RE: Sorting live human lymphocytesFrom: Mario Roederer
[roederer@drmr.com]
Sent: Monday, February 24, 2003 7:17 PM
To: Cytometry Mailing List
Subject: RE: Sorting live human lymphocytes

Never one to shy away, I shall rise to the bait.

Our standard of practice is to not sort untested human samples.  It
isn't difficult to get tested samples; the cost is not prohibitive
compared to the cost of everything else that you will do with the
specimen.  We will sort live tested samples in our "regular" (non
BSL-3) sorter.

Note, of course, that simply because the samples are "tested" does not
mean that they are "negative".  We still take universal precautions
with all human specimens.  We leave individual precautions (face mask)
up to the experimenter (gloves and lab coat are required).  It is my
opinion that the risk of infection from a tested negative sample is
low enough that requiring BSL-3 sorting is not required.  However, we
thoroughly train our users to understand that aerosolization
significantly increases the risk of infection.

For samples that are unknown or have been tested HIV+ or Hep+, we have
the luxury of having an additional sorter entirely contained in a
BSL-3 (negative pressure) environment.  Steve Perfetto, who manages
the facility here, has done an excellent job of developing a rapid SOP
for qualifying the containment on a daily basis (in press in
Cytometry; you can contact him at "perfetto@mail.nih.gov" for
details).

We do not feel that containment solely by the manufacturer's aerosol
containment unit is sufficient.  Thus, we require infectious sorts to
be performed by operators gowned in full suits accompanied by a HEPA
filtered breathing apparatus.  It is our opinion that only the triple
protection (instrument aerosol containment + personal suit + BSL-3) is
sufficient for this process, in that it allows for the unlikely
breakdown of any one component and still provides adequate
protection.  (It also protects against insufficient training: for
example, the requirement that the stream be shut off for at least 120
seconds prior to opening the sort chamber (following a clog, e.g.)
before all infectious particles are actually cleared by the
containment system!  Operators are often in such a hurry that they
neglect this simple step).

Note that HIV, while being a BSL-2 pathogen, when aerosolized becomes
BSL-3.  Therefore, it is entirely inappropriate to sort HIV+ human
samples viably in a BSL-2 environment (e.g., without negative pressure
rooms, etc.).  For those who are setting up "infectious sorters", you
probably should not rely solely on instrument containment but expect
to set them up in a separate room that is fully BSL-3 compliant
(including an anteroom to separate the area from standard laboratory
areas).

mr

(PS, the opinions expressed herein are mine, and in no way represent
the position, official or otherwise, of the United States Government.
Of course, if they paid more attention to me, that might be a
different story.)

At 7:45 PM -0600 2/21/03, Crowe, James wrote:
 I would be interested to hear an extension of this discussion from
Mario Roderer, Marty Bigos, and others who intentionally analyze or
sort live human lymphocytes from HIV-individuals for experimental
purposes. It seems to me the state-of-the-art for BL-3 sorting could
inform the development of good practice for BL-2 sorters.

 Jim Crowe

  -----Original Message-----
 From:   David Coder [mailto:d_coder@MSN.com]
 Sent:   Thu 2/20/2003 2:42 PM
 To:     Cytometry Mailing List
 Cc:
 Subject:        RE: Sorting live human lymphocytes [JPR-NGT5823]

 Paul,

 The proposed procedure--". . .sorting live human lymphocytes from
untested
 patients."--may perhaps (note word "perhaps") be permitted in a
facility
 that conforms to the requirements of the OSHA rule on handling human
blood
 or blood products. (ref. 29 CFR 1910.1030 - Occupational Exposure to
Blood
 borne Pathogens; for a text summary see:
 http://www.osha-slc.gov/needlesticks/needlesticks-regtxtrev.html.

 There should be a standard institutional policy that regulates work
with
 human tissues. For example such a policy should include:
 Written Exposure Control Plan;
 Exposure determination;
 Provision of free hepatitis B vaccine to employees with occupational
 exposure;
 Use of Universal Precautions when handling all human blood and other
 potentially infectious materials;
 Special procedures for HIV and HBV research;
 Annual training;
 Post-exposure follow-up;
 Documentation in the Exposure Control Plan of evaluation and
implementation
 of effective safer medical devices.

 As the cells are untested, you must assume that they are
contaminated.

 As a minimum, the laboratory should conform to Biosafety Level 2
 requirements (see the following for a brief overview:
 http://www.cdc.gov/od/ohs/symp5/jyrtext.htm).  Among a number of
other
 requirements, the sorter (a potential generator of aerosols that may
contain
 infective agents) must be in a Class II biosafety cabinet to meet
the
 requirements for aerosol containment.  Likely, you may have to
perform a
 validation study to demonstrate that no contaminants can escape from
the
 cabinet. (The ISAC Biosafety Committee is performing a correlation
study of
 the standard bacteriophage assay of aerosol detection with the
GloGerm
 assay. The latter may make such evaluations far more easy and rapid
to
 perform. We plan to submit for publication in Cytometry by the end
of
 summer.)

 As enforcement of the above is the responsibility of your local
 Environmental Health and Safety Officer. you might refer your client
the EHS
 Officer to plead his case; its not really your job.

 Dave
 Writing while wearing the hat of:
 Chair, ISAC Biosafety Issues Surveillance Committee
 ==================================
 David M. Coder, Ph.D.
 Consultant in Cytometry
 Seattle, Washington
 tel./message: 206-499-3446
 email: d_coder@msn.com

 -----Original Message-----
 From: J.Paul Robinson [mailto:jpr@flowcyt.cyto.purdue.edu]
 Sent: Wednesday, February 19, 2003 6:04 PM
 To: cyto-inbox

 Colleagues:

 I would like to get input into the following issue - this has been
discussed
 before, but I would like to put this topic into the summary page and
I also
 need some advice.

 There is a faculty member here who is insisting on sorting live
human
 lymphocytes from untested patients. His argument is that these are
from
 children or teenagers and therefore a low-risk group. He obtains the
 mateirals from a clinic and claims that he has no time to test the
samples.

 He is unbelievably insistent (my techs say he is rude and obnoxious)
and is
 very upset that I have told him that I need some time to research
this issue
 to see what we should do. Even after I stopped a sort from taking
place
 instructing my technicans not to sort the cells, he tried to
convince them
 to sort after I left for a meeting!!

 He claims that he has done dozens of similar live human sorts at
several
 major institutions (I am checking so I won't list the institutions
here!)

 He claims that "many of the major papers in the immunology
literature sort
 live human lymphocytes, so why can't you do that here? Other
institutions do
 it all the time...."

 Has anyone actually tracked the number of such sorts?

 So my questions are the following:

 1. What is your institution/lab policy on sorting live human
materials? 2.
 Does your institution list this policy on a web site 3.  How many of
these
 sorts do you do? 4. Do any of you have obnoxious faculty that treat
your
 techs like dirt? If not, we have one you can have!

 I will be happy to sumarize the discussion and post it to the new
summary
 page at http://www.cyto.purdue.edu/hmarchiv/cytomail.htm
 "view Summaries" link

 Regards
 Paul Robinson
 Purdue

 J.Paul Robinson, PhD             PH:(765)4940757
 Professor of Immunopharmacology
 Professor of Biomedical Engineering
 Purdue University          FAX:(765)4940517
 EMAIL:jpr@flowcyt.cyto.purdue.edu
 WEB: http://www.cyto.purdue.edu