From: Reed, Doug S Dr USAMRIID (Doug.Reed@DET.AMEDD.ARMY.MIL)
Date: Thu Nov 07 2002 - 09:17:29 EST
Allen, I saw your post on the Cytometry list. I hadn't responded because it was beyond the scope of what we do. We have an active interest in quantitating pathogens (virus and bacteria) in aerosols used for challenging animals to test vaccines and therapeutics. To that end, we've utilized flow cytometry to assist in that effort - but only to acquire data, not to sort. The key point being that our samples are fixed with formaldehyde before they're run on the cytometer. The reason I mention that is from what it sounds like you want to do is sort live pathogens. To me this seems like a very bad idea, primarily because of the potential for aerosolization. For example, if we were to do live pathogens on our FACSCalibur, I'd probably stick the whole thing under a specially built laminar flow hood where the operator could reach into the hood to run the instrument. However, I don't see how that could be done with a dedicated sorter (ala MoFlo or Vantage). The only way I see you being able to do that is to build a dedicated BSL-3 facility with negative air pressure (so air flows into the room from the environment and not the reverse). The operator(s) would then enter the room wearing a powered-air purifying respirator (PAPR, made by 3M). That's about the only way I could see you protecting the operator from potential exposure to the pathogen. Decontamination could be achieved with bleach as is typically done for sterile sorting, only in this case you'd be doing it post-sort as well as pre-sort. Surface sterilization of the instrument with bleach would probably also be a good idea. To decontaminate the instrument for maintenance/repairs, we typically drain any fluids from the machine, put the machine in an airlock, open everything up and then do an overnight exposure to a high concentration of formaldehyde. As you can imagine, this is not something you want to do on a routine basis because of the potential corrosion of the instrument. Alternatively you put the repair person in a PAPR and have them enter the facility to do the work - but at least so far the BD service reps are rather resistant to doing that in our facility. In addition, you've got to have some mechanism for preventing access to the room and an exit strategy - for example, for our BSL-3 and BSL-4 facilities we have to change clothes and shower out when we're done for the day. Not all BSL-3 facilities require that, but they do typically require you to change clothes. Handling different pathogens isn't that big a deal, as the safety precautions are pretty much the same for all, depending on the biosafety level of containment required. Because of the risk of aerosolization from the cytometer, everything would have to be treated as BSL-3. In one special case here, I've done samples from Ebola-infected monkeys and then even with fixed samples we ran the machine in BSL-4. One more thing to consider - if Tufts does not already possess licenses from the CDC for these pathogens, that will be a requirement as well. There are a lot of issues as regards these licenses - who has access to the pathogens, proper storage, security, disposal, etc. This would include flow cytometry personnel who handle the samples containing the pathogens. I was talking just this week at a meeting with a scientist from the University of Buffalo who was trying to get access to Francisella tularensis and was completely stonewalled by everyone he talked to. Francisella is of course an "A" list pathogen (of highest concern as a biological warfare threat) but even for "B" or "C" list pathogens you've pretty much got to have the approval of CDC. Those rules are still being set though, so you talking about a very fluid situation. Bottom line though is that your biggest headache/concern is going to be the safety of the operators, protecting them from potentially lethal exposure to live pathogens. To achieve that would require a pretty heavy outlay of cash. Good luck. You're gonna need it. -Doug Reed Douglas S. Reed, Ph.D. Microbiologist Respiratory & Mucosal Immunity Department of Aerobiology & Product Evaluation Division of Toxinology & Aerobiology U.S. Army Medical Research Institute of Infectious Diseases 1425 Porter Street, Fort Detrick Frederick, MD 21702-5011 301-619-6728 301-619-6911 fax doug.reed@det.amedd.army.mil -----Original Message----- From: allen parmelee [mailto:allen.parmelee@tufts.edu] Sent: Wednesday, November 06, 2002 7:02 PM To: cyto-inbox Subject: setting up a pathogen cell sorter.. Hi Howard Shapiro suggested that i contact you. we (several departments at Tufts University) are in the process of setting up a cell sorting facility (flow cytometry) for handling pathogens. i have some experience with flow, but none with pathogen handling. but since i run a core flow facility i've been recruited to help with the design and purchase of instrumentation and lab space. so i was wondering if you have any advice... an open ended question i know. some concerns i have are... >over all safety of the room >containment of the instrument >ability to decontam the machinery for repairs and maintainance >the over all goal of handling a multitude of pathogens (as opposed to a single one that could presumably be easier to monitor identify and prepare against). >i'm sure there should be more, but i haven't thought of them. thanks for any help! have a good one allen -- Tufts Laser Cytometry Allen Parmelee Manager 617-636-0464 allen.parmelee@tufts.edu 617-636-2990 (fax) a core facility offering: MoFlo (Cytomation), FACSCalibur (Becton Dickinson) , LSC (CompuCyte), LCM (Arcturus)
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