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Microbial Testing of Cell Therapy ProductsSummary of NIH Clinical Center StudiesElizabeth Read MDChief,Cell Processing Section Department of Transfusion MedicineNIH Clinical CenterBethesda,MD6/16/06化妆品 Study Design&GoalsParallel StudyCFR vs BacT/AlertCFR vs BactecUsing actual cell therapy products,compare use of automatedculture methods with CFR methodKhuu et at.Transfusion 2006,in press Seeded StudyCFRvs BacT/Alertvs BactecUsing mock MNC products,demonstrate that automated culture methods areequivalent to CFR methodKhuu et al.Cytotherapy 2004;6:183-195Seeded Study:DesignGoal:Evaluate organism detection and time to detectionMock mononuclear cell products from leukapheresis6 commonly used product mediaCitrated autologous plasmaPlasmaLyte A+HSAFreeze mix(DMSO/Pentastarch)RPMI 1640X-VIVO 20(contains gentamycin)RPMI 1640 w/multiple antibioticsEach sample seeded with 10 and 50 CFU of 10 different organisms CFR vs BacT/Alert vs BactecBoth BacT/Alert and Bactec were superior to CFR in overall organism detectionN=6x3x2=36;except AN,n=28CFR/USPBacT/AlertBactecBoth BacT/Alert and Bactec were superior to CFR in time to detectionEven for inocula of 10 CFU,time to detection was 7 days for both BacT/Alert and Bactec(but not for CFR)Multiple antibiotics in product medium impaired detection of organisms in all systemsN=10 x3x2=60In medium with multiple antibiotics,impaired detection was variable from one organism to the nextNo GrowthSA,YE,BS,ANNo GrowthSA,ML,BS,PBParallel Study:DesignGoal:evaluate field performance,false positives(true pos evaluation is best done by seeded study)Tested in process and final product samples from real cell therapy productsTimeframes12/1/02-5/16/04BacT/Alert vs CFR1125 samples5/17/04 12/31/05Bactec vs CFR492 samplesDefinition of positive resultsPositive results expressed asTrue positive=detection by system+confirmation by gram stain and/or subcultureFalse positive=detection by system,but could not confirm presence of organisms by gram stain or subcultureParallel Study:ResultsTrue positiveRates comparable for all systemsTime to detection:Automated systems were equivalent to,or faster than,CFR/USPFalse positiveHigh rates(7.1%)with CFR method vs almost none with automated methods(0.2%)Most related to high cell(RBC or WBC)counts in productParallel Study:Results by Product CategoryProduct CategoryDonor TypesCollection MethodsSampled Product TypesProcessing MethodsContainersMicrobial Culture ResultsApheresis Group AN=514Living,autologous or allogeneicApheresis in hospital apheresis unitPBSCMNCMinimal manipulation:RBC reduction,plasma reduction,cryopreservationBagsTrue pos 0.4%False pos 0.2%Apheresis Group BN=446Living,autologous or allogeneic,some pd research donorsApheresis in hospital apheresis unitPBSCMNCMinimal manipulation:immunomagnetic selection or elutriation in semiclosed systemsBags,vialsTrue pos 1.6%False pos 0.2%Apheresis Group CN=385Living,autologous or allogeneic,some pd research donorsApheresis in hospital apheresis unitPBSCMNCCultured for 3-15 days,some open processing steps,gentamycin used in most culturesProduct pooling in some casesBags,tubes,vials,flasksTrue pos 0.5%False pos 0.0%Parallel Study:Results by Product CategoryProduct CategoryDonor TypesCollection MethodsSampled Product TypesProcessing MethodsContainersMicrobial Culture ResultsBone marrowN=20Living,autologous or allogeneicPercutaneous needle aspiration of posterior iliac crests in operating roomBone marrowMinimal manipulation:automated cell concentration and washing in closed systemsBagsTrue pos 5.0%False pos 0.0%Umbilical cord bloodN=72Living,full term neonatePost partum umbilical venipuncture in room adjacent to delivery roomUmbilical cord bloodMinimal manipulation:RBC reduction and WBC concentration with some open processing stepsBags,tubes,vialsTrue pos 1.4%False pos 0.0%Pancreas HarvestN=52CadavericWhole organ removed from abdomen in operating roomPancreas transport mediumAddition of transport medium(UW solution with penicillin)to,and packaging of,organBagsTrue pos 36.5%False pos 0.0%Parallel Study:Results by Product CategoryProduct CategoryDonor TypesCollection MethodsSampled Product TypesProcessing MethodsContainersMicrobial Culture ResultsProcessed tissueN=107Cadaveric or living,autologousOrgan or tissue harvested in operating roomPancreatic islets,tumor infiltrating lymphsExtensive manipulation may include dissection,enzyme&mechanical digestion,washing,density gradient separation and culture,with many open stepsBags,tubes,flasksTrue pos 2.8%False pos 1.9%OtherN=21Living,autologous or allogeneicVariousPBSC,MNCVarious,may include samples taken after container failuresBags,vialsTrue pos 9.5%False pos 0.0%Organisms Detected in Cell Therapy Products*(not including pancreas and processed tissue)*most common organismsGram PositiveStaphylococcus epidermidisStaphylococcus capitisStaphylococcus,coag negativeProprionobacterium acnesActinomyces sppBacillus sppCorynebacterium sppCoryneform bacteriumEnterococcus faecalisEnterococcus sppPeptostreptococcusRothia sppStaphylococcus aureusStreptococcus mitsStreptococcus,alpha hemolyticStreptococcus,Group BGram NegativePseudomonas fluorescensPseudomonas putidaStenotrophomonas maltophiliaBrevundimonas dimunutaBacteroides sppProteus mirabilisDo all true positives represent actual product contamination?Highly unlikely,because we are frequently unable to demonstrate organism growth in samples from same product or product derived from same parent product and processed in parallelGiven limited volume and number of samples available for a given cell therapy product,this is a problem that is not easily resolved