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Aerosols and Cell Sorting:
Characterizing the Risk and
Evaluating Protection
Kevin L. Holmes, Ph.D.
Chief, Flow Cytometry Section,
National Institute of Allergy and
Infectious Diseases, NIH
Background: What Prompted this
Study
1. Clear that aerosols are a concern
2. Opening of new Biodefense/Emerging
Infectious Disease building at NIH
1
The C.W. Bill Young Center for Biodefense
and
Emerging Infectious Diseases (Bldg 33)
Op
ed
en
Ma
0
y2
06
• Non-Select Agents
– TB, HIV, SIV, West Nile Virus, Dengue
• Select Agents
– MonkeyPox, Anthrax, Avian Flu, 1918 Flu, JEV
• FCS maintain two BSL3 cell sorter labs
Background: What Prompted this
Study
1. Clear that aerosols are major concern
2. Opening of new Biodefense/Emerging
Infectious Disease building
3. Bridge the ‘gap’ between flow cytometry
community and the Division of Safety
2
Aerosols and Cell Sorting:
Characterizing the Risk and
Evaluating Protection
• Aerosols & Bioaerosols
– Infectious bioaerosols
• Measurement of aerosols produced by
Cell Sorter
• Protection from Aerosols
– Cell Sorter specific
– Personal protection
Aerosols
• Aerosol or bio-aerosols: definitions
– Aerosol: a suspension of solid or liquid
particles in a gas
– Bio-Aerosol: an aerosol comprising particles
of biological origin which may affect living
things through infectivity, allergenicity, toxicity,
pharmacological or other processes.
• Aerodynamic particle size of 0.5 to 100 μm
3
Aerodynamic Diameter
• For a given particle, the diameter of a
water droplet having the same
aerodynamic properties
– Standardizes for shape and density
Irregular
Particle
de = 5.0 μm
pp = 4000kg/m3
χ=1.36
de = 8.6 μm
pp =
1000kg/m3
VTS=2.2
mm/s
Aerodynamic
equivalent
sphere
VTS=2.2
mm/s
Infectious Bio-Aerosols
• An aerosol of bacterial, viral or fungal
origin capable of initiating an infectious
process in a susceptible host
– Although infectious agents have discrete size
ranges…
• Bacterial cells/spores: ~0.3-10μm
• Fungal spores: 2.0-5.0 μm
• Viruses: 0.02-0.3 μm
4
Infectious Bio-Aerosols
– Their size, in conjunction with the carrier
matrix generated from a source, changes as
they are aerosolized and exposed to
environmental factors:
• Relative Humidity and temperature may favor
desiccation or hygroscopicity
• Rapid desiccation results in smaller aerosols that
can remain airborne longer (droplet nucleii);
particles can increase in size due to hygroscopicity
(condensation)
Infectious Bio-Aerosols
• Infectivity related to
– particle size and shape, i.e. Aerodynamic
diameter
• lung deposition
– Microbe survival
– Microbe virulence
– Host factors
5
Size and Infectivity
• Lung Deposition
– Size determines retention and deposition
Hatch, T. 1961;
Bacteriol Rev
25:237
Size and Infectivity
• Size of infectious particles
– Human volunteer experiments (1963-70):
• Aerosolized rhinovirus, coxsackie virus and
adenovirus: infection associated with aerosols of
aerodynamic diameter of 1.0 to 2.0 μm
– M. tuberculosis patients:
• Fennelly, et al. (2004) characterized size
distribution of infectious aerosols from TB patients:
cough generated aerosols, mode size of 2.1 to 3.3
μm
6
Aerosols, Cell Sorters and
Biosafety
• BMBL, 5th Ed.:
– “General agreement among biosafety professionals…
that an aerosol generated by procedures…is the
probable source of many LAI’s”
• Observation: cell sorters produce aerosols
– ~40-200 μm plus 3-7 μm satellite droplets
– ‘secondary aerosols of various and undefined droplet
sizes’ produced during failures (clogs) (ISAC
biosafety guidelines)
• What is the size and concentration of aerosols
produced by a cell sorter during a failure?
What is the Size and Concentration
of Aerosols Produced by a Cell
Sorter?
• Aerodynamic Particle Sizer
– TSI Model 3314 UV-APS
– capable of measuring particles between
0.5μm to 20μm (aerodynamic diameter)
– Principle of operation
• Uses time of flight measurements to determine
aerodynamic particle size
• Incorporates UV laser for measurement of
biologicals
7
TSI Instruments UV-APS
355nm Laser
(diodepumped
Nd:YAG freq
tripled laser)
TSI Instruments UV-APS
Collection
optics:
430-580nm
8
Using the APS to Measure
Aerosols Produced by Cell Sorter
– Need to know that particles detected are
generated by sorter, not ambient air
• Measure inside biological safety cabinet
• Use UV-APS together with UV-excitable dye in
sheath fluid or sample to unambiguously determine
source of detected particles
Characterization of Aerosols
• FACS Aria in ‘fail mode’
– Deviate center stream to impact side of collection
trough
• Determine concentration and size distribution of
aerosols from Aria in fail mode
– Conducted in Baker BioProtect II class II BSC
– Variables
• Measurement location
• Sheath pressure
9
Measurement Locations
Position # Dist. From
stream
cm(in)
1
1.3 (0.5)
2
2.6 (1.0)
3
3.9 (1.5)
4
5.2 (2.0)
5
6.5 (2.5)
6
Position # Dist. From
stream
cm(in)
7
9.1 (3.5)
8
15.2 (6)
9
21.3 (8.4)
10
27.4 (10.8)
11
Hood grill
33.5
7.8 (3.0)
Measurement Locations
10
Sheath: 70 psi
Concentration (dw/dlogDp - Number)
10000
1000
Position 11
Position 10
Position 9
Position 8
Position 7
Position 6
Position 5
Position 4
Position 3
Position 2
Position 1
100
10
1
0.1
0
5
10
15
20
Mean Aerodynamic Particle Size
Sheath: 35 psi
Concentration (dw/dlogDp - Number)
10000
1000
Position 11
Position 10
Position 9
Position 8
Position 7
Position 6
Position 5
Position 4
Position 3
Position 2
Position 1
100
10
1
0.1
0
5
10
15
20
Mean Aerodynamic Particle Size
11
Sheath: 20 psi
Concentration (dw/dlogDp - Number)
1000
100
Position 11
Position 10
Position 9
Position 8
Position 7
Position 6
Position 5
Position 4
Position 3
Position 2
Position 1
10
1
0.1
0.01
0
5
10
15
20
Mean Aerodynamic Particle Size
Comparison of 20,35 & 70 psi
1.5in (3.9 cm) from chamber
Concentration (dw/dlogDp - Number)
10000
70 psi
35 psi
20 psi
1000
100
10
1
0.1
0.01
0
5
10
15
20
Range of alveolar
infectivity
Meandeposition
Aerodynamicand
Particle
Size
12
Aerosol Protection in Cell Sorters
• Biological Safety Cabinet
• Flow Cytometer specific
– Containment vs. Evacuation
– FACS Aria
• Aerosol management system (evacuation)
• Sort Chamber
– Is there aerosol evacuation from sort chamber?
Aria Sort Chamber
Sort Chamber
Door Closed
Collection
Chamber: AMS
evacuation
Aspirator Drawer
Closed
Tube Holder
w/rubber O-ring
Aspirator Drawer
Open
Sort Chamber
Door Gasket
13
Aria Sort Chamber Design
• Sealed Sort chamber door
• Tube holders sealed with o-ring below chamber
• Fail mode (clog):
– Stream turns off, sample unloads, aspirator door
closes
• What happens to aerosols in the sort chamber?
• Does this design permit evacuation of aerosols
after fail?
UV-APS Measurements of Aria
Sort Chamber
containment & evacuation
• Conditions:
– Induce fail mode by deviating stream to hit
side of waste trough
– After fail mode induced, shut off stream; wait
15 sec, open Sort Chamber door & take
measurement at sort chamber door opening
– Variables: AMS on vs. off, Aspirator door
open vs. closed,
– UV tracer dye as sample at 1:25 dilution (also
in Biological Safety Cabinet)
14
Risk Reactor Clear Blue Dye
Risk Reactor’s IFWB-C0 is used in:
Coatings.
Water Tracing.
Invisible fluorescent marking inks.
Folder-gluers.
Conformal Coatings.
UV Sensor and automation markings.
Non-destructive testing.
Printing Inks.
Leak detection.
Man-made fibers.
Special Effects.
Color shifter.
Household Articles.
Coating Coverage.
Public power utilities.
Military facilities.
Engineering firms.
Testing companies and laboratories.
Tank constructors and installers.
Electrical equipment manufacturer.
Assembly operations.
Quality control during manufacturing.
Product tracking.
Technical Information:
Shelf Life: Minimum 12 months.
Emission Wave Length: 460-470 nm
Excitation Wave Length: 350 nm
Risk Reactor also carries EPA Approved,
NSF Certified Water Based Tracers for
drinking water applications.
Aerosol Detection From Opened
Sort Chamber Door
Conditions:
•UV tracer dye as
sample
•Fail mode (stream
deflection)
•Sort chamber door
open
•Measure 6.5cm from
chamber
•Performed in Biosafety
Cabinet
15
UV-APS Measurements of Aria
Sort Chamber Containment &
Evacuation
TEST 1: CLOSED CHAMBER
1. Tube holder in place
2. Sort chamber closed
3. Aspirator door closed
4. Fail mode – turn off stream-wait
15 sec
5. No AMS.
6. Open sort chamber and take
measurement
18x10e3
UV-APS Measurements of Aria
Sort Chamber Containment &
Evacuation
TEST 2: OPEN ASPIRATOR DOOR
WITH AMS
1. Tube Holder in place.
2. Sort chamber closed
3. Aspirator door open
4. Fail mode – turn off stream-turn
on AMS, wait 15 sec
5. Open sort chamber and take
measurement
450
16
Problem With ‘Containment’ Design
Even with aspirator door open,
Tube holder o-ring and tubes in
place prevent air flow from sort
chamber
Sort Chamber is sealed
prevented air flow exchange for
evacuation
Inefficient evacuation of sort
chamber: potential exposure
when chamber is opened
Modifications of Aria Tube Holder
and Sort Chamber Door
Threaded
hole for
Luer
fitting
Holes in
tube
holder
0.22 μm
syringe
filter
17
Modifications of Aria Tube Holder
and Sort Chamber Door
UV-APS Measurements of Aria
Sort Chamber Containment &
Evacuation
TEST 3: MODIFICATION OF TUBE
HOLDER AND SC DOOR;
CLOSED ASPIRATOR DOOR
WITH AMS
1. Tube Holder in place.
2. Sort chamber closed
3. Aspirator door closed
4. Fail mode – turn off stream-turn
on AMS, wait 15 sec
5. Open sort chamber and take
measurement
1.5 x 10e3
18
UV-APS Measurements of Aria
Sort Chamber Containment &
Evacuation
TEST 3: MODIFICATION OF TUBE
HOLDER AND SC DOOR;
OPEN ASPIRATOR DOOR
WITH AMS
1. Tube Holder in place.
2. Sort chamber closed
3. Aspirator door open
4. Fail mode – turn off stream-turn
on AMS, wait 15 sec
5. Open sort chamber and take
measurement
6. Conclusion: Simple
modification permits
evacuation of aerosols from
Sort chamber
3
Published Methods of Containment
Assessment in Cell Sorters
• Bacteriophage T4 test
• GloGerm bead test
– Can we measure Glo-Germ beads with the UV-APS
and…
– What frequency of total aerosols produced in fail
mode contain GloGerm beads?
– Can we use the UV-APS to validate the GloGerm
procedure for routine testing?
19
GloGerm: Can We Measure Them
Using the UV-APS?
GloGerm from spray bottle
GloGerm stream fail mode
18% UV+
0.3% UV+
Why So Few GloGerm Positive
Particles?
• Positive control for Glo-Germ tests routinely
recover >50 beads/slide field
• Aerotech collects 35L/min for 5 min
– 175L total
• UV-APS collects 1L/min for 20 sec
– 0.333L total
•
•
•
•
175/0.33=525
Or 500 fold more air sampled with Aerotech
0.3%X65261=173UV+ (GloGerm) particles
173x500=8.65x104 in 5 minutes at increased
rate
20
UV-APS and GloGerm
• GloGerm are measurable by UV-APS
• Differences in beads detected explained
by collection rates and times
• Possible to validate GloGerm procedure
with the UV-APS
Aerosol Protection
• Biological Safety Cabinet
• Flow Cytometer specific
• PPE (Personal Protective Equipment)
– Respirators, Surgical Masks & Helmets,
PAPR’S
21
Filtration Theory of Fiber Filters
– Misconception that aerosol filters are
microscopic sieves
– Mechanisms of deposition on fiber filters:
•
•
•
•
•
1. Interception
2. Inertial impaction
3. Diffusion
4. Gravitational settling
5. Electrostatic attraction
Filtration Theory of Fiber Filters
0.3 μm
particles have
highest
penetration
22
Respirators
(filtering facepiece)
– Standards: US NIOSH 42 CFR Part 84:,
European EN149: 2001 & Australian Standard
AS1716.
• Validation: NaCl particles used to test for filtration
efficiency
• NIOSH: N-95, -99, -100 respirators
– % penetration of most penetrating particles, i.e. 5%
penetration (95% efficiency) of 0.3μm NaCl particles for
N-95 filters
Does NaCl Particle Penetration
Represent Equivalent Protection to
Biologics?
• Published experiments have demonstrated
no difference in filtration of biological
aerosols and non-biologics
– Brosseau et al, 1997; Chen et al., 1994;
McCullough et al., 1997; Qian et al., 1998;
Willeke et al., 1996
– Conclusion: non-biological particles with the
same size, shape, and density are
appropriate surrogates for biological aerosols.
23
Respirator Fit Testing
Requirements
• For Respirators to work, they must fit your face!
– In US, OSHA requires annual fit testing for all
respirator wearers
– In Europe, CEN standard includes a ‘total inward
leakage’ test
– In UK, Approved Code of Practices of COSHH
requirement, but only 1st time for wearer
• Fit testing does not guarantee proper fit every
time it is used
– Requires the wearer to confirm correct fit
– Note: Respirators cannot be used with facial hair
(beards)
Other PPE
• Surgical Masks
– Not NIOSH certified as respirators
– Numerous reports of poor filtration efficiency
e.g.. Penetration of 20 – 100% for particles in
the 0.1 to 4.0 μm aerodynamic diameter
range have been observed
24
Other PPE
Surgical Helmet Systems
• Developed for orthopedic surgery, where
aerosols and splatter is common.
• Not certified as respirators; may have BFE or
VFE >99.9% but use 3.0μm particles with
bacteria or viruses (70% or < with 0.3 μm NaCl
particles)
– Some cite ASTM standard, but
meant for clothing not a respirator
Standard
PAPR’s
• Powered Air Purifying Respirators
– HEPA filter
– NIOSH certified
– Equivalent to N-100 respirator rating
– Can be helmet or half-face respirators
– Do not require fit testing
– Can be worn with facial
hair
25
Conclusions
• Using the UV-APS, Aerosols generated by cell
sorters can be characterized & the range of
aerosol size include the most potentially
infectious
• Modifications to the sort chamber in a FACS Aria
reduces risk of aerosol exposure
• The APS should help to validate current
methods of containment testing
• PPE is essential to operator safety, but must be
aware of types and their limitations
Acknowledgements
• The Flow Cytometry
Section, NIAID:
–
–
–
–
–
–
–
–
Mehrnoosh Abshari
Calvin Eigsti
Bishop Hague
Carol Henry
Tom Moyer
Michele Racine
Dave Stephany
Elina Stregevsky
• The Division of
Occupational Health
and Safety, NIH
– Jeff Potts
– Gino Begluitti
26
THANK YOU!
27