The Effectiveness of
HEPA filters on DNA

Kara F. Held1, Clark Rundell2, Robert Thibeault1, Daniel Ghidoni1,Daniel Magoon2, Trinh Nguyen2, Alyson Fisher2, Bretna Parker2,
Tania Spenlinhauer2, Joan Gordon2, and Steven Nesbitt2
The Baker Company, Sanford, ME, USA;
Maine Molecular Quality Controls, Inc., Saco, ME, USA1
2ABSTRACTMost laboratory techniques utilize
biosafety cabinets (BSCs) in order toprovide contamination control of theexperiments. BSCs depend on airflow
and HEPA filters to remove contaminantsfrom the environment. Aerosols may
be created constantly by common
lab practices, such as centrifugation,pipetting, and opening tubes. HEPA filters
are very effective at removing various
sized contaminants, but do not prevent
gasses and vapors from penetrating
through them.

At some particular size of particle,
HEPA filters will not be effective in theirremoval. Researchers have speculated
that DNA may not be captured by HEPA
filters, allowing for contamination ofsubsequent experiments by aerosolized
DNA. Here we determined that DNA iscaptured by a HEPA filter at the same
efficiency as the filter is rated anddiscovered DNA cannot be dislodged
from a HEPA filter.

INTRODUCTIONHEPA filters are the main line of defense
against contamination in Biosafety
Cabinet (BSC) construction. HEPA (HighEfficiency Particulate Air) filters arecomposed of one continuous pleatedsheet of borosilicate fibers woven intoa crosshatched design. This sheet has
spacers in between the pleats to holdthe shape and is completely sealed withtwo part polyurethane to a sturdy frame(NSF International Standard 49, 2016).
This rigid shape and structure shownin Figure 1B is critical for directing
the air through the filter material toensure particulate removal.

of a wide range of sizes, with the mostpenetrating particle size of 0.21μm.
When tested at 0.3μm, near its worst, theHEPA filter has to still remove 99.97% or
better of all particulates. NSF requires
HEPA filters to be of Type C or better,removing a minimum of 99.99% of 0.3μm particulates (NSF International
Standard 49, 2016).

HEPA filters remove particles by threedifferentmechanisms:
Impaction,Interception, and Diffusion. As seen inFigure 1A, all three of these methods
lead to very high particulate removal

October 2017

1Many
commonpractices
withinmicrobiological procedures such as
pipetting, opening capped tubes, andcentrifugation, have the capacity tocreate aerosols. These aerosols cancontain cross-contaminating DNA that
may affect further experimentation.

FIGURE 1.
HEPA filter function and design.
(A) Shows the efficiency of a HEPA filterof removing particles of a particular size.
(B) Depicts the folded structure of theborosilicate sheets of a HEPA
filter cross section. These pleats
help direct air through the filterand ensure a greater surface areafor filtration.

METHODSAB

It has long been unknown to researchers
and laboratory personnel whether DNA
can be captured in a HEPA filter whenprocedures are conducted within a BSC
due to the unique properties and size ofDNA. Plasmid DNA has been estimatedto be 200nm or 0.2μm in diameter
when supercoiled (Tsoi et al., Biophys
Chem, 2010), however the ability of
DNA to exist in multiple conformations
leads to uncertainty. If the DNA stays
in suspension, it has a greater chanceof being captured, however, as thedroplets dry, if the DNA will act the sameis unknown.

Here we test the ability of DNA to becaptured by a HEPA filter in multiple
scenarios: (1) direct application to a
HEPA filter and (2) circulating in theairstream of a Class II Type A2 BSC (themost common style found in laboratories,
a partially recirculating cabinet). We thentested whether DNA can be dislodged
from a HEPA filter after a physical shockor an electrical power failure, and as
well as if DNA is destroyed by afull cabinet decontamination withchlorine dioxide (CD).

Direct Application to the HEPA filter
Plasmid DNA (60mL, 1x109 copies/mLin TE buffer) was placed in a Collison
nebulizer which generates a defined
droplet size of ~1μm (May, 1973).
A Class II Type B2 BSC (BioChemGARD,Baker) was installed with new HEPA
filters, adjusted to setpoint of 105 fpm
intake air with an 8 inch viewscreen
opening, and cleaned with 10% bleachand 70% ethanol. The worksurface wasremoved to expose the two side by side
exhaust HEPA filters. The nebulizer wasplaced directly in front of either side ofthe HEPA filters at a height of 6, 9, and 12
inches, secured by a ring stand (Figure 2).
Pre-experiment samples were collected
to ensure no background contamination.
The nebulizer was then run for 1 minuteOctober 20172

on each side, with and without theHEPA filters installed (Figure 2A and 2B,respectively). Samples were collected bycotton swab on glass slides and the direct
metal surface, and Whatman filter paper.
Before and after weight measurements
were used to determine how much DNA
was introduced in each condition.
The BSC was thoroughly cleaned with
10% bleach and 70% ethanol in between
samples and replicates. within the HEPAfilter. Electrical power failure on theBSC and exhaust blower system wassimulated three successive times to testfor unintentional DNA contamination. In
both situations, samples were captured
downstream of the HEPA filter.

FIGURE 2.
Experimental design for direct
application of the aerosolized DNA to
the under worksurface HEPA filters ofa Class II Type B2 BSC. Depicted are
the filters in place (A) and removed(B). C. Schematic of a Class II Type B2BSC showing airflow arrows, positive
and negative pressure areas (+ and- , respectively), HEPA filter locations(zigzag lines) including one below theworksurface, motor/blower, and thesampling locations denoted by red Xs.

ABC

EQUATION 1.

(1–(copies detected
copies introduced*% sampled

October 2017

DNA collection in a BSC airstream.

)) * 100%

A Class II Type A2 BSC (SterilGARD,Baker) was installed with new supply
and exhaust HEPA filters, adjusted
to a setpoint of 105 fpm intake air,
50 fpm downflow velocity with an 8
inch viewscreen opening, and cleaned
with 10% bleach and 70% ethanol.
Plasmid DNA (60mL, 1x109 copies/mL inTE buffer) in a Collision nebulizer wasplaced 8 inches away from the bottomof the viewscreen and run for 15 minutes(Figure 3A).

3Samples were collected by cotton swab
on glass slides or directly on the metal
surface, and on Whatman filter paper
throughout the BSC: on the worksurface,
below the worksurface, upstream from the
supply HEPA filter, directly downstream
from the supply HEPA filter, and directly
downstream from the exhaust HEPAfilter (Figure 3B). Before and after weightmeasurements were used to determine
how much DNA was introduced. The BSC
was thoroughly cleaned with 10% bleachand 70% ethanol in between samples
and replicates. Capture efficiency wascalculated by Equation 1.

FIGURE 3.
DNA in the BSC airstream. (A) The
nebulizer layout in front of the BSC.
(B) Diagram of a Class II Type A2 BSC
showing airflow arrows, positive andnegative pressure areas (+ and - ,respectively), HEPA filter locations(zigzag lines), motor/blower, and thesampling locations denoted by red Xs.

RESULTS

October 2017ABDNA destruction by Chlorine Dioxide.

DNA quantification.

After aerosolization of the plasmid DNA
into the Class II Type A2 BSC (SterilGARD,Baker) in the previous experiment, thecabinet was prepared for a standard
biodecontamination
withchlorine
dioxide using the DRS Laboratories Mini
Chlorine Dioxide System per themanufacturer’s instructions.

Samples collected with swabs wereextracted by eluting in 150µL of TE.
Samples collected using filter papers
were hole punched into 150µL of TE.
Both types of samples were vortexed,
centrifuged and 5µl of sample added toqPCR reaction. DNA quantification wasperformed by using qPCR with MMQCI’sinternal assay and plasmid based
standards on Roche LightCycler 2.0.

Direct application of the aerosolized
DNA to the HEPA filter (pictured inFigure 2A) resulted in a complete
capture of all DNA containing particles(Table 1) as demonstrated by placing
the nebulizers directly upstream of theHEPA filters located directly below theworksurface on the Class II Type B2
BSC and collecting the samples directly
downstream of these exhaust HEPA
filters (Figure 2C). After attempting toremove the DNA captured in the HEPA
filter through mechanical and electricalmeans by simulating a power failure of
the cabinet and external exhaust system,no DNA was detected (Table 1).

Using airflow calculations previously
determined (Stuart et al., 1983; Held etal., 2016), it is known that the mass flow
velocity of any compound added to theairstream by the intake air is equal tothe concentration of compound in thedownstream air and exhaust air. Combinedwith the known amount of DNA added tothe airstream (Volume displaced*DNA
density = copies introduced) and the
proportion of the surface area tested
as compared to the full HEPA face area,
the capture efficiency can be calculated
by Equation 1. All downstream HEPA
samples exhibited better than 99.99%
DNA removal efficiency (Table 2).

When DNA was aerosolized within
a Class II Type A2 BSC as depicted inFigure 3A, DNA was detected at significant
quantities throughout the cabinet as
expected based on airflow: under theworksurface and upstream from the
supply HEPA filter that provides clean air
to the worksurface (Table 2, Figure 3B).
However, unlike the direct application
above, downstream from both the supply
and exhaust HEPA filters, low quantitiesof plasmid DNA were detected.

Chlorinedioxide(CD)wasapplied to determine if a gaseous
biodecontamination
protocolwouldremove DNA from the metal surfaces
within the BSC. As shown in Table 2, CD
resulted in the removal of 99.7% of thecontaminated surface. After further
cleaning with bleach and isopropanol,
all traces of plasmid DNA were removed.

4

TABLE 1.
Direct application of aerosolized DNA toa HEPA filter using a Class II TypeB2 BSC.

TABLE 2.
Aerosolized DNA detected throughout
a Class II Type A2 BSC and the capture
efficiencies of the HEPA filters.

CONCLUSIONS

October 2017

Filter IN/OUTLocation

Avg Copies ofDNA detected

IN

Left0

IN

Right0OUT

Left

2.5x107

OUT

Right

3.3x107

Power Failure

Left

0

Power Failure

Right0Location

Avg Copies ofDNA detected

HEPA Capture
Efficiency (%)

Pre-cleaning0

Under worksurface

2.17x107

Worksurface

65

99.9997%

Upstream Supply HEPA

6.25x10

Downstream Supply HEPA

159

Downstream Exhaust HEPA

2.37x10Pre-decontamination

3.31x10

Post-decontamination

1.07x107

Post-cleaning

0499.9945%

9Upon direct application of DNA to theHEPA filter by the nebulizer inside theClass II Type B2 BSC, it was observed thatall DNA aerosols were captured, with novariation observed when the nebulizer
was placed 6, 9, or 12 inches above theexhaust HEPA filters (Table 1, Figure 2A,2B, and 2C). However, when the DNA wasintroduced to an entire BSC as simulated
in the Class II Type A2 BSC (Figure 3A),
DNA was detected both upstream and,to a lesser extent, downstream of boththe supply and exhaust HEPA filters(Table 2). DNA was expected to be
detected upstream of the HEPA filter(under the worksurface and pre-HEPA
samples, Figure 3B), as these samples
were exposed to the DNA-contaminatedroom air introduced by the nebulizer.
Controlling for the area sampled ascompared to the total amount of
DNA introduced into the airstream(Equation 1), allows for the determinationof the efficiency of DNA removal by
the HEPA filter.

599.9991%
3

All three areas where DNA was detected
post-HEPA had efficiencies greaterthan 99.99%, as specified by NSF
Standard 49 (NSF International, 2016,
Table 2). The disparity between theresults from the Class II Type B2 and Type
A2 BSCs may be explained by particle
size. Direct aerosolization produces
droplets ~1μm/1000nm in size. Theseparticles are an ideal size to be captured
by a HEPA filter. However, as the droplets
move through the airstream, they will
dry, as a droplet has a “wet time” of 0.6s
(May, 1973), and this will decrease theDNA aerosol size to the most penetrating
size of the HEPA filter, ~0.2μm or 200nm(Figure 1).
Chlorine Dioxide was able to remove
99.7% of the surface contaminated DNA,but not all. The most thorough cleaning
option was bleach and isopropanol,
however this solution only pertains toeasily reachable surfaces.

RECOMMENDATIONS

Here we have shown that direct
application of DNA aerosols will be
captured completely by a HEPA filter, but
as the droplets move through the BSC,
there can be up to 0.01% contamination.
To eliminate this potential contamination,
under worksurface or redundant HEPA
filters, or a 100% exhausted BSC design
could be employed depending on wherethe concern for DNA contamination is(e.g. the samples, the worker, the lab).

It could also be helpful to use a more
stringent filter, such as an Ultra Low
Particulate Air (ULPA) filter with
99.999% particulate removal efficiency
or a Super Ultra Low Particulate Air(SULPA) filter with 99.9999% particulateremoval efficiency.

REFERENCES

NSF International. (2016) NSF/ANSI
49 – 2016 Biosafety Cabinetry: Design,Construction, Performance, and Field
Certification. Ann Arbor, MI.

May, K. R. (1973) The Collison Nebulizer:
Description,PerformanceandApplication. Aerosol Science. 4: 235-243.

Tsoi, M., Do, T.T., Tang, V., Aguilera, J.A.,
Perry, C. C., and Milligan, J.R. (2010)
Characterization of condensed plasmid
DNA models for the studying the direct
effect of ionizing radiation. Biophys
Chem. 147(3): 104-110.

October 2017

6Stuart, D.G., First, M.W., Jones, Jr., R.L.,
and Eagleson, J.M. (1983) Comparison ofChemical Vapor Handling by Three Typesof Class II Biological Safety Cabinets.
Particulate & Microbial Control. 2:18–24.
Held, K.F., Ghidoni, D., Hazard, G., andEagleson, D. (2016) Volatile Chemicals
and Class II Type A2 “Recirculated” BSCs:How Much is Safe? The Baker Company.