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flow-cytometry-for-clinical-microbiology.pdf

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Flow cytometry (FCM) is a technique for the rapid, opti- cal analysis of individual cells. Measurements are made by an array of detectors as the cells flow in a fluid stream through a laser (or arc lamp) beam [Figure 1]. At the sample interrogation point, light is scattered by the cells; the extent of light scatter provides information on the size and structure of the cell. In addition, fluorescence may result from the absorption and re-emission of light by chemicals t
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  Flow cytometry (FCM) is a technique for the rapid,opti-cal analysis ofindividual cells.Measurements are madeby an array ofdetectors as the cells flow in a fluid streamthrough a laser (or arc lamp) beam [Figure 1].At thesample interrogation point,light is scattered by the cells;the extent oflight scatter provides information on thesize and structure ofthe cell.In addition,fluorescencemay result from the absorption and re-emission oflightby chemicals that are either naturally present within thecell (autofluorescence),or which have been added to thesample prior to analysis.FCM has many advantages over conventional cytometry.Firstly,since acquisition rates ofup to 10,000 cells.sec-1can be achieved (depending on the instrument used),flow cytometric data sets often represent measurementsofin excess of100,000 cells.In contrast,measurementsby microscopy often involve only a few hundred cells.The increased sample throughput ofFCM leads to theacquisition ofstatistically significant results and thedetection ofrare cell types.Secondly,since FCM usesvery sensitive electronic detectors to measure the intensi-ty ofscattered light or fluorescence at a given wavelength,different intensities oflight scatter/fluorescence can bedistinguished.By calibrating an instrument with samplesofknown size or fluorescent intensity,it is possible toobtain quantitative measurements.Thirdly,by usingdichroic filters to optically separate light ofdifferentwavelength,flow cytometric measurements can be madeon several different characteristics ofeach cell.Typicalcommercial flow cytometers allow 5-10 different param-eters (e.g.size,protein content,DNA content,lipid con-tent,antigenic properties,enzyme activity,etc.) to be col-lected for each cell,allowing the operator to distinguishbetween different cell types.Finally,since measurementsare made on single cells,heterogeneity within the popu-lation can be detected and quantified in a way that can-not be achieved by other means.Whilst all commercial flow cytometers have the advan-tages described above,some specialised instruments (cellsorters) are able to physically separate cells on the basis of user-defined characteristics.Depending on the instru-ment,cells may be bulk-sorted or individual cells may besorted onto microscope slides or microtitre/agar plates.Providing that appropriate cell staining and samplepreparation methods have been used to maintain viabil-ity,sorted cells can be grown to give clonal colonies orbroth suspensions for con-firmation ofidentity viastandard clinical microbi-ology methods.Over recent years a num-ber ofreviews ofFCMhave been published [seeexamples in reference 1].The purpose ofthis review is to highlight the value of FCM for clinical samples,with particular referenceto microorganisms. Clinical applications of microbial detection by FCM The detection ofbacteria or yeasts in body fluids is important for the diagnosis ofanumber ofdifferent diseases.Urine may contain a variety ofparticulates,including red and white blood cells,epithe-lial cells,bacteria and inorganic chemical crystals.Thepresence and concentrations ofthese particulates can beused for the diagnosis ofa range ofdiseases and disorders.Flow cytometers designed specifically for urinalysis areavailable commercially and these allow the simultaneousdetermination ofmany different cell types [2].Thesedevices have been shown to be more sensitive than manu-al microscopic methods [3].In comparison to the relatively straightforward detectionofbacteria in urine samples,blood is a much more chal-lenging sample type to use.In clinical infections such asbacteraemia,concentrations ofthe contaminants may beofthe order of10 bacteria in 1 mL ofblood,whilst thenumber ofred blood cells is >109per mL.The high 'back-ground' cellular load ofblood makes the detection ofbac-teria by microscopic methods all but impossible.Consequently,although bacteraemia is a potentially life-threatening condition,diagnosis relies in many casesupon the growth ofbacteria in media inoculated withsamples ofwhole blood.However,methods are availableto selectively lyse the erythrocytes in a blood sample,leav-ing a sufficiently low cell concentration to allow the rapidsample throughput capabilities ofthe flow cytometer tobe utilised for the detection ofbacteria.A number of products are now available commercially to achieve this,for example,CyLyse from Partec GmbH,Münster,Germany.Mansour and colleagues [4] developed a model system inwhich they used ethidium bromide labelling to non-specifically detect Escherichia coli  in blood at concentra-tions of10 - 100 cells.ml-1.The sensitivity was 100 to1000-fold better than that achieved using microscopy techniques,and took just 2 hours to perform,includingsample preparation.In clinical presentations where bac-terial concentrations are less than 10 per mL,a short pre-incubation step prior to flow cytometric analysis may beenvisaged to increase the bacterial load ofthe sample to alevel where it may be detected.The detection ofspecific pathogenic microorganisms inclinical samples has been much improved by the avail-ability ofmonoclonal antibodies.These antibodies canbe fluorescently labelled (either directly or indirectly) toenable them to be detected flow cytometrically.A variety offluoresecent labels are available,the most common isfluorescein isothiocyanate (FITC).This has the advan-tage ofbeing well-excited by the 488 nm Argon ion laserwhich is used as standard in most flow cytometers.Other(spectrally-distinct) molecules such as allophycocyanin,Texas Red and phycoerythrin allow multiple targets to bedetected simultaneously.The labelled-antibody approachhas proven to be useful for the detection ofmycobacteri-al species from clinical (sputum) specimens [5].Yi andcolleagues showed that Mycobacteria could be detected Flow Cytometry Flow cytometry for clinicalmicrobiology as published in CLI February/March 2004 Flow cytometry (FCM) is a rapid technique for the analysis of individual cells. Light scattering and fluorescence properties ofcells are analysed as the cells pass through a laser beam and, in specialised instruments, cells with specific characteristics canbe isolated. This review article describes FCM and discusses recent advances that may be expected to increase its use in clin-ical microbiology. New applications include susceptibility testing, where FCM allows death or damage to microorganisms tobe identified without the necessity to observe microbial growth, as well as monitoring the status and extent of infection inHIV-positive patients. by Dr. Hazel Davey    l  a   b    t  e  c   h  n  o   l  o  g  y DichroicFiltersFlowcellWasteBandpassFilters Lasers & Lamps Figure 1.Schematic drawing ofa generalised flow cytometer.Modified with permission froma drawing by Robert Murphy,Carnegie Melon University,Pittsburgh,PA,USA.(The Purdue Cytometry CD-ROM Volume 4,J.Watson,Guest Ed.,J.Paul Robinson,Publisher.Purdue University Cytometry Laboratories,West Lafayette,IN,USA.1997,ISBN 1-890473-03-0).  in as little as 3 hours;since Mycobacteria grow very slow-ly in laboratory culture,a detection method that does notrely on growth is very advantageous for clinical diagnos-tic purposes.The method described used a rabbit poly-clonal antibody against Mycobacterium species togetherwith a goat anti-rabbit IgG secondary antibody labelledwith R-phycoerythrin,and detected several differentMycobacterium species.However,use ofa species-specif-ic antibody as the primary antibody would allow themethod to be used to detect M.tuberculosis  specifically. Susceptibility testing  In an era ofworrying and increasing levels ofantibiotic-resistant pathogens,it is not surprising that understand-ing the interactions between microorganisms and thedrugs designed to kill them has become another impor-tant area for the clinical application offlow cytometricmethods.A variety offluorescentstains for assessing the viability of microorganisms have been identi-fied [Table 1,see also reference 6]and these are particularly usefulfor determining the efficacy of antimicrobial compounds.Microorganisms exposed toantibiotic or antifungal com-pounds (either in vivo  or in vitro  )are compared to control (untreat-ed) samples and appropriatestains are used to identify changesin nucleic acids,proteins,mem-branes,etc.Antibiotics disrupt cellular activi-ties and the particular mode of action can be determined flow cytometrically.For example,antibiotic-induced damage to cell membranes can bedetected by the entry offluorescent compounds (such aspropidium iodide) which are normally excluded by theintact cell membrane.Alternatively,to deter-mine the response ofcells to an antibiotic,which affects nucleic acid synthesis,one coulduse a stain such as DAPI,which binds to DNA,or pyronin Y,which binds to RNA.In addition,FCM permits subpopulations withvarying resistance to be identified and accurateassessment ofthe dose-response curve can alsobe performed as part ofthe assay [see examplesin reference 7].Flow cytometric susceptibility testing thus allows death or damage ofmicroor-ganisms to be identified without the necessity toobserve microbial growth (or lack thereof).Flow cytometric susceptibility testing can beperformed in a few hours [Figure 2] and conse-quently this method has the potential to con-tribute to the decision ofwhich drug or drugcombination would be most appropriate for aparticular patient. HIV FCM has been used to great effect for monitor-ing the status and extent ofHIV infection.Whilstviral antigens can be detected by FCM [8],monitoring of HIV infection usually relies on regular quantitation of lymphocyte populations.The absolute numbers ofCD4+lymphocytes and their percentage values within the totallymphocyte populations are good indicators ofthe dis-ease and its progression.Fluorescently-labelled antibod-ies can be used to selectively label different types oflym-phocytes and thus FCM has an important role to play notonly in disease surveillance,but also in determining theefficacy oftreatment.Ideally analysis ofblood samplesshould be performed within hours ofcollection.Unfortunately,the majority ofHIV-infected individualsare not within easy reach ofthe specialised laboratoriescapable ofperforming these tests.A mobile flow cytom-etry laboratory has recently been developed to addressthis issue (Partec GmbH,Münster,Germany).TheCyFlow flow cytometer is installed in an off-road 4-wheeldrive car and is powered using 12 V DC car batteriescharged by solar panels [Figure 3].The system has advan-tages over many flow cytometers in that lymphocyte pop-ulations can be simultaneously identified and quantifiedwithout the addition ofreference controls [9].Detectionofthe different lymphocyte populations is achieved usingmonoclonal antibodies targeted against the appropriateCD markers.The cells in a fixed volume (200 mL) of sample are counted;counting is switched on and off using an electrode to sense the depth offluid in the sam-ple tube.The combined detection and counting not only simplifies the procedure,thus reducing the potential forerror,but also minimises costs. Future prospects A recent development that may be expected to promotethe use ofFCM for the analysis ofclinical samples is theAmnis ImageStream System (www.amnis.com),whichpermits images ofindividual cells to be captured alongwith their multiparametric flow cytometric data.Thusdots on a flow cytometric data plot can be directly linkedto an image ofthe cell.This has particular use when abnormal signals are detected by FCM - the operatorcan relate these signals back to up to six separate imagesofthe cell to check for the presence ofcell doublets,con-taminating cell types or to verify the result ofscreeningtests.Over the last few years,kits designed specifically for theflow cytometric analysis ofmicroorganisms have becomeavailable (see e.g.www.bdbiosciences.ca /downloads/hot-lines/Cell_Viability_HL_Fall2003.pdfandwww.probes.com/ handbook/sections/1503.html).Thegrowing popularity ofsuch kits reflects,at least in part,their ease ofuse.Whilst this is to be welcomed,there issome danger that the kits may be adopted without analysisofproper control standards.Despite the names ofthesekits,distinguishing live and dead bacteria and yeasts is notalways straightforward and care in interpretation oftheresults is still ofgreat importance.In conclusion,FCM offers many advantages for clinicalmicrobiology.Recent developments are likely to open upfurther possibilities ofnew applications,as well as increas-ing the use ofexisting flow cytometric techniques. as published in CLI February/March 2004 Flow Cytometry Stain BacLight Kit: Molecular Probes – www.probes.combis-(1,3-dibutylbarbituricacid) trimethine oxonol(DiBAC4(3))Calcofluor White5-cyano-2,3-ditolyltetra-zolium chloride (CTC)Fluorescein diacetate/ Carboxy-fluorescein diacetateRhodamine 123TO-PRO-3 / Propidiumiodide Mode of Action Propidium iodide excludedby intact membranes. Allcells take up SYTO9Uptake by dead cellsUptake by dead cellsRespiratory activityEnzymic activityUptake by live cellsExcluded by intact cellmembrane Results Live cells are green, deadcells are red.Dead cells appeargreen/yellow.Dead cells appear blue.Live cells appear red.Live cells appear green.Live cells appear green.Dead cells appear red. Table 1.Some fluorescent dyes used for determination ofviability by FCM. Untreated40 min.1 hour3 hoursRed Fluorescence    G  r  e  e  n   F   l  u  o  r  e  s  c  e  n  c  e   G  r  e  e  n   F   l  u  o  r  e  s  c  e  n  c  e   G  r  e  e  n   F   l  u  o  r  e  s  c  e  n  c  e   G  r  e  e  n   F   l  u  o  r  e  s  c  e  n  c  e Red FluorescenceRed FluorescenceRed Fluorescence Figure 2.Antimicrobial susceptibility testing using flow cytometry.Two colour fluorescence histograms of Enterococcus faecium treated with vancomycin and stained with the FAST-2 kit (BioRad).With increasing exposure time,an increase in the number ofdead and dying cells (events present in quadrants 2,3,and 4) was observed.Data collected by Kuo-Ping Chiu and colleagues at BioRad,printed with permission (The Purdue Cytometry CD-ROM Volume 4,J.Watson,Guest Ed.,J.Paul Robinson,Publisher.Purdue University Cytometry Laboratories,West Lafayette,IN,USA.1997,ISBN 1-890473-03-0).Figure 3.The CyFlow flow cytometer,image kindly provided by Partec,GmbH.  References 1.Davey HM,Kell DB.Flow cytometry and cell sorting of heterogeneous microbial populations-the importance of single-cell analyses.Microbiological reviews1996;60(4):641-696.2.Delanghe JR,Kouri TT,Huber AR,Hannemann-PohlK,Guder WG,Lun A,Sinha P,Stamminger G,Beier L.Therole ofautomated urine particle flow cytometry in clini-cal practice.Clinica Chimica Acta 2000;301(1-2):1-18.3.Hannemann-Pohl K,KampfSC.Automation ofurinesediment examination:A comparison ofthe sysmex UF-100 automated flow cytometer with routine manual diag-nosis (microscopy,test strips,and bacterial culture).Clinical Chemistry and Laboratory Medicine1999;37(7):753-764.4.Mansour JD,Robson JA,Arndt CW,Schulte TE.Detection ofEscherichia coli in blood using flow cytome-try.Cytometry 1985;6:186-190.5.Yi WC,Hsiao S,Liu JH,et al.Use offluorescein labelledantibody and fluorescence activated cell sorter for rapididentification ofMycobacterium species.BiochemBiophys Res Commun 1998;250(2):403-8.6.Davey HM,Kaprelyants AS,Weichart DH,Kell DB.Estimation ofmicrobial viability using flow cytometry.Current Protocols in Cytometry.New York:Wiley;1999.p11.3.1-11.3.20.7.Pore RS.Ketoconazole susceptibility ofyeasts by theFCST method.Current Microbiol.1991;23:45-50.8.McSharry JJ.Uses offlow cytometry in virology.Clinical microbiology reviews 1994;7(4):576.9.Greve B,Cassens U,Westerberg C,Jun WG,SibrowskiW,Reichelt D,Gohde W.A new no-lyse,no-wash flow-cytometric method for the determination ofCD4 T cellsin blood samples.Transfusion Medicine andHemotherapy 2003;30(1):8-13. The author Hazel M.Davey,Ph.D.,Postdoctoral Research Assistant,Institute ofBiological Sciences,University ofWales,Aberystwyth,Ceredigion,SY23 3DD,Wales,U.K.Tel.: +44 1970 621829 Fax: +44 1970 622307 Email: hlr@aber.ac.uk Website: http://qbab.aber.ac.uk/home.html  as published in CLI February/March 2004 Flow Cytometry
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