From: Mario Roederer (roederer@drmr.com)
Date: Mon Feb 24 2003 - 19:17:03 EST
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>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>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>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>mailto:jpr@flowcyt.cyto.purdue.edu] >Sent: Wednesday, February 19, 2003 6:04 PM >To: cyto-inbox Subject: Sorting live human lymphocytes > > > >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>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>http://www.cyto.purdue.edu
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