[Cytometry] TB Sorting

Reed, Douglas S dsreed at pitt.edu
Sun Feb 1 18:56:06 EST 2009

You should also consult the BMBL. The 5th edition, which is the latest, has this to say about containment of tuberculosis:

BSL-2 practices and procedures, containment equipment, and facilities are required for
non-aerosol-producing manipulations of clinical specimens such as preparation of acidfast
smears. All aerosol-generating activities must be conducted in a BSC. Use of a slidewarming
tray, rather than a flame, is recommended for fixation of slides. Liquifaction and
concentration of sputa for acid-fast staining may be conducted safely on the open bench
by first treating the specimen in a BSC with an equal volume of 5% sodium hypochlorite
solution (undiluted household bleach) and waiting 15 minutes before processing.109,110
BSL-3 practices, containment equipment, and facilities are required for laboratory
activities in the propagation and manipulation of cultures of any of the subspecies of the
M. tuberculosis complex and for animal studies using experimentally or naturally
infected NHP. Animal studies using guinea pigs or mice can be conducted at ABSL-2.111
BSL-3 practices should include the use of respiratory protection and the implementation
of specific procedures and use of specialized equipment to prevent and contain aerosols.
Disinfectants proven to be tuberculocidal should be used. See Appendix B for additional

Manipulation of small quantities of the attenuated vaccine strain M. bovis Bacillus
Calmette-Guérin (BCG) can be performed at BSL-2 in laboratories that do not culture M.
tuberculosis and do not have BSL-3 facilities. However, considerable care must be
exercised to verify the identity of the strain and to ensure that cultures are not
contaminated with virulent M. tuberculosis or other M. bovis strains. Selection of an
appropriate tuberculocidal disinfectant is an important consideration for laboratories
working with mycobacteria. See Appendix B for additional information.

Here's the link: http://www.cdc.gov/OD/ohs/biosfty/bmbl5/sections/SectionVIIIA-BacterialAgents.pdf

You should have an Institutional Biohazards Committee (IBC). They should also be consulted before any work is initiated.

I am curious what you mean by a BSL-2+ lab that makes it safe to sort unfixed, infectious cells. In my mind, any cytometer being used to analyze or sort unfixed samples which might contain infectious pathogens should be housed INSIDE a class II biological safety cabinet. It might also be prudent to have laboratory personnel wearing the proper personal protective equipment such as a N95 mask with a face mask and gloves. Particularly something that is KNOWN to be highly infectious by aerosol (such as TB). As has been recently pointed out on this list, you cannot trust the Aria (no matter how it is configured) to protect you from anything being aerosolized.

Doug Reed

Douglas S. Reed, Ph.D.
Aerobiology Manager
Regional Biocontainment Laboratory
Assistant Professor
Department of Immunology
University of Pittsburgh
8037 BST3, 3501 Fifth Avenue
Pittsburgh, PA 15261
(412) 648-9290
(412) 648-0050 lab
(412) 648-8917 fax
dsreed at cvr.pitt.edu

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Perfetto, Steve (NIH/VRC) [E]
Sent: Saturday, January 31, 2009 6:31 PM
To: hms at shapirolab.com; Kelly, Elizabeth
Cc: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] TB Sorting


Howard is absolutely correct, please consult the the attached ISAC standards of Infectious cell sorting for additional details.



On 1/31/09 1:12 PM, "Howard Shapiro" <hms at shapirolab.com> wrote:

Elizabeth Kelly-McKnight wrote-
> Our Flow Core has recently had an inquiry about sorting Tuberculosis-infected cells.  Our Aria is situated in a modified BSL2 lab (BSL2+) so that we are able to sort unfixed, infectious cells.  Because the machine is not officially in a BSL3 lab, we are not sure if it is safe to sort TB cells.
> Does anyone have any insight or experience with this?  Thanks!
Mycobacterium tuberculosis is a BSL3 organism, although an increasing
number of labs are now working with auxotrophs that can be handled at
BSL2 and the attenuated strain M. tuberculosis H37Ra can also be handled
at BSL 2. These could probably be dealt with in a BSL2+ sorting
facility. It would *not*, however, be safe to sort cells infected with
known virulent strains (e.g., H37Rv) or clinical isolates in your BSL 2+
facility; in fact, it might have very serious consequences for your


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Stephen P. Perfetto, MS.,MT. (ASCP)
Director, Core Flow Cytometry Facility
Vaccine Research Center, NIH
Building 40
40 Convent Dr., Room 5507
Bethesda, MD 20892-3015

email: sperfetto at nih.gov
Phone: (301) 594-8659


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