Hi fellow flowers, First, thanks to all who responded to my query about setting up a FACSCalibur/flow cytometer for GLP/GMP, it was indeed enlightening. Second, I have been asked to summarize the responses so others can possibly do the same thing, so here they are. The first consensus is that there is no consensus/standard procedure for validating the instrument like Analytical Chemists do for their machines. So, here are some excerpts from the responses as it seems like individual users need to tailor their set ups and validation protocols. 1. All clinical flow cytometers have a "certificate of validation" that validate the hardware and software. I know this because I was required to produce it for the GLP auditors. Our instrument set up only involves running a bead to confirm alignment (DNA Check), and a second bead to shown that each day they fall within the same fluorescence channel (Standard Brite). (If you're doing relative fluorescence intensity, then probably showing amplifier linearity and multiple level fluorescence checks might be needed. Like a Rainbow Beads. I have never used these.). The rest of our protocol QCs each reagent (how can you detect a defect in lysis, how do you know each antibody stains as it should, etc.) We use Cyto-Trols and CD Chex (preserved PMBC with assay values) to QC each antibody in our panel. Then there is the whole issue of calibrating pipets, scales, etc. Also important was recording lot numbers and expriation date on every reagent we used. We use a sheet for each day's run that records all of this information and becomes part of our results. 2. I have been doing GLP studies in flow cytometry for 5 years. Depending on what your GLP study is, will depend on how in depth your QC needs to be!!! Example: Immunophenotyping - Calibrite beads including APC monthly QC for alignment Daily check off list (usually provided by vendor) Antibody titers and parallels for new lots Basically you need to show that your detectors and lasers are working properly and consistently from day to day. 3. We are running a FACSCalibur for our quality control.(GMP) Prerequisites are quite simple: 1. a service contract for the instrument 2. regular performance of internal quality control by FACSComp 3. validation of methods (CD34 quantification) and essential reagents (antibodies) 4. standard operating procedures (SOP) 4. We routinely use a FACSCalibur for GLP studies. We used to run both GLP and non-GLP studies on the same machine. In that instance each assay was audited by QA. You will have to maintain records of when the machine was used, user name, whether the user was trained and certified for FACScalibur use, daily QCing (FACSCOMPing basically), printouts from the FACSComp, SOP for the assay you wish to run under GLP, reagents date of mfr, expiry, records of reagent use, so on and so forth. 5. f you are just starting out, visit http://www.cap.org/html/ftpdirectory/checklistftp.html and download the Flow Cytometry Checklist and others. 6. I have used our Coulter XL to perform GLP studies. I think what the chemist is referring to is that your QC procedure must include analyzing known standards that span the expected range (multiple levels) of your specimens. For a chemist, this would mean, for example, analyzing control reangents known to contain 10, 100 and 1000 IU of alkaline phosphatase, to show that your assay matches these values. In flow cytometry, for example, you can assay CD-Chex which have assay values (in the normal range) for the major lymphocyte subsets and CD-Chex LOW which have assay values for where CD4 counts are below normal. I've gone to battle with GLP inspectors because there are few QC reagents for flow and less with "mulitple levels". However, for most of our studies, the GLP inspectors were content that we run an assay with each monoclonal antibody tested on one level of control cells (Cyto-trol or CD-Chex) and show that our results fall within the expected range. Again, as it is obvious to even those of us just starting down this path, there are no real guidelines/requirements which we can easily follow. The CAP checklist does give a little help in providing the questions to be answered, but no answers to the question of what standards/controls to use, only to use them. If any more information becomes available, I will of course post it to the list. Randy Fischer TherImmune Research Corporation 9700 Great Seneca Hwy Rockville, MD 20850 (240) 453-6256 RFischer@therimmune.com
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