cytometry Facility.

1.1 Introduction The BSL-2+ facility involves moderate to high-risk agents and therefore requires a strict adherence to BSL-2 containment with BSL-3 work practices and procedures. It is important that all personnel that work in the BSL-2+ facility understand and adhere to the proper procedures and techniques outlined in this manual. Failure to adhere to appropriate practices and procedures may endanger others.

1.1.1 Information about agent in use. Unfixed human cells from blood, body fluid, or other tissues are used in the facility. All untested human samples should be considered potentially infectious for HIV, HBV and other bloodborne pathogens, which can infect humans through exposure of mucosal membranes to aerosol, broken skin or aerosol inhalation. Retroviral and lentiviral transfected cells are used in this facility. The retroviral and lentiviral vectors are replication-defective, however, they will be inserted with some genes that may be oncogenic. The viruses will be psuedotyped with the vesicular stomatitis virus G-glycoprotein (VSV-G), which can infect human cells through direct contact with blood or mucous membranes. The agents described above will be run through a high-speed cell sorter (MoFlo), which operates under high pressure (30-60 psi) and is capable of generating aerosol under normal operating conditions, especially when samples are sorted onto microscope slides. Although the fluidics of the MoFlo are contained within a Class I safety cabinet (CytoShield), we require all personnel to observe universal precautions when handling live human cells. Since hepatitis B virus is a bloodborne pathogen, immunization is strongly recommended.

1.2 BSL-2+ personnel requirements

All individuals working in the BSL-2+ facility must be trained according to the compliance policies of the University of Pittsburgh. Personnel receive annual



6

updates, or additional training as necessary for procedural or policy changes. Additionally, the facility Directors will conduct training for all personnel in the area, covering the potential hazards associated with the work, the necessary precautions to prevent exposures, exposure evaluation procedures, and the standard operating procedures of a BSL-2+ laboratory. Facility Directors are responsible for training employees concerning the hazards of the agents they will be working with and the proper laboratory techniques to use to avoid injury and illness. New employees must be trained prior to assignment in the lab. This training is to be documented in the employee's file with a listing of the session agenda, the name of the person providing the training, and the date and signature of the person trained. This record should be retained for the duration of employment and at least 3 years after. Specialized training is required for the person operating the MoFlo high-speed cell sorter. This training will be provided by the manufacturer of the MoFlo or by cytometrists with extensive cell sorting experience. Training is to be determined by the Facility Director.

1.3 BSL-2+ facility, layout, and air handling

BSL2+ facility is located on the first floor of the Hillman Cancer Center, room 1.45. There are multiple instruments inside the lab: MoFlo high-speed cell sorter (MoFlo), 2 CyAn bench-top flow cytometers and an Amnis ImageStream100, a Beckman Coulter Gallios and a Cellomics ArrayScan. There are 2 daily working benches and a Class II Biosafety Cabinet for the lab staff. There is one entry door to the facility and a door to an adjoining room that houses the UserLab, nitrogen and the water chilling equipment required for operation of the MoFlo. Doors will be locked during sorting of unfixed, unknown human and non-human primate cells or lentivirally-transfected cells, as well as after working hours and on weekend. The airflow in the laboratory is regulated centrally by Facilities Management to maintain a specified number of air changes per hour. The room air pressure is negative with respect to the corridor. The MoFlo CytoShield hood is vented to the house air handling system. The CytoShield exhausts through a HEPA filter.

1.4 Medical requirements, hygiene, and good lab practices

1.4.1 Medical Requirements All individuals present in the laboratory during the operation of the MoFlo must meet the following medical requirements:



7

Those with exposure to animals and/or their body fluids, fresh tissues, bedding, or caging are required by the University to enroll in the Animal Exposure Surveillance Program. Initial enrollment can be completed by downloading the enrollment form (available at http://www.ehs.pitt.edu/assets/docs/AESPform2007.pdf) and faxing it to 412-647-1993. An update form is required to be completed every three years (available at http://www.ehs.pitt.edu/assets/docs/AESPUpdateForm.pdf)..

All faculty and staff with exposure to human bloodborne pathogens are required to complete the following:

Those initiating work with materials potentially containing bloodborne pathogens are required to enroll in the University of Pittsburgh Bloodborne Pathogen Exposure Control Program upon hire, and annual completion of BBP training.

Serum Surveillance Program participation is optional through Employee Health Services (647-3407)

Hepatitis B vaccination is strongly recommended and available without charge to individuals enrolled in the Bloodborne Pathogen Program.

1.4.2 Basic hygiene It is mandatory that all personnel wear personal protective equipment (lab coats and gloves) when handling human specimens or working on the instrument. When cleaning the inside of the Class I biosafety cabinet on MoFlo (CytoShield hood), a face protection shield or goggles should be worn. All personnel must wash their hands after removing gloves. Eating, drinking, storing food, handling contacts, and applying cosmetics are not permitted in the laboratory. Food must not be stored in refrigerators or freezers in the laboratory. 1.4.3 General good laboratory practices All unfixed specimens to be run on the MoFlo cell sorter should be handled in a Class II safety hood using universal precautions. Mandatory laboratory practices include use of mechanical pipetting, use of plastic instead of glass, minimizing the use of sharps (see Section 3.1.8 for Sharps policy), labeling equipment with appropriate biohazard stickers, and minimizing work with infectious substances on the open bench.

2. Location of storage and use of BSL-2+ agents

Purpose: To provide a list of all agents used in the BSL-2+ lab and their storage and use locations:

Agents used in the BSL-2+ lab

http://www.ehs.pitt.edu/assets/docs/AESPform2007.pdf

http://www.ehs.pitt.edu/assets/docs/AESPUpdateForm.pdf



8

Unfixed or fixed human cells from blood, body fluid or other tissues

Retroviral or lentiviral transfected cells Cells from animal tissues, e.g. mouse, rat Human or animal cell lines from variety sources

Beads for instrument alignment or QC Murine or rat derived monoclonal antibodies DNA stains (DAPI, PI, Hoechst 33342, DRAQ5) Formaldehyde (10% EM grade)

Location of storage of agents

Beads, monoclonal antibodies and DNA stains are stored in the refrigerator (40C). Formaldehyde is held in small quantities on the reagent shelf. Generally, all the cells listed above are used immediately when they are brought to the lab. If the cells cannot be used immediately for some reason, they will be temporarily stored in the refrigerator.

All reagents are eventually used on the MoFlo high-speed cell sorter or analytical cytometers. After running unfixed, unknown human and non-human primate cells or lentivirally-transfected cells on MoFlo or CyAn, sample lines must be flushed with 10% bleach for at least 5 minutes followed by at least 5 minutes with DI water.



9

3. Standard Operating Procedures for the BSL-2+ facility Purpose: To provide safe handling procedures and operations for personnel working in the BSL-2+ lab.

3.1 General BSL-2+ laboratory practices

3.1.1 Facility entry.

Access is restricted to those persons whose presence is required for experimental or support purposes. The entry door is locked when sorting is in progress and during non-working hours. The Facility Director and Supervisor have the final responsibility of assessing each circumstance and deciding who may enter or work in the laboratory. Only the door to the corridor will be used to enter and exit the laboratory, except as noted in 1.3. 3.1.1.a Visitors. Other individuals permitted to enter the laboratory when sorting is not in progress include personnel from Housekeeping who remove waste and clean the laboratory, and from Facilities Management and Information Services Division (ISD). Other visitors to the laboratory must be pre-approved by the Facility Director or Supervisor. Only approved laboratory workers may enter the laboratory during sorting of unfixed, unknown human and non-human primate cells or lentivirally-transfected cells.

3.1.1.b Contractors and vendors. Field service engineers from Beckman Coulter and Amnis, technical supporting personnel from the local maintenance department, and Filtech, Inc. are allowed to enter the lab for service purposes, as requested by lab staff, except when unfixed, unknown human and non-human primate cells or lentivirally-transfected cells are being sorted.

3.1.1.c Emergency medical personnel. Emergency medical personnel may enter in the lab if they are notified that there is a medical emergency inside the facility. Efforts to secure all biological agents prior to emergency medical personnel entering the laboratory will be made.



10

3.1.2 Training requirements. All personnel must complete the following training prior to working in the BSL-2+ laboratory. Training includes two Environmental Health and Safety training sessions (Bloodborne Pathogens and Chemical Hygiene training). These training sessions are held bimonthly. Bloodborne Pathogen training is required on an annual basis and Chemical Hygiene training is required every 3 years. A special training will be required for the operators of the MoFlo high-speed cell sorter. This special training will be provided by the manufacturer of the MoFlo or by cytometrists with extensive cell sorting experience. Workers cannot work in the BSL-2+ lab unless all the training requirements have been met, they have read and signed off on the manual, and received approval from the Facility Director and Supervisor.

3.1.3 Personal protective equipment. Personal protective equipment (PPE) is designed to protect the worker from contact with biohazardous agents as well as to protect the work from contamination by the worker. PPE is considered a secondary line of defense against the infection. The primary line of defense is the use of Universal Precautions and good laboratory techniques. Mandatory PPE includes lab coats and gloves. When cleaning inside the hood, eyewear (safety goggles or face shield) must be worn.

3.1.4 Biosafety cabinet (BSC).

There are 2 biosafety cabinets inside the BSL2+ facility. They include the Class II biosafety cabinet in the sorting lab and the Class I safety cabinet (CytoShield) on the MoFlo high-speed cell sorter.

3.1.4.a Signup sheet. There is no mandatory sign up policy for the use

of BSCs. 3.1.4.b Use of biosafety cabinets, and decontamination.

3.1.4.b.1 BSC’s are the most commonly used containment devices for

preventing the escape of biohazardous materials into the laboratory environment. Four classes of BSC’s are recognized: Class I, Class II-Type A, Class II-Type B, and Class III. All four classes are suitable for work with biohazardous materials in BSL 1 to BSL 3. The Class III BSC is required for BSL 4 work. The sample manipulations (e.g. filtering cells to remove clumps) prior to sorting or analysis on the MoFlo will be conducted inside



11

the Class II biosafety cabinet. Prior to use, the blower and the lights of the BSC must be turned on. Make sure that the opening of the hood is at a safe operating level (10 inches). Minimize the working materials inside of the hood and make certain that they are placed well behind the ventilation unit in the front of the cabinet. Do not block the airflow. Keep contaminated materials separate from clean materials. After using the BSC, make sure to disinfect the hood as detailed in 3.1.4.b.2. When finished using the BSC, turn off the blower, close the door to the hood, and turn on the UV light.

3.1.4.b.2 The CytoShield Class I biosafety cabinet operates continuously.

Make sure the face velocity indicator (above the hood on the right side) reads greater than 75ft/sec. The operator must uncap or recap samples inside the MoFlo hood. 1:64 dilution of Vesphene IIse phenolic disinfectant (Steris, Mentor OH) is used as disinfectant for the surface of biosafety cabinets and the surface of the MoFlo instrument. Vesphene IIse solution is typically wiped or sprayed onto a surface and allowed to air dry. After Vesphene usage, an alcohol (70% EtOH) rinse of the surfaces should be completed followed by a sterile water rinse to prevent corrosion of the stainless steel surfaces.

3.1.5 Decontamination of biological waste.

3.1.5.a Liquid waste. All liquid wastes generated during BSL-2+ experiments should be immediately decontaminated by mixing with household bleach (6% sodium hypochlorite, final dilution not greater than 1:50) for at least 30 minutes contact time. The solution may then be disposed of in the sink; however, the sink must be washed and decontaminated after.

3.1.5.b Aspiration of liquid waste. No vacuum utility is available in the

BSL2+ sorting facility.

3.1.5.c Solid waste. Solid wastes generated during sorting unfixed unknown human and non-human primate cells or lentivirally-transfected cells (e.g. pipettes tips, tubes) will be deposited into a beaker containing 10% household bleach to be located within the CytoShield hood. Waste materials must soak in bleach for at least 15 minutes before removing from the hood. Bleach-decontaminated waste will be strained in the sink. Decontaminated waste and used



12

gloves will be disposed in a biohazard bag in a labeled cardboard container. Full containers are sealed and removed by Housekeeping for autoclaving.

3.1.5.d Autoclave. Wastes disposed of in red biohazard bags are

routinely autoclaved prior to disposal.

3.1.6 Transportation of Agents. Potentially infectious material transported into or out of the facility must be placed in a closed, leak-proof container labeled with a biohazard sticker.

3.1.7 Centrifugation of Agents.

There is one centrifuge in the BSL-2+ sorting laboratory. Prior to use, make sure that all samples are properly placed inside the centrifuge with balanced carriers and carrier safety caps. The centrifuge is to be decontaminated with Vesphene at the end of the day (on days that it is used). EH&S requires loading the centrifuge carriers/cups inside of the biological safety cabinet. After centrifuge cycle is completed, the carrier/cups should be opened in a biological safety cabinet.

3.1.8 Sharps Policy.

The use of needles in the BSL2+ sorting laboratory should be minimized. Glass-slides will be used for the MoFlo set up and sorting. Broken glass slides must be disposed of into a designated and labeled box for broken glass. The box is located in BSL2+ sorting laboratory. If Sharps (needles, scalpels, etc.) must be used in the BSL-2+ sorting laboratory, University Guidelines require the use of Safety-Engineered Sharps. EH&S should be contacted to provide information and trial devices if Sharps are required in the laboratory.

3.1.9 Exit Procedures.

Before leaving the work area, all solid and liquid wastes are to be disposed of in the proper manner (see 3.1.5 a and c). All equipment exposed to potentially biohazardous materials will be disinfected and returned to their correct place in the lab. Laboratory coats and gloves must be removed and disposed of in the biohazard solid waste container before leaving the lab.



13

3.2 Equipment maintenance.

3.2.1 Biosafety cabinets. BSCs must be cleaned after each use. The surface of the BSC is wiped down with a 1:64 diluted Vesphene IIse solution. The Oakland Dispatch Center (412 647-3370) will provide service as needed. Filtech, Inc will certify BSCs once a year.

3.2.2 Centrifuges.

The centrifuge is to be cleaned and disinfected with Vesphene at the end of the day (on days that it is in use).

3.2.3 Incubators. There are no incubators in the BSL2+ sorting facility.

3.3 Spill response in the BSL-2+ lab.

Spill control procedures are posted in the lab.

Spills of biological materials are decontaminated, contained, and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious or potentially infectious material. Spills and accidents that result in overt exposures to BSL-2+ materials are immediately reported to the Facility Directors. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are kept.

Spills in Biosafety Cabinets Remove any contaminated clothing and change gloves. If needed, use the emergency shower and/or eyewash station located at the end of the workbench in the facility. Cover with paper towels, surround the spill with disinfectant solution, and let mix for 20 minutes. Wipe down with a second application of disinfectant. Dispose all paper towels and PPE in a biohazardous waste bag. Leave the cabinet fans on.

Spills outside Biosafety Cabinets Evacuate the area, close all the doors, and call the Facility Director or Supervisor. Contact University of Pittsburgh EH&S to clean up the spill.

3.6 References.

1. “Biosafety in Microbiological and Biomedical Laboratories – 4th Edition”,

CDC/NIH, U.S. Government Printing Office, Washington, D.C., 1999.

2. University of Pittsburgh Biosafety Manual.



14

4. Emergency Response Guidelines Purpose: To provide safe procedures for handling medical and facility emergencies in the BSL-2+ facility.

4.1 Emergency contact information. In the event of an emergency, the following contact information is posted near the phone in the BSL2+ facility.

Title, name Phone number Facility Director 3-3256 or 3-7780 Lab Supervisor 3-3282

Medical Emergency 3-3131

UPMC Presbyterian Emergency Department 9-412-647-3333

FIRE 3-3131

Chemical Spill 3-2990

Biological Spill 9-412-624-9505

Environmental Health and Safety (Pitt) 9-412-624-9505 UPMC EH&S if applicable 9-412-647-6409

University Police 9-412-624-2121 Employee Health 9-412-647-3695

4.2 Medical emergencies.

In the case of medical emergency, call 911 for help and file any on-the job injuries reports as required.



15

4.3 Injuries.

Describe appropriate procedures in case of an injury.

4.3.1 On-the-job injury, bloodborne pathogen injury or sharps injury. Report for treatment at: Minor Injuries or Medical Surveillance During normal working hours (7 am – 3:30 pm Mon-Fri) Employee Health Services/UPMC Work Partners In Shadyside: Aiken Medical Building, Suite 209 412-623-1920 Medical Emergencies at any Time or Minor Injuries After 3:30 pm or on Saturday / Sunday Shadyside Hospital Emergency Department 412-623-2063 Minor Injuries or Medical Surveillance During normal working hours (7:30 am – 4:00 pm Mon-Fri) Employee Health Services/UPMC Work Partners In Oakland: Medical Arts Building, Fifth Floor 412-647-3695 Medical Emergencies at any Time or Minor Injuries After 4:00 pm or on Saturday / Sunday Presbyterian University Hospital Emergency Department 911 or 647-3333

Worker’s compensation information. Employee and Supervisor will need to fill out Employer's Report of Occupational Injury or Disease Form (ODIC-344) and file within 48 hours with the Worker's Compensation Specialist in the Office of Human Resources (100 Craig Hall).

4.4 Facility emergencies.

4.5.1 Electrical failures. In case of a power outage, the back-up generators of

the building will turn on, if not, find the emergency flashlight. If it happens during the sorting, stop sorting.

4.5.2 Exhaust failures. In case of an exhaust failure or change in negative air

pressure, stop the ongoing jobs, secure open containers of biological agents, and leave the facility immediately and close the door behind you. Call for assistance. The laboratory should be signed “Do Not Enter – Emergency Personnel Only”

4.5.3 Fire emergencies. In case of a fire, walk to the nearest exit of the facility

and go to the nearest staircase. Walk down and exit the building.



16

5. Revision History

Revision Change Rationale Standards Start Date

End Date

0 Creation Commissioning of CytoShield 29 CFR 1910.1030

11/02/04 12/07/04

1 Final recommendations of EHS

Implementation n/a 12/07/04 1/19/04

2 Update of instruments, lab organization

3 Environmental Health and Safety edits (Mark DiNardo)

Update of EH&S information n/a 1/20/04 12/10/09

4 Deletion of SortMaster related procedures, Changes to SOP for operation of the MoFlo.

SmartSampler allows for remote operation. Sorting permissible on samples containing BSL2 pathogens as determined in a case by case basis. Addition of “Decontamination after sorting.”

n/a 12/11/09

6. Appendices

SOP for operation of the MoFlo high-speed cell sorter

Validation of the Cytoshield hood using Glo-Germ fluorescent beads

“SAFE SORTING IN THE CORE FACILITY AS A PHYSICS PROBLEM” Abstract of poster presented at the International Society of Cytology, May 2008.



17

Appendix I

SOP for MoFlo high-speed cell sorter at University of Pittsburgh Cancer Institute

Introduction The MoFlo high-speed cell sorter manufactured by Beckman Coulter is a jet-in air cell sorter. It operates under relative high pressure (60 psi) and is capable of generating generate aerosols under normal operating condition. The risk of a high-pressure aerosol is greatly increased when the sorter’s nozzle tip becomes obstructed. The MoFlo high-speed cell sorter at University of Pittsburgh Cancer Institute is equipped with a Cytoshield hood that operates as a Class I biosafety cabinet and is designed to contain aerosols generated in the sorter. In order to sort unfixed unknown human and non-human primate cells or lentivirally-transfected cells, the MoFlo is located inside a BSL2+ laboratory. This SOP is designed to provide an operational guideline for sorting potentially biohazardous samples in the BSL2+ environment.

Personal requirements for operators Proper training for instrument operation

Training in Bloodborne Pathogens, Chemical Hygiene, and participation in the Animal Exposure Surveillance Program (mandatory for animal workers)

Participation in the Serum surveillance program (optional)

Hepatitis B virus immunization (optional, strongly recommended).

Laboratory practice No food and drink allowed in the lab

Limit unnecessary entry to the lab during the sorting

Lock the door during the sorting

A sign of “ sorting in progress” on the door during the sorting

A lab coat is required for all the persons involved in sorting

Gloves are required for all the persons handling sorting samples

Sample acceptance criteria Samples known to contain BSL3 pathogens are not acceptable for sorting.

Some BSL2 pathogens (considered as BSL3 in practice as described in “Biosafety Guidelines for Sorting Unfixed cells”, Ingrid Schmid et al, Cytometry 28:99-117 (1997)) require case by case evaluation by the Facility Director in consultation with the Facility Supervisor and the Biosafety Officer.

Sample must be contained in a leak proof container and clearly labeled with a sample identifier and a biohazard symbol.

Sample must be clump free and in an appropriate tube for sorting (12 x 75 mm snap cap tube)

Sample must not contaminate the outside of the tube



18

Sample handling The operator and other individuals handling the specimen must wear liquid

barrier gloves.

All individuals present during a sort must wear a lab coat.

If the sample requires transfer or filtration, this must be done in the BSC2 using universal precautions

The sample must be uncapped inside the CytoShield hood by operator.

The sample must be recapped inside the CytoShield hood prior to removal

The sorted cells must be capped or covered inside the CytoShield hood prior to removal.

Unused cell samples and sorted cells must be placed in caped, labeled tubes. It is the responsibility of the laboratory that provided the sample to remove unused cell samples and sorted cells from the Flow Facility.

Routine procedure to start a sort Make sure that the Cytoshield hood is on and functioning normally prior to turning

on the MoFlo. During normal operation the face velocity indicator reads >75 ft/sec and the red light (located next to the Cytoshield on-off switch) is NOT illuminated. The red filter-indicator light indicates that the HEPA barrier filters located in the plenum of the hood require service. The Cytoshield must not be operated if the filter indicator light in illuminated.

Turn on the MoFlo according to the manufacturer’s instructions.

Make sure the sheath tank is full.

Empty the waste tank into the sink only after the waste liquid has been exposed to 10% bleach for at least 30 minutes. Pour concentrated bleach into the tank to make sure that there is about 10% bleach when the tank is full.

Align the instrument before sorting.

Make sure sample is free of clumps before acquisition. Filter if necessary in the BSC 2.

Replace the removable glove box cover prior to initiating a sort.

The CytoShield hood must be on with the face velocity >75 ft/sec during sorting of unfixed unknown human and non-human primate cells or lentivirally-transfected cells.

Operator should never leave the sample unattended while sorting unfixed unknown human and non-human primate cells or lentivirally-transfected cells.

The operator must terminate the sort if The operator detects a clog or stream irregularity on the video monitor.

An alarm is triggered because the face velocity of Cytoshield hood drops below 75 ft/sec. This will occur if the house exhaust system fails.

The red filter-indicator light is illuminated due to a clog in the HEPA filter or an exhaust failure in the building.

Routine procedure to stop a sort Terminate sort



19

Don liquid barrier gloves and open sort chamber door

Wait 2 minutes before removing sample

Wipe sample tube with 70% EtOH

Wash sample line by flushing for at least 5 minutes with a 10% dilution of bleach stock in DI water, followed by at least 5 minutes with DI water at > 1.0 sample pressure differential.

Disinfect surface of inside CytoShield hood and the sorting chamber with 1:64 diluted Vesphene IIse.

Wear face shield or goggle when cleaning inside of CytoShield hood

In case of a clog while running unfixed unknown human and non-human primate cells or lentivirally-transfected cells

Operator will terminate the sort

Turn off sort plate charge

Stop the stream by pushing unclog button on Auto-Sample station

Wait at least 5 minutes before opening the sort chamber door

Don liquid barrier gloves, with extended cuffs, face shield or safety goggles and open sorting chamber door

Wipe collection tubes with 1:64 diluted Vesphene IIse and take out after capping

Follow standard procedure to remove the clog

Clean the surface of inside CytoShield and sorting chamber with 1:64 diluted Vesphene IIse

Replace the collection tubes

Continue with sort

Routine Decontamination after Sorting unfixed unknown human and non-human primate cells or lentivirally-transfected cells

Clean the surface of inside biosafety cabinets by using 1:64 diluted Vesphene IIse. Allow Vesphene to sit for 5-10 minutes, then wipe with a paper towel.

Decontaminate sample line by running Vesphene solution like a cell sample, followed by a deionized water rinse.

Place an open Petri dish containing 10 mL of 10% formaldehyde inside the glovebox of the Sorter.

Close glovebox with supplier cover.

Seal the CytoShield hood with the supplied gas tight door.

Allow to sit overnight.

When Cytoshield door is removed in the morning, all formaldehyde gas will be evacuated automatically through the hood exhaust.

Routine Maintenance Daily routine clean the surface of inside biosafety cabinets by using 1:64 diluted

Vesphene IIse



20

Check sheath line and waste line daily for wet due to leaking. Replace any leaking lines immediately

Rinse sheath tank with clean sheath if there is debris or crystals inside the tank

Certify Cytoshield hood annually

References

1. CDC. Recommendations for prevention of HIV transmission in health-care settings. MMWR 1987;36 (suppl no. 2S).

2. CDC. Update: Universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR 1988;37:377-388.

3. CDC. Guidelines for prevention of transmission of human immunodeficiency virus and hepatitis B virus to health-care and public-safety workers. MMWR 1989;38(S-6):1-36.

4. Schmid I, Nicholson JK, Giorgi JV, Janossy G, Kunkl A, Lopez PA, Perfetto S, Seamer LC, Dean PN. Biosafety guidelines for sorting of unfixed cells. Cytometry. 1997 Jun 1;28(2):99-117.

http://www.cdc.gov/mmwr/preview/mmwrhtml/00023587.htm









21

Appendix II

Validation of the MoFlo class I safety cabinet (CytoShield) for aerosol containment using Glo-Germ particles

Introduction The class I safety cabinet (CytoShield) on the MoFlo high-speed cells sorter is designed to serve as a secondary aerosol containment barrier. Aerosols are generated during normal sorting and especially during a clog condition and may potentially spread infectious agents, if present in the sample. To validate that the Class I cabinet is able to contain aerosols, we adapted the “Glo-Germ” test described by Oberyszyn and Perfetto. Glo-Germs are 5μm melamine copolymer resin beads developed for teaching the importance of hand washing to food and healthcare workers. Glo-Germ particles are highly fluorescent under near ultraviolet illumination and can easily be detected with a fluorescent microscope. Before proceeding to validation, the Class I cabinet must be tested and certified by Filtech (West Homestead, PA).

Procedures Make 2.5% suspension of Glo-Germ in PBS + 2% FBS as described[1,2].

Prescreen microscope slides for absence of fluorescent debris under the Nikon Eclipse E400 Fluorescent Microscope. Only the slides without any background will be used for the Glo-Germ test.

Carefully lay 11 slides at the specified location inside or outside the CytoShield hood (Table 1).

Table 1 Description of the location for each slide

Slide 1 Left, bottom of window of Cytoshield

Slide 2 Middle, bottom of window of Cytoshield

Slide 3 Right, bottom of window of Cytoshield

Slide 4 Left, top of window of Cytoshield

Slide 5 Middle, bottom of window of Cytoshield

Slide 6 Right, bottom of window of Cytoshield

Slide 7 Left, 3-inches apart from window of Cytoshield

Slide 8 Middle, 3-inches apart from window of Cytoshield

Slide 9 Right, 3-inches apart from window of Cytoshield

Slide 10 Top of receptacle

Slide 11 Top of sorting chamber



22

After confirming proper operation of the Cytoshield (face velocity > 75 ft/sec, HEPA filter functioning properly), run Glo-Germ on the MoFlo at ~20,000 events per second. MoFlo should be properly aligned.

Test aerosol generation at 4 conditions during normal sort mode and failure mode. The failure mode is created to mimic the clogging situation by deflecting the waste stream to hit the edge of the waste catcher as described[1,2]. The 4 test conditions are as follows:

I: Normal sort mode, CytoShield on, sorting chamber door closed II: Normal sort mode, CytoShield on, sorting chamber door opened III: Failure mode, CytoShield on, sorting chamber door closed IV: Failure mode, CytoShield off, sorting chamber door closed

Carefully check for Glo-Germ particles on each set of slides collected under the 4 test conditions, using the fluorescent microscope.

Record the number of Glo-Germs detected on each slide. Detection of 1 or more Glo-Germ particles indicates that aerosol is reaching the position represented by the slide. Care must be taken to distinguishing Glo-Germs from adventitious fluorescent debris. Glo-Germs are heterogeneous, but can usually be distinguished from fluorescent dust by shape.

Test evaluation No Glo-Germs should be detected on the slides outside the CytoShield hood.

Validation results from June 9th, 2004 Results (Table 2) indicated that no Glo-Germ particles were detected on the slides outside the CytoShield or on the edges of Cytoshield window during normal sort mode or failure mode. However, Glo-Germ particles were detected during normal sort mode and failure mode inside the sorting chamber.

Table 2 Number of Glo-Germ particles detected on slides

Condition I Condition II Condition III Condition IV

Slide 1 0 0 0 0

Slide 2 0 0 0 0

Slide 3 0 0 0 0

Slide 4 0 0 0 0

Slide 5 0 0 0 0

Slide 6 0 0 0 0

SSlliiddee 77 0 0 0 0

Slide 8 0 0 0 0

Slide 9 0 0 0 0

Slide 10 >>50 >>50 >>>50 >>>50

Slide 11 2 0 0 3



23

Conclusion Based on the results of this validation procedure, the certified Class I safety cabinet (CytoShield) on the UPCI Flow Facility MoFlo is working properly to contain aerosol generated during normal use and in failure mode.

References 1. Oberyszyn AS, Robertson FM. Novel rapid method for visualization of extent

and location of aerosol contamination during high-speed sorting of potentially biohazardous samples. Cytometry. 2001 Mar 1;43(3):217-22.

2. Perfetto SP, Ambrozak DR, Koup RA, Roederer M. Measuring containment of viable infectious cell sorting in high-velocity cell sorters. Cytometry. 2003 Apr;52A(2):122-30.



24

Appendix III Presented at ISAC 2008, Budapest, Hungary E.M.Meyer, I.Kim, Xio-Lun Wu and A.D.Donnenberg SAFE SORTING IN THE CORE FACILITY AS A PHYSICS PROBLEM An increasing awareness of laboratory biosafety has sharply limited the use of high speed flow-based cell sorting for separation of potentially infectious human and primate cells and transfected cell lines. Modern sorters operate at pressures between 30 and 60 psi and are capable of generating powerful aerosols when the nozzle is partially obstructed. Although vendors provide containment systems, none are certified by the manufacturer to protect the operator from potentially biohazardous aerosols. It is our view that safe sorting, like all work involving human tissues and body fluids, is simply a matter of exercising universal precautions as defined by the Center for Disease Control. Accordingly, we have isolated the fluidics units of our MoFlo sorter in a Class I Safety Cabinet (BSC1). When combined with standard BSL 2+ practices, this approach reduces our biosafety problem to one of basic physics. Namely, do the aerosols generated by the sorter under the most extreme conditions have sufficient mass and velocity (kinetic energy) to overcome the forces imposed by the medium through which they travel, gravity, and the opposing airflow of the hood? To answer this question we directly measured the size and velocity of aerosol particles generated under a variety of conditions designed to simulate worst case aerosols. Aerosol images were captured at 3,800 frames per second with 40 microsecond exposure using a Phantom v.5 camera with Phantom software (VisionResearch, Wayne NJ). Using a 70 micron tip at a sheath pressure of 60 psi, the Initial particle velocity formed a log normal distribution ranging from 0.02 – 8.5 (mean 3) m/sec. Given an airflow of 0.5 m/sec opposing the aerosol, the calculated maximum distance that a droplet could travel in the direction of the hood sash ranged from 2 to 9 cm for droplets ranging from 25 microns (the average) to 57 microns (the radius of a sort droplet). Since the minimum distance from the aerosol source (nozzle) to the hood sash is 30 cm, we concluded that aerosol droplets could not physically escape the safety cabinet when the hood is properly functioning. We conclude that cell sorting within a BSC1 is consistent with universal precautions and is safe, in the context of fully implemented standard BSL 2+ practices.