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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 thesize and structure of the cell. In addition, fluorescence

may result from the absorption and re-emission of light

by chemicals that are either naturally present within the

cell (autofluorescence), or which have been added to the

sample prior to analysis.

FCM has many advantages over conventional cytometry.

Firstly, since acquisition rates of up to 10,000 cells.sec-1

can be achieved (depending on the instrument used),

flow cytometric data sets often represent measurements

of in excess of 100,000 cells. In contrast, measurements

by microscopy often involve only a few hundred cells.

The increased sample throughput of FCM leads to the

acquisition of statistically significant results and the

detection of rare cell types. Secondly, since FCM uses

very sensitive electronic detectors to measure the intensi-ty of scattered light or fluorescence at a given wavelength,

different intensities of light scatter/fluorescence can be

distinguished.By calibrating an instrument with samples

of known size or fluorescent intensity, it is possible to

obtain quantitative measurements. Thirdly, by using

dichroic filters to optically separate light of different

wavelength, flow cytometric measurements can be made

on several different characteristics of each cell. Typical

commercial 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 distinguish

between different cell types. Finally, since measurements

are 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 (cell

sorters) 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 be

sorted onto microscope slides or microtitre/agar plates.

Providing that appropriate cell staining and sample

preparation methods have been used to maintain viabil-

ity, sorted cells can be grown to give clonal colonies or

broth suspensions for con-

firmation of identity via

standard clinical microbi-

ology methods.

Over recent years a num-ber of reviews of FCM

have been published [see

examples in reference 1].

The purpose of this review

is to highlight the value of

FCM for clinical samples,

with particular reference

to microorganisms.

Clinical applications of

microbial detection

by FCM

The detection of bacteria or

yeasts in body fluids is important for the diagnosis of a

number of different diseases. Urine may contain a variety

of particulates,including red and white blood cells,epithe-lial cells, bacteria and inorganic chemical crystals. The

presence and concentrations of these particulates can be

used for the diagnosis of a range of diseases and disorders.

Flow cytometers designed specifically for urinalysis are

available commercially and these allow the simultaneous

determination of many different cell types [2]. These

devices have been shown to be more sensitive than manu-

al microscopic methods [3].

In comparison to the relatively straightforward detection

of bacteria in urine samples, blood is a much more chal-

lenging sample type to use. In clinical infections such as

bacteraemia, concentrations of the contaminants may be

of the order of 10 bacteria in 1 mL of blood, whilst the

number of red blood cells is >109 per mL.The high 'back-

ground' cellular load of blood makes the detection of bac-teria by microscopic methods all but impossible.

Consequently, although bacteraemia is a potentially life-

threatening condition, diagnosis relies in many cases

upon the growth of bacteria in media inoculated with

samples of whole blood. However, methods are available

to selectively lyse the erythrocytes in a blood sample,leav-

ing a sufficiently low cell concentration to allow the rapid

sample throughput capabilities of the flow cytometer to

be utilised for the detection of bacteria. A number of

products are now available commercially to achieve this,

for example, CyLyse from Partec GmbH, M

Germany.

Mansour and colleagues [4] developed a model syst

which they used ethidium bromide labelling to

specifically detect Escherichia coliin blood at conc

tions of 10 - 100 cells.ml-1. The sensitivity was

1000-fold better than that achieved using micro

techniques, and took just 2 hours to perform, inc

sample preparation. In clinical presentations wher

terial concentrations are less than 10 per mL, a sho

incubation step prior to flow cytometric analysis m

envisaged to increase the bacterial load of the samp

level where it may be detected.

The detection of specific pathogenic microorgani

clinical samples has been much improved by the

ability of monoclonal antibodies. These antibodi

be fluorescently labelled (either directly or indirec

enable them to be detected flow cytometrically. A vof fluoresecent labels are available, the most comm

fluorescein isothiocyanate (FITC). This has the a

tage of being well-excited by the 488 nm Argon io

which is used as standard in most flow cytometers.

(spectrally-distinct) molecules such as allophycoc

Texas Red and phycoerythrin allow multiple targets

detected simultaneously. The labelled-antibody app

has proven to be useful for the detection of mycob

al species from clinical (sputum) specimens [5]. Y

colleagues showed that Mycobacteria could be de

F low Cytometry

Flow cytometry for clinicalmicrobiology

as published in CLI February/March 200

Flow cytometry (FCM) is a rapid technique for the analysis of individual cells. Light scattering and fluorescence properti

cells are analysed as the cells pass through a laser beam and, in specialised instruments, cells with specific characteristics

be isolated. This review article describes FCM and discusses recent advances that may be expected to increase its use in

ical microbiology. New applications include susceptibility testing, where FCM allows death or damage to microorganism

be identified without the necessity to observe microbial growth, as well as monitoring the status and extent of infectio

HIV-positive patients.

by Dr. Hazel Davey

labt

echnology

DichroicFilters

Flowcell

Waste

BandpaFilters

Lasers & Lamps

Figure 1. Schematic drawing of a generalised flow cytometer. Modified with permissio

a drawing by Robert Murphy, Carnegie Melon University, Pittsburgh, PA,USA. (The P

Cytometry CD-ROM Volume 4, J. Watson, Guest Ed., J. Paul Robinson, Publisher. P

University Cytometry Laboratories,West Lafayette, IN, USA. 1997, ISBN 1-890473-03


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in as little as 3 hours; since Mycobacteria grow very slow-

ly in laboratory culture, a detection method that does not

rely on growth is very advantageous for clinical diagnos-

tic purposes. The method described used a rabbit poly-

clonal antibody against Mycobacterium species together

with a goat anti-rabbit IgG secondary antibody labelled

with R-phycoerythrin, and detected several different

Mycobacterium species. However, use of a species-specif-

ic antibody as the primary antibody would allow the

method to be used to detect M. tuberculosisspecifically.

Susceptibility testing

In an era of worrying and increasing levels of antibiotic-

resistant pathogens, it is not surprising that understand-

ing the interactions between microorganisms and the

drugs designed to kill them has become another impor-

tant area for the clinical application of flow cytometric

methods. A variety of fluorescent

stains for assessing the viability of

microorganisms have been identi-

fied [Table 1, see also reference 6]

and these are particularly useful

for determining the efficacy of

antimicrobial compounds.

Microorganisms exposed to

antibiotic or antifungal com-

pounds (either in vivoor in vitro)are compared to control (untreat-

ed) samples and appropriate

stains are used to identify changes

in 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 be

detected by the entry of fluorescent compounds (such as

propidium iodide) which are normally excluded by the

intact cell membrane. Alternatively, to deter-

mine the response of cells to an antibiotic,

which affects nucleic acid synthesis, one could

use a stain such as DAPI, which binds to DNA,

or pyronin Y, which binds to RNA.

In addition, FCM permits subpopulations with

varying resistance to be identified and accurate

assessment of the dose-response curve can also

be performed as part of the assay [see examples

in reference 7]. Flow cytometric susceptibility

testing thus allows death or damage of microor-

ganisms to be identified without the necessity to

observe microbial growth (or lack thereof).

Flow cytometric susceptibility testing can be

performed in a few hours [Figure 2] and conse-

quently this method has the potential to con-

tribute to the decision of which drug or drug

combination would be most appropriate for a

particular patient.

HIV

FCM has been used to great effect for monitor-

ing the status and extent of HIV infection.Whilst

viral antigens can be detected by FCM [8], monitoring of

HIV infection usually relies on regular quantitation of

lymphocyte populations. The absolute numbers of CD4+

lymphocytes and their percentage values within the total

lymphocyte populations are good indicators of the dis-

ease and its progression. Fluorescently-labelled antibod-

ies can be used to selectively label different types of lym-

phocytes and thus FCM has an important role to play not

only in disease surveillance, but also in determining the

efficacy of treatment. Ideally analysis of blood samples

should be performed within hours of collection.

Unfortunately, the majority of HIV-infected individuals

are not within easy reach of the specialised laboratories

capable of performing these tests. A mobile flow cytom-

etry laboratory has recently been developed to address

this issue (Partec GmbH, Mnster, Germany). The

CyFlow flow cytometer is installed in an off-road 4-wheel

drive car and is powered using 12 V DC car batteries

charged by solar panels [Figure 3]. The system has advan-

tages over many flow cytometers in that lymphocyte pop-

ulations can be simultaneously identified and quantified

without the addition of reference controls [9]. Det

of the different lymphocyte populations is achieved

monoclonal antibodies targeted against the appro

CD markers. The cells in a fixed volume (200 m

sample are counted; counting is switched on a

using an electrode to sense the depth of fluid in th

ple tube. The combined detection and counting no

simplifies the procedure, thus reducing the potent

error, but also minimises costs.

Future prospects

A recent development that may be expected to pro

the use of FCM for the analysis of clinical samples

Amnis ImageStream System (www.amnis.com),

permits images of individual cells to be captured

with their multiparametric flow cytometric data

dots on a flow cytometric data plot can be directly

to an image of the cell. This has particular use

"abnormal" signals are detected by FCM - the op

can relate these signals back to up to six separate i

of the cell to check for the presence of cell doublet

taminating cell types or to verify the result of scr

tests.

Over the last few years, kits designed specifically f

flow cytometric analysis of microorganisms have b

available (see e.g. www.bdbiosciences.ca /download

lines/Cell_Viability_HL_Fall2003.pdf

www.probes.com/ handbook/sections/1503.html)

growing popularity of such kits reflects, at least in

their ease of use. Whilst this is to be welcomed, t

some danger that the kits may be adopted without an

of proper control standards. Despite the names of

kits, distinguishing live and dead bacteria and yeasts

always straightforward and care in interpretation

results is still of great importance.

In conclusion, FCM offers many advantages for c

microbiology. Recent developments are likely to op

further possibilities of new applications,as well as in

ing the use of existing flow cytometric techniques.

as published in CLI February/March 20F low Cytometry

Stain

BacLight Kit: MolecularProbes www.probes.com

bis-(1,3-dibutylbarbituricacid) trimethine oxonol(DiBAC4(3))

Calcofluor White

5-cyano-2,3-ditolyltetra-zolium chloride (CTC)

Fluorescein diacetate/Carboxy-fluoresceindiacetate

Rhodamine 123

TO-PRO-3 / Propidiumiodide

Mode of Action

Propidium iodide excludedby intact membranes. Allcells take up SYTO9

Uptake by dead cells

Uptake by dead cells

Respiratory activity

Enzymic activity

Uptake by live cells

Excluded 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 of viability by FCM.

Untreated 40 min.

1 hour 3 hours

Red Fluorescence

Green

Fluorescence

Green

Fluorescence

Green

Fluorescence

Green

Fluorescence

Red Fluorescence

Red Fluorescence Red 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 of dead 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.


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References

1. Davey HM, Kell DB. Flow cytometry and cell sorting of

heterogeneous microbial populations-the importance of

single-cell analyses. Microbiological reviews

1996;60(4):641-696.

2. Delanghe JR, Kouri TT, Huber AR, Hannemann-Pohl

K, Guder WG,Lun A,Sinha P, Stamminger G,Beier L. The

role of automated urine particle flow cytometry in clini-

cal practice. Clinica Chimica Acta 2000;301(1-2):1-18.3. Hannemann-Pohl K, Kampf SC. Automation of urine

sediment examination: A comparison of the sysmex UF-

100 automated flow cytometer with routine manual diag-

nosis (microscopy, test strips, and bacterial culture).

Clinical Chemistry and Laboratory Medicine

1999;37(7):753-764.

4. Mansour JD, Robson JA, Arndt CW, Schulte TE.

Detection of Escherichia coli in blood using flow cytome-

try. Cytometry 1985;6:186-190.

5. Yi WC, Hsiao S, Liu JH,et al. Use of fluorescein labelled

antibody and fluorescence activated cell sorter for rapid

identification of Mycobacterium species. Biochem

Biophys Res Commun 1998;250(2):403-8.

6. Davey HM, Kaprelyants AS, Weichart DH, Kell DB.

Estimation of microbial viability using flow cytometry.

Current Protocols in Cytometry.New York: Wiley;1999. p11.3.1-11.3.20.

7. Pore RS. Ketoconazole susceptibility of yeasts by the

FCST method. Current Microbiol.1991;23:45-50.

8. McSharry JJ. Uses of flow cytometry in virology.

Clinical microbiology reviews 1994;7(4):576.

9. Greve B, Cassens U, Westerberg C, Jun WG, Sibrowski

W, Reichelt D, Gohde W. A new no-lyse, no-wash flow-

cytometric method for the determination of CD4 T cells

in blood samples. Transfusion Medicine and

Hemotherapy 2003;30(1):8-13.

The author

Hazel M. Davey, Ph.D.,

Postdoctoral Research Assistant,

Institute of Biological Sciences, University of Wales,

Aberystwyth, Ceredigion, SY23 3DD,Wales, U.K.

Tel.: +44 1970 621829

Fax: +44 1970 622307

Email: [email protected]

Website: http://qbab.aber.ac.uk/home.html

as published in CLI February/March 20F low Cytometry

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