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Biosafety Level 2 plus (BSL-2+) Safety
Manual
UPCI Cytometry Facility
Room 1.45, Hillman Cancer Center
Revision 4, December 11, 2009
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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
Albert D. Donnenberg, Ph.D.
Date
Manager
E. Michael Meyer
Date
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
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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
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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
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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 nonhuman 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:
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 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
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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 (4 0C).
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.
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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 preapproved 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.
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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 highspeed 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
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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
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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.
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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.
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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.
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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.
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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
recommendatio
ns 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.
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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
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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 lentivirallytransfected 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
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





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 nonhuman 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
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


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.
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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
Slide 2
Slide 3
Slide 4
Slide 5
Slide 6
Slide 7
Slide 8
Slide 9
Slide 10
Slide 11
Left, bottom of window of Cytoshield
Middle, bottom of window of Cytoshield
Right, bottom of window of Cytoshield
Left, top of window of Cytoshield
Middle, bottom of window of Cytoshield
Right, bottom of window of Cytoshield
Left, 3-inches apart from window of Cytoshield
Middle, 3-inches apart from window of Cytoshield
Right, 3-inches apart from window of Cytoshield
Top of receptacle
Top of sorting chamber
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



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
Slide 1
Slide 2
Slide 3
Slide 4
Slide 5
Slide 6
Slide 7
Slide 8
Slide 9
Slide 10
Slide 11
Condition I
0
0
0
0
0
0
0
0
0
>>50
2
Condition II
0
0
0
0
0
0
0
0
0
>>50
0
Condition III
0
0
0
0
0
0
0
0
0
>>>50
0
Condition IV
0
0
0
0
0
0
0
0
0
>>>50
3
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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.
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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.
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