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Validation of biological safety cabinet (BSC)

Research · May 2015

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Khaled Elnagar
National Institute of Standards (Egypt)
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 By
Prof. Dr. Khaled E. Elnagar
National Institute for Standards
Tersa Str., Haram, Giza, Egypt
P.O.Box 136 Giza, Code No. `12211
Email: khnagare@hotmail.com
Fax: 00202-33867451
Cell phone: 00201222800620/01090711488

5/2/2015 Page 1 of 19
 Table of contents

Title Page
Cleaning and disinfection 2
Responsibilities 4
1- HEPA filter leak test (LT) 5
2- Inflow velocity test (IV) 7
3- Downflow velocity test 10
4- Airflow pattern test 12
5- Light Test 13
5- Noise Test 14
Safety Instruction and General recommendation 15
Definitions of Class II biological safety cabinet types 16
Operation qualification checklist 18
Performance qualification checklist 19

Warning
 It is not allowed to take a copy of any part of this document unless there is a written
permission from the author.
 The Health safety precautions are must for any test
 All Measurements and evaluations should be carried by professional personnel and
approved by National metrological institute or accredited lab. According to the
requirements of ISO/IEC 17025:2005
 The continuity of correct performance should be planned by regular testing at least once a
year.
 The manufacturer instructions must be followed for installation and proper use of any
equipment.
 Manufacturer Quality Control checks list shall be reviewed before unpacking and the first
installation.
 Changing the position of the equipment inside the lab or to the other lab needs revalidation.
 The criteria limits in this protocol valid only at the issue time. Accepting criteria may be
varied according to the requirement of the lab or the standard used.

5/2/2015 Page 2 of 19
 Protocol for the Validation of
Biological Safety Cabinet

Equipment Biohazard Safety Cabinet

Model: JSCB-1200SB 1200x600x650
Type: BSA2
Manufacturer: JS Research Inc- Korea
Reference EU (EN 12469)
NSF/ANSI Standard 49 – 2007
Test required 1- Filter Leak Test (Impulsion/downflow and Exhaust)
2- Inflow Test
3- Downflow test
4- Airflow pattern (smoke test)
5- Light
6- Noise

Cleaning and disinfection:

 Check electrical requirement on the name plate before use.
 Turn UV lamp at least 30 minutes before use. Be sure to turn it
off during the operation.
 For worktable and sides, sue sterile or non-use cloths, which do
not shed particles or fibers.
 To disinfect use cloths damped with a disinfecting solution which
do not damage or affect the paintwork, stainless steel or glass. A
previous clean to disinfecting can also be done with water and
soap.

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Responsibilities
End-User – The ultimate user or otherwise appointed personnel in the lab is responsible
to ensure the BSC is installed and operating properly. This document can assist in that
validation. This document cannot however anticipate every application or unique
situation encountered with the installation and operation. It is therefore essential that
users, lab managers and safety officers work together to broaden the scope of this
document through cautious forethought.

End-User Employer – The employer is responsible for supporting the validation through
adequate resources and training. The organization shall also ensure the validation process
has been fully carried out prior to use of the BSC. Records should be stored in a safe,
easily retrievable location. The location of the BSC, preventive maintenance and
certification schedules should be documented in the company’s quality system.

Cabinet Certifier – All BSC’s must be certified prior to use. A qualified certifying
technician must do this process with calibrated instruments. The cabinet must be certified
upon installation, on a scheduled annual basis and whenever the cabinet is moved to a
new location. Certification is the key requirement of this protocol.

Manufacturer – It is recommended to be certified ISO-9001, is responsible to fully test

the Delta Purifier BSC to NSF 49 requirements prior to shipment. The manufacturer must
retain these records. Their staff of Product Service Representatives and Product
Specialists can assist with information on the purchase, delivery and installation..

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 1- HEPA filter leak test (LT)
Purpose:
This test is conducted to check the HEPA impulsion/downflow and exhaust
filters, the filter housing, and the mounting frames for possible leakage.

Tools:
Aerosol generator (cool or hot) (DEHS, DOB or PAO are recommended)
Aerosol Photometer with ex Tended logarithmic scale
Accuracy: particle counter for individual particles with a dilution range
capable of detecting a penetration of 0.01% or less of particle exceeding 0.3
µm (HEPA filter of purity class 100).

Test conditions
The sample chamber of the safety cabinet must be completely empty,
widows and doors of the operating room must be closed (no draft).

Methodology:
1. Turn on the photometer and adjust it for the measurement are in
accordance to the manufacture’s instruction
2. Introduce aerosol to the airflow before the filter (entrance of the BSC)
3. With the probe nozzle approximately at 2.5 cm from the surface, scan
the downstream side of HEPA filter, including the perimeter of the
filter passing the photometer probe over the entire surface.
4. Scan the entire periphery of the filter and junction between the filter
and the frame a speed not exceed 5cm/s.

Acceptation criteria:
The leak (impulsion/downflow or exhaustion) should be not more than
0.01% of the specific before the filter (escaping volume of the aerosol).

Trouble shooting: replace downflow filter/ replace exhaust filter

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 HEPA Filter

Photometer probe
2.5 cm apart
Scan speed 5 cm/s

Aerosol entrance

Figure 1: LEAK TEST of the HEPA filter (Impulsion/downflow)

Photometer probe
2.5 cm apart
Scan speed 5 cm/s HEPA Filter

Aerosol entrance

Figure 2: LEAK TEST of the HEPA filter (Exhaust)

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 2- Inflow velocity test (IV)
Purpose:
 This test is conducted to determine the nominal value of the inflow
velocity (air entered to the BSC)
Value to be tested: inflow velocity V1 (m/s or ft/min)
Reference: ISO/IEC 17025:2005 NSF 49 (Annex F) and EU EN 12469
Test method:
 The inflow velocity (V1) is calculated using the values of the exhaust
volume and of the sample chamber inflow surface.
 If the BSC is connected to technical ventilation, inflow velocity can
also be determined at the work aperture.
Test Conditions:
 For all test methods, the sample chamber of the safety cabinet must be
completely empty, windows and doors of the operating room must be
closed (no draft).
Testing equipment:
 Suitable anemometer measure the velocity (m/s or ft/min)

Accuracy of testing equipment

 Accuracy of ± 0.01 m/s or maximal deviation 3% from the indicated
airflow velocity.

Calibration of test equipment

 The equipment must be calibrated in an accredited laboratory or
national metrological institute in accordance to the requirement of
ISO/IEC 17025:2005.

Methodology
 The inflow velocity is measured at several measuring points in the
work aperture. For this purpose, the front window is moved up to
height A=7.3 cm. The anemometer is installed directly to the inside of
the work aperture front window. The measuring points are located at
distance C of 3.8 cm immediately at the inside of the front window
lower edge and at distance B of 10 cm to each other. The lateral
distance to the work aperture edges is also 10 cm.

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The data can be tabulated in the following table:
Velocity Points
1 2 3 4 5 6 7 8 9 10 11 12
(m/s)
Average velocity (m/s)=V1=

2-1 Other values to be calculated

i- Average value for exhaust velocity V2
ii- Exhaust airflow volume R1
iii- Average value for inflow velocity V1

i- Average value for exhaust velocity V2

the direct exhaust velocity (V2) is measured using an anemometer at
distance of 10 cm above the exhaust aperture. The dimensions of the exhaust
aperture dimensions (depth and width)
Nine measuring points are located along the width at an even distance of ‘A’
to each other and to the lateral edges of the exhaust aperture. The measuring
points along the depth are located at an even distance ‘C’each other and to
the lateral edges of the exhaust aperture.

Methodology
 Install the anemometer to the following grid point:
A A A A

C
T
C

Figure 3: Test grid above the exhaust aperture

 Move the front window to the measuring position and operate the air
system blowers at full speed for approx 20 min.
 Perform measurement at all nine measuring points above the exhaust
aperture for a minimum of one minute for each measuring point.

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  Evaluate the individual measurements.

Calculations
The exhaust velocity can be tabulated in the following table to calculate the
average exhaust velocity (V2)

Velocity Points
1 2 3 4 5 6 7 8 9
m/s
Average exhaust velocity= sum of nine velocities/9= V2=

Average value for the inflow velocity V1 (m/s) = V2XA2 /V1

Where A1= 0.279 m2 (for 1200 mm BSC)
A2= 0.080 m2
V2= calculated value from Exhaust velocity.

Exhaust air flow volume test= V1x A1

Acceptation Criteria
 The nominal value is 0.45 m/s. The average value must be within
±10% of the nominal value.
 Exhaust airflow volume =580m3/h

Troubleshooting
 Raise exhaust bower speed
 Check exhaust blower control voltage
 Replace exhaust filter
 Check installed exhaust system for correct function.

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 3- Downflow velocity test
Purpose of the test
 This test is conducted to check the nominal value of the downflow
velocity (displacement airflow) in the work area of the safety cabinet.
Value to be tested: Downflow velocity (m/s or ft/min)
Reference: ISO/IEC 17025:2005 NSF 49 (Annex F) and EU EN 12469
Test Conditions:
 For all test methods, the sample chamber of the safety cabinet must be
completely empty, windows and doors of the operating room must be
closed (no draft).
Testing equipment:
 Suitable anemometer measure the velocity (m/s or ft/min)
 The anemometer measuring head must be secured tightly at the
measuring point to allow accurate measurements.
 The distance between the measuring points must be correct; the grid
must not be distorted.

Accuracy of testing equipment

 Accuracy of ± 0.01 m/s or maximal deviation 3% from the indicated
airflow velocity.

Calibration of test equipment

 The equipment must be calibrated in an accredited laboratory or
national metrological institute in accordance to the requirement of
ISO/IEC 17025:2005.
Methodology:
 Move the door to the working position.
 Install the measuring head at 10 cm 1bove the door edge
 For BSC of 1200 mm, leave 1/8x120 from all sides. Draw the grid and
define the measuring point as the following grid.

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 L=120 cm

15.75 cm

31.5 cm

15.75 cm

L/8 2L/8 2L/8

L/8

Figure 4: Downflow velocity test arrangement in accordance to EN 12469

 Calculate the average value for the downflow velocity

Calculating the average value for the downflow velocity

Average= Sum of measured values/number of measuring points

Accepting criteria
 The average value must be within ±10% of the nominal value (0.36
m/s).
 The difference between measured values at the individual measuring
points must not exceed 20%.

Troubleshooting
 Raise downflow blower speed
 Check exhaust blower control
 Replace downflow and or exhaust filters
 Check installed exhaust system for correct function.

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 4- Airflow pattern test
Purpose of the test
 This test is conducted to check the behavior of the airflows in the
sample chamber. Check to see if the displacement airflow passes
along the entire work area, if the vertical passage is correct, and of air
escapes through joints or seals of the housing and of the front window.

Reference: ISO/IEC 17025:2005 NSF 49 (Annex F) and EU EN 12469

Test Conditions:
 For all test methods, the sample chamber of the safety cabinet must be
completely empty, windows and doors of the operating room must be
closed (no draft).
 Inflow and downflow should be adjusted to the nominal accepted
values.
Testing equipment:
 Smoke tube or any safe smoke producer.

Methodology:
 Move the door to the working position.
 Using a smoke tube, scan the work area along its centerline from one
end to the other. Hold the smoke tube so that its tip is at a distance of
approximately =15cm above the work surface.

Accepting criteria
The smoke shows smooth downward flow with no dead spots or reflux. No
smoke escapes through the work aperture.

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 5- Light Test
Purpose of the test
 This test is to check the level and uniformity of the lighting system is
the correct one to guarantee a safety working area inside the cabinet.

Reference: ISO/IEC 17025:2005 NSF 49

Test Conditions:
 For all test methods, the sample chamber of the safety cabinet must be
completely empty, windows and doors of the operating room must be
closed (no draft).
Testing equipment:
 Light meter
Calibration of test equipment
 The equipment must be calibrated in an accredited laboratory or
national metrological institute in accordance to the requirement of
ISO/IEC 17025:2005.
Methodology:
 An imaginary division of the working surface will be done in equal
parts (not more than 8 and less than 6)
 The illuminating level will be measured in each of these parts, placing
the light meter over the working area.
 The average of the measures carried out will be calculated as well as
the safety cabinet illumination level.

Accepting criteria
 The illumination level average is not less than 750Lux
 Any Individual reading can not be less than 70% of the average
illumination level.

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 5- Noise Test
Purpose of the test
 This test is to verify the noise level of the cabinet is in the allowed
limits.

Reference: ISO/IEC 17025:2005 NSF 49

Test Conditions:
 For all test methods, the sample chamber of the safety cabinet must be
completely empty, windows and doors of the operating room must be
closed (no draft).
Testing equipment:
 sound meter (dBA)
Calibration of test equipment
 The equipment must be calibrated in an accredited laboratory or
national metrological institute in accordance to the requirement of
ISO/IEC 17025:2005.
Methodology:
 The sound meter is placed 1m from the front of the cabinet and 1.5 m
high. With the cabinet running, 2,3 readings will be taken for different
duration’s (10 and 30 sec).

Accepting criteria
 Less than 60 dBA.

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 Safety Instruction and General recommendation
1. The bench working area must not be used as a laboratory equipment store. This
may cause an unnecessary presence of dust, which endangers sterility.
2. Even if the working area in a clean sterile laminar flow, the bench surroundings
may be contaminated. The contaminating sources must be detected in order to
avoid them while introducing the arms, material, etc.
3. All the material necessary for the work must be particle free and previous cleaned.
4. Do not put inside the working area materials such as: paper, wood, cardboard,
pencils, erasers, etc. since they give off a lot of particles.
5. It is recommended to wash arms, hands and finger nail with a germicidal soap
before and after working. The users must avoid touching their mouth and eyes.
6. It is recommended to use long sleeved overalls with tight cuffs, and, which
special work, protecting gloves. (Alternatively use mittens). Both the overalls and
the mittens must be made of a material which gives off as few fibers and particles
as possible.
7. Start the bench 10 minutes before beginning your work with it. Thus the working
area and the material introduced are scavenged and the particles removed.
8. Whenever pipettes are used, they must be the mechanical suction type. Never use
them with mouth suction since you are very likely to inhale the aerosol originated
by the suction.
9. Whenever platinum handles must be used, we suggest the use of electrical
incinerators and even disposable ones, if possible.
10. If the work to be done requires the use of a Bunsen burner, or similar gas flame,
we recommend those with a constant pressure button. Remember that an
excessively long flame might burn the absolute filters and bend the light diffuser.
11. The shadows and whirls caused by the objects, equipment and material
surrounding the working area must be examined before starting the operation, in
order to determine their possible effects. Remember that the airflow does not go
back to its laminar condition possible effects. Remember that the airflow does not
go back to its laminar condition before a distance 2.5 times the diameter of the
object causing the obstruction.
12. When vials and tubes are used, we recommend the use of screw caps instead of
cotton ones, the latter giving off a lot of particles.
13. Once the operation is finished, all disposable products (handles, Petri Plates, etc)
and the culture medium, samples, tubes, vials, etc., will be removed from the
bench inside impermeable and sterilizable bags, if necessary.
14. When working with the bench or during its cleaning operations, special care must
be taken not to damage the HEPA filters by hitting them, pouring liquids,
splashing, etc.
15. When using the biological safety cabinet, adjust the seat to adopt a correct
position will allow the user to work within safety conditions and moreover to
avoid possible physical damage (sight, cervical, back, etc.)
16. Avoid locating the biological safety cabinet in a current that can be produce
turbulences in the laminar flow.

5/2/2015 Page 15 of 19
 Definitions of Class II biological safety cabinet types

Volatile toxic
Class II Minimum intake
Construction Airflow pattern chemicals (gases or
BSC Type velocity (fpm)
vapors) permitted

Downflow and
May have biologically
inflow air mix in
contaminated ducts and
plenum
a common,
A1 75 approximately 70 No
plenums under positive
percent recirculated
pressure to the room
as downflow,
exhausted
30 percent
Biologically No—when exhaust air
Downflow and
contaminated ducts and is vented back into the
inflow air mix in a
plenums must be under room
common plenum,
negative pressure to Yes—minute quantities
A2 100 approximately 70
the room, or allowed when canopy
percent recirculated
surrounded by connected and
as downflow, 30
negative-pressure exhausted to the
percent exhausted
ducts and plenums outdoors
Approximately 60
percent of of
Biologically
downflow air
contaminated ducts and
exhausted through
plenums must be under
a dedicated duct;
negative pressure to Yes—minute quantities
B1 100 the remainder of
the room, or allowed
downflow air
surrounded by
(approximately 40
negative-pressure
percent) mixes with
ducts and plenums
intake air and is
recirculated
Downflow air drawn
Contaminated ducts from laboratory;
and plenums must be inflow and
under negative downflow air
Yes—as an adjunct to
B2 100 pressure to the room, exhausted with no
microbiological work
or surrounded by recirculation in the
negative- pressure cabinet or return to
ducts and plenums the laboratory (100
percent exhausted)

5/2/2015 Page 16 of 19
 Figure 5: Class II BSC airflow schematic

Reference:
 David C. Eagleson, The Baker Company, Inc., Certification of Class II biological safety
cabinets With proper field certification and commissioning practices, BSCs will keep
hazardous materials away from personnel and ensure environmental cleanliness. Reprinted
with revisions to format, from the April 2008 edition of CLEANROOMS, Copyright 2008 by
Pennwell corporation
 NSF International, NSF/ANSI Standard 49 – 2007, Class II (laminar flow) biosafety
cabinetry, adopted Jan. 24, 2007.

5/2/2015 Page 17 of 19
Operational Qualification
Step Description Specification or Acceptance Criteria Result
yes no
1 Certification Prior to use, has a qualified certifier tested the
cabinet prior to the NSF 49 or European
standards the cabinet was listed under?
Has the certifier labeled the BSC with the
successful certification date?
Certification should be done annually. Has the
next required certification been added to your
quality system’s preventive maintenance or
certification schedule?
2 Training
User Training Have all users been properly trained on the
safety, theory of operation and limitations of
the BSC?
Do all users understand techniques for:
 Cleaning & disinfection of the abinet’s
interior
 Loading supplies and equipment,
 Avoiding cross contamination,
 Not disturbing the laminar flow,
 Spill control and clean up,
 Shutting down the cabinet?
Are users aware of ergonomic factors that can
cause unnecessary fatigue or personal
discomfort? (Refer to the Safety and Comfort
section of the User’s Manual.)
3 Cleaning
Exterior Cleaning Has the exterior of cabinet been cleaned of
dust that accumulated throughout installation
Interior Cleaning Have the BSC’s interior surfaces been
cleaned and disinfected appropriately for the
work that is about to be performed in it?
Has the towel catch screen located in the rear
under the work surface been checked for any
foreign debris requiring removal?
4 Others Defined by the end users

All theses steps should be checked with yes

5/2/2015 Page 18 of 19
 Performance Qualification
NOTE: This Performance Qualification section is only a recommendation of some basic items to
consider for your protocol. Your protocol should include tests and inspections that are pertinent to
the applications performed within the equipment.

Step Description Suggested Criteria

1 Periodic
Certification
Cabinet  Certification should be done at a minimum annually. An
Performance experienced certifier can verify the cabinet’s
performance.
 Is the BSC’s current certification within the acceptable
timeframe set by your organization?
 Has there been a procedure established if a cabinet is
found to have exceeded its certification due date?
Is the next required certification noted in your quality
system’s preventive maintenance or certification schedule?
2 Maintenance
U. V. Lamp If equipped, the U.V. Lamp should be replaced at least
annually to remain effective.
Towel Catch The towel catch screen located in the rear under the work
surface should be checked for any foreign debris when the
cabinet is cleaned.
Fluorescent Regular maintenance should ensure that the Fluorescent
Lamp or D65 Lamp is operating properly.
3 Others Defined by the end users

5/2/2015 Page 19 of 19

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