Standardization, Calibration, and Control in Flow Cytometry

UNIT 1.3Standardization, Calibration, and Controlin Flow CytometryLili Wang1 and Robert A. Hoffman2

1Biosystems and Biomaterials Division, NIST, Gaithersburg, Maryland2Independent Consultant, Livermore, California

Because flow cytometers are designed to measure particle characteristics, par-ticles are the most common materials used to calibrate, control, and standardizethe instruments. Definitions and cautions are provided for common terms toalert the reader to critical distinctions in meaning. This unit presents extensivebackground on particle types and cautions and describes practical aspects ofmethods to standardize and calibrate instruments. Procedures are provided tocharacterize performance in terms of optical alignment, fluorescence and lightscatter resolution, and sensitivity. Finally, suggestions follow for analyzingparticles used for calibration. C© 2017 by John Wiley & Sons, Inc.

Keywords: standardization � calibration � control � particles � MESF(molecules of equivalent soluble fluorochrome) � ERF (equivalent numberof reference fluorophores) � sensitivity � resolution

How to cite this article:Wang, L. and Hoffman, R.A. 2017. Standardization, calibration, and

control in flow cytometry. Curr. Protoc. Cytom. 79:1.3.1-1.3.27.doi: 10.1002/cpcy.14

INTRODUCTIONStandardization, control, and calibration

provide different degrees of certainty aboutthe data acquired with an instrument. Eachprocess is aimed at ensuring that results fromthe instrument have the quality required forthe intended purpose (Horan et al., 1990; NC-CLS, 1998a; Muirhead, 1993a,b; Schwartzand Fernandez-Repollet, 1993; Owens and Lo-ken, 1995; Schwartz et al., 1996; Owens et al.,2000). The purpose may be an individual re-search experiment or a clinical result that de-termines the longitudinal course of patient’sresponse to treatment. In the terminology usedin this commentary, an instrument is standard-ized at certain time points and subsequentlyoperated under quality control conditions [seeUNITS 3.1 & 3.2 (Hurley, 1997a,b)]. These pro-cesses maintain the instrument within prede-termined bounds and ensure that results willvary only within certain limits. If results arealso calibrated when instruments are standard-ized, then future results can be objectively andquantitatively compared to future calibratedresults or to calibrated results in other labora-tories. Quantitation of results should be con-

sidered. Most results from flow cytometers areexpressed either in terms of “percent posi-tive” or in qualitative terms such as “dim” or“bright.” These terms are relative: what is con-sidered “negative,” “dim,” and “bright” in onelaboratory may be quite different in anotherlaboratory. When visualizing fluorescence us-ing a fluorescence microscope, such relativeterms are mostly sufficient, though perfor-mance benchmarking with control materialson microscopes has recently been tested (Hal-ter et al., 2014). Flow cytometers can measurethe amount of fluorescence and provide moreobjective criteria for expressing results.

As particles are the most common materialsused to calibrate, control, and standardize theinstruments, this commentary describes howvarious types of particles are used for thesepurposes. It also briefly reviews the status ofstandardization and quality control for flowcytometry (see Chapter 3 for further discus-sion of quality control). UNIT 1.4 (Wood, 2009)covers calibration of detection system com-ponents (e.g., linear and logarithmic ampli-fiers) to ensure linearity of the flow cytometerresponse.

Current Protocols in Cytometry 1.3.1-1.3.27, January 2017Published online January 2017 in Wiley Online Library (wileyonlinelibrary.com).doi: 10.1002/cpcy.14Copyright C© 2017 John Wiley & Sons, Inc.

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The first section of this unit focuses on howthe term “standard” has been used in flowcytometry (see Standards, Standardization,and Jargon). The intent is to alert readers offlow cytometry literature that they must al-ways interpret critically how “standard” is be-ing used in a particular context. The next sec-tion defines terms and also includes commentsto put the term in context or to highlight issues(see Definitions).

After providing extensive background onparticle types and cautions (see Overview ofStandardization in Flow Cytometry), this unitdescribes practical aspects of methods to stan-dardize and calibrate flow cytometers (e.g., interms of optical alignment, fluorescence andlight scatter resolution, and sensitivity; seeStandardization and Calibration section). Fi-nally, suggestions are given for analyzing par-ticles used as calibrators, including how to as-sign to fluorescent beads a value for moleculesof equivalent soluble fluorochrome (MESF)and equivalent reference fluorophores (ERF)and how to determine the inherent fluores-cence coefficient of variation (CV) of a dimbead sample (see Characterizing Particles forCalibration and Control of a Flow Cytometer).

STANDARDS, STANDARDIZATION,AND JARGON

It is common in flow cytometry to combinewords that describe use of a particle with theword “standard.” Examples are “calibrationstandard” and “alignment standard” (Horanet al., 1990; Schwartz and Fernandez-Repollet,1993; Schwartz et al., 1996; Shapiro, 2003).Rarely is there any indication of who has setthe “standard” and by what authority or con-sensus.

There can be many levels of “standards,”depending on the size and authority of thegroup that establishes them. For example, anindividual laboratory or investigator may havestandard practices or materials. A large clinicalor research study may have standard practicesand materials that are agreed to by all inves-tigators involved in the study. A professionalorganization may establish standard methodsor identify standard materials for specific pur-poses. If the word “standard” is not modifiedby a term such as “laboratory,” “clinical trial,”or “study XYZ,” it may imply something thatis generally and widely accepted by acknowl-edged authorities. In that authoritative sense,however, there are few “standards” in flowcytometry.

Clear and common understanding of whatis meant by a term is important, especially as

flow cytometry is used by increasing numbersof investigators. The verb “standardize” meansto cause to be without variation. Early use ofthe noun “standard” in flow cytometry seemsto have been in the sense of a particle usedto standardize (make consistent) one instru-ment in one laboratory (Fulwyler, 1979). Thisis much different from the authoritative senseof “standard.” In this commentary, other termsare used to describe more specifically whattype of particle or material is being used fora particular purpose. For example, “calibra-tion particle” or “calibrator” is used instead of“calibration standard,” and “alignment parti-cle” rather than “alignment standard.”

DEFINITIONSConcern with terminology and its evolution

is not just semantics, but reflects what has beenimportant in flow cytometer technology andhow the technology has grown and changed.More precise and generally accepted terminol-ogy should clarify communication and under-standing among flow cytometrists as well asscientists in other fields. Definitions related toquantitative fluorescence cytometry are pro-vided by Henderson et al. (1998).

The definitions below should be considereda reasonable point along the way toward au-thoritative and broadly accepted and under-stood terminology. Some definitions includecomments and references that may help putthem in context.

Accuracy: degree to which a measurementagrees with the true or expected value.

Alignment particle: particle with uniformsize, fluorescence, and light scatter character-istics that is used to check the alignment (or, insome instruments, adjust the alignment) of theexcitation and emission optics in the flow cy-tometer. It is desirable that the alignment parti-cle emit fluorescence in all detector channels,as this allows all channels to be checked simul-taneously. Alignment of the optics is optimalwhen signals from the particles have maxi-mum intensity and minimum variation or CV.The more uniform the particles, the better thedegree to which small deviations from optimalalignment can be detected. Optimal alignmentis most critical for measuring DNA, because ofthe very low inherent variation in DNA contentfrom cell to cell.

Antibody binding capacity (ABC): num-ber of antibodies of a particular type that canbind to a cell under saturating staining condi-tions. Researchers also use the term “ABC” tostand for “Antibodies Bound per Cell.” Thisterm may not always imply a requirement

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for saturating staining partly due to interfer-ence caused by simultaneous staining of dif-ferent kinds of antibodies on the same cellpopulation.

Autofluorescence: inherent fluorescencefrom a cell or particle to which no stain orfluorochrome has been added. Manufacturedparticles (such as plastic beads) can be pre-pared to have nearly the same autofluorescenceas lymphocytes.

Background (noise, fluorescence, scatter):signal present when no particles are flowingin the sample stream. Background noise isone factor that limits the sensitivity of fluo-rescence detection (see definitions of fluores-cence sensitivity and light-scatter sensitivitybelow). Depending on how low the signals arethat one is trying to detect in the sample, dif-ferent factors are dominant contributors to thebackground. When no light is coming from theflow cell (e.g., lasers turned off), detector noiseis the background limit. For photomultipliertubes (PMTs), the detector background noiseis called dark current and is due to randomemission of electrons from the photocathode.For photodiodes and other solid-state detec-tors, which have no or low signal amplifica-tion, the limiting factor under best conditionsis noise from the amplifier required to raisethe signal to a useful level. Sources of flu-orescence noise include Raman scatter fromwater and optical components; fluorescencefrom unbound fluorochrome, reagent, or con-taminants in the sample or sheath stream; andfluorescence from optical components.

Calibration: process of adjusting an instru-ment so that the analytical result is accuratelyexpressed in some physical measure.

Calibrator: material that has been manu-factured or assayed to have known, measuredvalues of one or more characteristics. The as-sayed values are provided with the material.Fluorescent manufactured particles can be as-sayed for diameter or for the amount of flu-orescence they produce. A practical measureof particle fluorescence is the number of flu-orochrome molecules in solution that producethe same amount of fluorescence as one bead(see definition of MESF and ERF).

Coefficient of variation (CV): statisticalmeasurement of the broadness of a distributionof values, usually defined as CV = σ /μ, wherethe standard deviation σ = [�(xi–μ)2/(N–1)]1/2, with the sum over N measurements ofxi (where xi is the ith measurement of vari-able x), and the mean μ = (� xi)/N. Shapiro(2003) gives an excellent discussion of CV and

other, more robust statistics for flow cytome-try. Another excellent reference for statisticalmethods is Bevington (1969). A robust CVexcludes outlier data and is typically definedusing ranges of data including a specified per-centage (e.g., 95%) of the data. In the casewhere there are no significant outliers, the ro-bust CV is equal to the CV using the standarddefinition.

Control particle or material: stable mate-rial (e.g., sample of manufactured particles)that gives reproducible results when analyzed.Particles used to set up a flow cytometer areused as a control even if they do not have anassayed value assigned to a physical character-istic. Controls can be used to monitor the sta-bility of an instrument and determine whetherit is acceptably within calibration. A calibratorcan be used as a control material, but a con-trol material does not have to have an assignedvalue for a characteristic.

Control sample: sample prepared in thesame or nearly same way as a test or unknownsample and which should give an expected,predetermined result. In immunofluorescenceanalysis, a positive control sample may useknown cells (characterized for reactivity to apanel of antibodies) and the same antibodyreagents as the test sample. A negative controlsample may use the test cells but without an-tibody reagent or with an irrelevant antibodyreagent. Also, the positive control sample cancontain some negative cells which functionas an internal negative cellular control. Thiscan also be referred to as an internal isotypecontrol.

Fluorescence sensitivity: In flow cytometry,there are two different aspects to the notion ofsensitivity: threshold and resolution. The firsthas to do with the smallest amount of lightthat can be detected (Wood, 1993; Owens andLoken, 1995; Schwartz et al., 1996; Shapiro,2003). This notion has also been given thename “detection threshold” (Schwartz et al.,1996). The second has to do with the abilityto resolve dimly stained cells from unstainedcells in a mixture (Brown et al., 1986; Horanet al., 1990; Shapiro, 2003). These concepts donot measure the same thing. The second no-tion incorporates a measure of the broadnessof the fluorescence distributions for dim andunstained particles, not just the average fluo-rescence. Two instruments can have the samedetection threshold but differ significantly inability to resolve a dimly stained population.This is illustrated by example later (see Stan-dardization and Calibration Section). Flow Cytometry

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1. Degree to which a flow cytometer canmeasure dimly stained particles and distin-guish them from a particle-free background(threshold). Threshold is important when themean fluorescence of a dimly fluorescent pop-ulation is measured. The greater the num-ber of particles analyzed, the more accuratelyand precisely will the mean fluorescence bemeasured.

2. Degree to which a flow cytometer candistinguish unstained and dimly stained pop-ulations in a mixture of particles (resolu-tion). Resolution is important for immunoflu-orescence analysis of subpopulations and isstrongly affected by the measurement CVs fordim and unstained particles.

Inherent sample CV: actual variability inthe characteristics of a sample; for example,the actual variation in the amount of fluo-rochrome per bead in a sample of beads. Be-cause the measurement process is not perfectand itself adds variation, the CV of the mea-sured fluorescence will be greater than the in-herent sample CV. The inherent CV of a sam-ple can be estimated within a small uncertaintyif the measurement variability added by theflow cytometer is well characterized (see De-termining Inherent Fluorescence CV of a DimParticle Sample).

Light-scatter sensitivity: degree to whichsmall particles can be detected above “particle-free” fluid. In practice, forward-scatter sensi-tivity is usually limited by optical noise causedby the excitation source, and side-scatter sensi-tivity is usually limited by submicron particlesin the sheath fluid.

Limit of detection: the lowest amount ofanalyte in a sample that can be detected butnot quantified as an exact value.

Limit of quantitation: the lowest amount ofanalyte in a sample that can be quantitativelydetermined with acceptable precision and ac-curacy under stated experimental conditions.

Manufactured particles (beads, plas-tic beads, latex particles, microspheres,microbeads): particles made of syntheticpolymers (plastics). Sizes range from submi-cron to over 100 μm, which generally coversthe range of cells analyzed in flow cytometry.Most manufactured particles are made by bulkpolymerization, but very uniform beads can bemade employing the same droplet generationprinciple used for flow cytometric cell sorting(Fulwyler et al., 1973). Colored or fluorescentparticles can be made by staining the beadswith dyes or fluorochromes. Nonfluorescentbeads, as well as many fluorescently stained

beads, seem to be stable for many years.Two methods, namely, solvent (or “hard”)dying and surface staining, are used to stainparticles. In solvent staining, non-water-soluble dyes are mixed with the particlesin an organic solvent. The particles take upthe dye and are then suspended in aqueoussolution. The dye is trapped in the beads,which essentially become a “hard-dyed”plastic material. In some cases, hard-dyedparticles can be synthesized directly usingfluorescent monomers (Rembaum, 1979).As most dyes or fluorochromes used tostain cells are water soluble, solvent stainingcannot generally be used for them. Whensolvent staining is possible for water-solublefluorochromes, the spectral characteris-tics can differ significantly from those offluorochrome in aqueous solution. Sur-face staining allows many commonfluorochromes—especially those used astags on fluorescent antibodies—to be used forparticle staining. In this case a chemical groupon the particle surface (e.g., amino group) iscovalently bound to a reactive group on thefluorochrome.

MESF (molecules of equivalent soluble flu-orochrome) and ERF (equivalent number ofreference fluorophores): measure of particlefluorescence in which the signal from a fluo-rescent particle is equal to that from a knownnumber of molecules in solution. The ERF unitis different from MESF in that the fluorophoresattached to particles and the fluorophores in so-lution can be very different and may have verydifferent molar absorptivities. Hence, MESF isa special case of ERF where the labeling flu-orophore and fluorophore in solution are thesame. This is a practical measure because aknown concentration of particles can be com-pared directly with a solution of fluorochromein a spectrofluorometer (see Calibrating Parti-cle Fluorescence in ERF).

Nonfluorescent particle: particle whose flu-orescence distribution is the same as that of aparticle-free sample. In practice, the concept ofnonfluorescence is dependent on the sensitiv-ity of the instrument making the measurement.A particle that is not measurably fluorescent inone instrument may be so in a more sensitiveinstrument. Fluorescence (or other lumines-cence or Raman scatter) from otherwise un-stained manufactured particles depends on thematerial and treatment with which the beadsare made. With all other factors equal, the “flu-orescent” signal from microbeads will be pro-portional to the volume of a single bead.


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Precision or reproducibility: degree towhich repeated measurements of the samething agree with each other. In flow cytom-etry, precision of a measurement is estimatedby the CV obtained when measuring a sam-ple of particles (biological or nonbiologi-cal) with very uniform characteristics multipletimes.

Resolution: degree to which a flow cytom-etry measurement parameter can distinguishtwo populations in a mixture of particles thatdiffer in mean signal intensity. Fluorescencesensitivity (see above) can be considered a spe-cial case of fluorescence resolution for whichthe signals are very dim. Note that the reso-lution will appear different when data are ac-quired and/or displayed on a logarithmic ratherthan linear intensity scale. Depending on themaximum number of channels into which thesignal intensity is acquired (e.g., 256 or 1024),a logarithmic display of the data may not havesufficient resolution to display populations thatcan actually be resolved by the instrument.

Standard: 1. noun. (a) acknowledged mea-sure of comparison for quantitative or qualita-tive value; (b) something recognized as correctby common consent or by those most compe-tent to decide.

2. adj. (a) serving as a standard of mea-surement or value; (b) commonly used andaccepted as an authority.

Standardize: (a) cause to conform to a givenstandard; (b) cause to be without variation.

Test pulse–triggered background fluores-cence: measurement of background fluores-cence in a flow cytometer by using anelectronic pulse to trigger the pulse detectionelectronics and acquire data from the fluores-cence detector(s) (see UNIT 1.4; Wood, 2009).As no particle is present to emit light, the flu-orescence signals acquired are due only to in-strument background light and noise, and thusestablish the lowest signal that can be mea-sured. The duration of a test pulse usually sim-ulates a signal from a particle of typical size.Larger particles would have signals of longerduration and produce more background signaland noise. If equipped with a test pulse func-tion, the flow cytometer can provide a mea-surement equivalent to running a sample oftruly nonfluorescent particles. The backgroundfluorescence distribution produced by a testpulse should provide a measure of the “detec-tion threshold” described by Schwartz et al.(1996). In many instruments, the test pulsesignal produces a pulse of light from a light-emitting diode that is detected and processedby only one detector. When the test pulse

signal is applied only to the forward scatterdetector, the response of all other detectorsto background light and noise can be mea-sured. When a sample is run under normalconditions, any signal from particles abovethis background and noise level actually comesfrom the particles. There is no guarantee, how-ever, that the particle signal is from particlefluorescence; for example, light scattered bythe particles may not be totally blocked bythe optical filters, or in some cases, the lightscatter may actually induce the filter to fluo-resce. The possibility of scatter-induced lightdetected as fluorescence signal can be checkedby running unstained cells and looking for asignal in the fluorescence channel. Becausesuch a signal can also come from autofluores-cence, one should also look at the side scatterversus fluorescence histogram for a strong cor-relation between side scatter and fluorescencefrom unstained cells.

OVERVIEW OFSTANDARDIZATION ANDPERFORMANCECHARACTERIZATION IN FLOWCYTOMETRY

Standardization (see Definitions) is thefoundation of flow cytometry and allows in-vestigators to have confidence in instrumentperformance and measurements. This sectionsurveys characteristics of particles used in flowcytometry, for example, to standardize im-munofluorescence and to check alignment andmeasurement precision (see Types of Parti-cles). Specific types of particles are compared.Standardization can be complicated, however,by factors other than particle type (see Gen-eral Cautions for Using Particles in Standard-ization and Calibration; see What the Instru-ment Cannot Control: Sample, Reagent, andData Analysis), but prospects for formaliz-ing flow cytometry standards are encourag-ing (see Standard-Setting Organizations). Thenext section (see Standardization and Calibra-tion) reviews various parameters of flow cy-tometers that can be standardized, such as reso-lution and sensitivity, and the final section (seeCharacterizing Particles for Calibration andControl of a Flow Cytometer) describes proce-dures and cautions for characterizing particles.

Where appropriate in the discussion of stan-dardization, approaches to characterizing in-strument performance will be included. Per-formance characterization is different fromstandardization in that it provides informa-tion about limitations or limits of performance.


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Examples included fluorescence sensitivityfor resolution of dim populations and lin-earity limitations of computed fluorescencecompensation.

Types of ParticlesManufactured particles and biological par-

ticles may be used to standardize flow cy-tometers. Beads may be spectrally matched tothe fluorochromes used to stain cells, or theymay simply fluoresce to a useful extent in thespectral range of interest. Spectrally matchedbeads allow standardization or even calibra-tion across instruments that do not have ex-actly the same emission filters and/or excita-tion wavelengths. Biological particles may bestained with the same fluorochromes used inexperiments to stain cells. Examples of datafor some of the more common types of parti-cles follow. Classification schemes for varioustypes of particles used for standardization inflow cytometry have been proposed (Schwartzet al., 1996, 1998).

Comparison of spectrally matched andunmatched fluorescent particles

Figures 1.3.1 and 1.3.2 show emission spec-tra from three types of particles: fluorochrome-tagged beads (CaliBRITE beads, BDBiosciences), stained with either fluoresceinisothiocyanate (FITC) or phycoerythrin (PE);broad-spectrum hard-dyed beads (Rainbowbeads, Spherotech); and glutaraldehyde-fixedchicken red blood cells (gCRBC, BioSure).Figure 1.3.1A compares FITC-stained Cali-BRITE beads with Rainbow beads andgCRBC, which are not spectrally matched toFITC. Figure 1.3.2A makes the same com-parison with PE-stained CaliBRITE beads.Figure 1.3.1B compares quantitatively thefluorescence signal of each particle throughfilters of differing spectral bandwidth placedin front of PMT1, with data being normalizedto the signal from FITC CaliBRITE beads foreach filter. The gCRBC varied by about 35%over the range of filters used. Rainbow beads,however, varied by nearly 200% with the samefilters. The differences in relative fluorescencewith different filters should be consideredwhen comparing different instruments. Evenfor a particular flow cytometer type or model,filters and other factors affecting spectralresponse vary slightly due to manufacturingtolerances. Figure 1.3.2B shows a similarcomparison for the relative fluorescence withdifferent filters placed in front of PMT2. Inthis case, both gCRBC and Rainbow beadsvary only slightly from PE-stained CaliBRITE

beads. The fluorescence could be standardizedwith a maximum difference of 40% with anyof these particles.

A large, multi-laboratory, multi-instrumentstudy has been performed to estimate the vari-ability of instrument calibration and standard-ization using several different hard-dyed beadscompared to beads surface stained with an-tibody conjugated to four different commonfluorophores. (Hoffman et al., 2012). Across135 different flow cytometers the fluorescenceof various hard dyed beads had considerablevariation compared to fluorescence of beadsstained with antibodies conjugated to spe-cific fluorophores (FITC, PE, APC, and Pa-cific Blue). Figure 1.3.3 serves as an exam-ple and shows the variability of hard dyedbead fluorescence in the detector channel de-fined as FITC channel. Within a particular in-strument model, most hard-dyed beads hadfluorescence variation of at least 20% refer-enced to fluorophore-conjugated antibody flu-orescence. This study concludes that the spec-trally unmatched hard-dyed beads can be usedas fluorescence calibrators but have to be veri-fied for every instrument model and bead. Cal-ibrators that are stained with the specific fluo-rophore of interest, e.g., FITC, PE, etc., aremore suitable for reproducible fluorescencecalibration and standardization across differ-ent instrument platforms.

“Nonfluorescent” and autofluorescentparticles

Figure 1.3.4 shows emission spectra forparticles with very low fluorescence. Un-stained CaliBRITE beads have fluorescencecomparable to autofluorescence from lym-phocytes. Osmium-fixed chicken red bloodcells (CRBC) had no fluorescence detectableabove background in the fluorometer. Such“negative” particles are useful for estimat-ing how well low-level signals can be de-tected, as discussed later (see Sensitivity orSignal/Noise for Dim Fluorescence). An al-ternative type of “non-fluorescent” particle issubmicron polystyrene beads (e.g., 0.5 µm di-ameter), which are easily triggered by sidescatter, but, because their volume is at least100 times less than typical particles used forstandardization, have also 100 times lower aut-ofluorescence, which (with the possible excep-tion of using UV or violet excitation) is essen-tially below the detection threshold of com-mercial flow cytometers. As the sensitivity ofinstruments improves, “nonfluorescent” par-ticles that had not previously been detectablemay no longer serve as as “nonfluoresent.” The


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Figure 1.3.1 (A) Fluorescence emission spectra of FITC CaliBRITE beads (BD BioSciences),Rainbow beads RFP-30-5K (Spherotech), and glutaraldehyde-fixed chicken red blood cells(gCRBC, BioSure). Excitation at 488 nm was used. (B) Percentage of fluorescence signal throughdifferent optical filters for Rainbow beads and gCRBC normalized to signal from FITC CaliBRITEbeads. Data in B are also scaled relative to the signal through the 505 to 525–nm filter.

only true nonfluorescent event is an event withno particle present, which can be achieved us-ing an electronic or optical signal to triggerdata acquisition without providing any signalto the fluorescence detector.

Comparison of particles forstandardizing immunofluorescenceanalysis

Figure 1.3.5 shows light-scatter dot plots(panel A) and green (515 to 545 nm) fluo-rescence histograms (panels B-F) for severaltypes of particles used to standardize flowcytometers for immunofluorescence analysis.Fluorescence from the stained particles is inthe range observed for immunofluorescencefrom cell-surface markers. All data were ac-quired using the same instrument settings,and panels A-D were obtained from the samesample acquisition of a mixture containing

(1) unstained (autofluorescence) and FITC-stained CaliBRITE beads, shown in regionR1 in panel A; (2) a combination of un-stained and multiple levels of stained Rain-bow beads, shown in region R2 in panel A; (3)gCRBC, shown in region R3; and (4) forward-scatter (FS) test pulses (no particle, R4 inpanel A). Fluorescence histograms in Figure1.3.5 are from unstained and FITC CaliBRITEbeads (panel B); Rainbow beads (panel C);FS test pulses and gCRBC (panel D); Quan-tum 24 beads (Flow Cytometry Standards;panel E); and osmium- and glutaraldehyde-fixed CRBC (panel F). Panels B, D, and Fillustrate different pairs of particles or sig-nals at the low and high ranges of a scale forimmunofluorescence. The Quantum 24 beadsshown in Figure 1.3.5E had calibration val-ues for the stained beads (upper four peaks inthe histogram) of 4,201, 16,936, 37,466, and


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Figure 1.3.2 (A) Fluorescence emission spectra of PE CaliBRITE beads (BD Biosciences), Rain-bow beads RFP-30-5K (Spherotech), and glutaraldehyde-fixed chicken red blood cells (gCRBC,BioSure). Excitation at 488 nm was used. (B) Percentage of fluorescence signal through differentoptical filters for Rainbow beads and gCRBC normalized to signal from PE CaliBRITE beads. Datain B are also scaled relative to the signal through the 564- to 606-nm filter.

65,797 fluorescein MESF (molecules of equiv-alent soluble fluorochrome) provided by thevendor.

In the same multi-laboratory, multi-instrument study whose results are illustratedin Figure 1.3.3, four different bead manu-facturers used identical protocols and mate-rials to assign ERF values to beads. Consid-erable variation in results was found amongmanufacturers compared to values measuredby the National Institute of Standards andTechnology (NIST; Hoffman et al., 2012). Toprovide reproducible and traceable fluores-cence intensity assignments to beads, NISThas established a flow cytometry quantitationconsortium under which a service of the ERFintensity value assignment to beads will beprovided to bead manufacturers.

Particles for aligning and checkingmeasurement precision

Figure 1.3.6 shows scatter and fluorescencedata for a uniform 2.49-μm-diameter fluores-cent bead that is useful for checking or adjust-ing optical alignment. All fluorescence CVswere <2%. A low fluorescence CV is an im-portant performance characterization, partic-ularly for DNA content measurements wherefor non-replicating cells the biological varia-tion is essentially zero.

General Cautions for Using Particlesin Standardization and Calibration

There are two important factors to remem-ber when using manufactured particles ratherthan cells in a flow cytometer. First, beads arenot cells and do not necessarily scatter light


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Figure 1.3.3 Box and whisker plots of the normalized ratio of the MFI of the indicated hard-dyedbeads to the MFI of the FITC fluorophore standard bead for 10 different flow cytometer models.The box shows the 25th to 75th percentiles, and the line in the box indicates the median value.Horizontal bars outside the box indicate 10th and 90th percentiles and the circles indicated 5th and95th percentiles. The percentile markers indicate the percentage of instruments for which the crosscalibration was within the indicated normalized range. The number of instruments represented foreach instrument model is noted after the model name on the x axis of each plot. (This figure isfrom Hoffman et al., 2012, Cytometry Part A, 81A, 785).

as cells do. Second, fluorescence from a beadmay be similar to that from a cell stained with aparticular dye, but it is almost never identical.

Regarding the first point, light scatter frombeads usually differs greatly from scatter fromcells of the same size. This is primarily due todifferences in the optical refractive indexes ofbeads and cells. Beads with high water con-tent and consequently low refractive index,

such as Sephadex chromatography beads, givelight scatter results similar to those from cells(Sharpless et al., 1977). However, such com-mercially available beads have a wide distri-bution of diameters and are difficult to use instandardizing an instrument.

Regarding the second point, fluorochromesused in hard-dyed beads are rarely the sameas those used to stain cells. Even when the


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Figure 1.3.4 Fluorescence emission spectra of unstained CaliBRITE beads (BD Biosciences)and osmium-fixed chicken red blood cells (CRBC, BioSure). Excitation at 488 nm was used, andconcentrations of the two types of particles were the same. No fluorescence from the osmium-fixedCRBC could be measured above background noise in the spectrofluorometer.

fluorescence emission spectra of a hard-dyedbead and a fluorescently stained cell areclosely matched, it will be rare that the excita-tion spectra also match unless bead and cell arestained with the same fluorochrome. Also, oneneeds to be aware of a possible nonlinear rela-tionship between the fluorescence signal andthe intensity of light used to illuminate the par-ticles [UNIT 1.4 (Wood, 2009) discusses systemlinearity]. The relative fluorescence of two dif-ferent fluorochromes can differ considerablywith the intensity of excitation light (Bohmeret al., 1985).

Fluorochromes such as FITC and PE,which are used to tag antibodies, are availableon surface-stained beads, and such beadsclosely match the fluorescence characteristicsof cells stained with tagged antibodies. Thesurface-stained beads have the same excitationspectrum and sensitivity to excitation lightintensity as do cells stained with the samefluorochromes. However, surface-stainedbeads in suspensions are less stable comparedto hard-dyed beads, and, hence, have limitedshelf life. Freeze-dried surface-stained beadshave a longer shelf life.

What the Instrument Cannot Control:Sample, Reagent, and Data Analysis

It is important to keep in mind the factorsthat affect results but are beyond the control ofthe flow cytometer. A well-calibrated instru-ment and careful quality control cannot correct

for samples and reagents that are not properlymaintained, prepared, and used (see Chapter4 for information on molecular and cellularprobes; see Chapter 5 for specimen handling,storage, and preparation). Good data producedby the instrument cannot guarantee correct re-sults if data analysis is wrong (see Chapter 10on data processing and analysis). The flow cy-tometer hardware is only one part of the systemthat must work correctly to give good results.Owens and Loken (1995) provide an excellentand instructive introduction to the entire rangeof factors that affect results of flow cytomet-ric analyses commonly performed in clinicallaboratories.

Standard-Setting OrganizationsThe Clinical and Laboratory Standards

Institute, an international clinical labora-tory standards–setting organization, has estab-lished several guidelines specifically for flowcytometry or that apply to flow cytometry. Thelist (at the time of this writing) includes H42-A2 (Enumeration of Immunologically De-fined Cell Populations by Flow Cytometry;Approved Guideline- Second Edition); H43-A2 (Clinical Flow Cytometric Analysis ofNeoplastic Hematolymphoid Cells; ApprovedGuideline- Second Edition); and H52-A2 (RedBlood Cell Diagnostic Testing; ApprovedGuideline- Second Edition).

The CLSI guideline H42-A2 (CLSI, 2008)takes a conservative approach in stating “There


1.3.10


Figure 1.3.5 Light scatter and green fluorescence distributions from several types of standard-ization particles or sources. All data were acquired with identical instrument settings, and pulseheight was measured. (A) Forward scatter (FS) versus side scatter (SS) dot plot of mixture ofbeads and test pulse signals. Each type of particle or the test pulse is enclosed by a region inthe dot plot: region R1 contains unstained and FITC CaliBRITE beads or Quantum 24 beads;R2 contains the Rainbow bead mixture RCP-30-5K; R3 contains glutaraldehyde-fixed chickenred blood cells (gCRBC). Region 4 contains forward-scatter test pulses, which allow backgroundnoise from all other parameters to be measured. (B) Green fluorescence histogram of unstainedand FITC CaliBRITE beads. Unstained beads have nearly the same autofluorescence as lympho-cytes. (C) Green fluorescence histogram of Rainbow bead mixture containing unstained beadsand five levels of stained beads. (D) Green fluorescence histogram of background noise from testpulse–triggered acquisition (region R4 in panel A) and gCRBC (region R3 in panel A). (E) Greenfluorescence from mixture of Quantum 24 FITC beads from Flow Cytometry Standards. (F) Greenfluorescence histogram of osmium-fixed CRBC (low population) and gCRBC (high population).

are at present no standards which can be usedto check the accuracy of flow cytometric testresults. Hence, verifying reproducibility of in-strument performance is an essential elementof daily quality assurance for the flow cytom-etry laboratory. Instrument performance mustbe monitored under the same conditions as

are used to run test samples.” The guidelineproposes a two-step procedure for instrumentquality assurance. First, establish that the in-strument performance is acceptable at a par-ticular point in time. Then, monitor perfor-mance with stable materials under test-specificinstrument conditions. No specific criteria for


1.3.11


Figure 1.3.6 Scatter (FS and SS) dot plot (A) and fluorescence histograms (B-D) of 2.49-μm-diameter beads stained with Nile red.

calibration or control materials are given. Theguideline instead provides a process that a lab-oratory can follow by using materials recom-mended or supplied by the instrument manu-facturer or by establishing independent criteriaand materials. [Further discussion of the prin-ciples of quality control is given in UNIT 3.1

(Hurley, 1997a); applications of quality assur-ance in phenotyping and in nucleic acid analy-sis are covered in UNIT 6.1 (Hurley, 1997c) andUNIT 7.2 (Darzynkiewicz, 2011)

A guideline for validation of clinical testsdeveloped in-house by clinical laboratorieswas written by a working group sponsoredby the International Clinical Cytometry So-ciety (ICCS) and the International Council forStandardization of Haematology (ICSH). Thisguideline addresses instrument and analyticalissues (Tanqri et al., 2013) and assay perfor-mance criteria (Wood et al., 2013).

NIST has developed a method for deter-mining particle MESF (Gaigalas et al., 2001;Schwartz et al., 2002) and has produced a stan-dard fluorescein solution (Standard ReferenceMaterial 1932). Practical issues to considerin using MESF in quantitating fluorescencehave been addressed in detail by NIST (Wanget al., 2002). Because of the increasing de-mand for multiparameter flow cytometric mea-

surements, e.g., 12 color clinical cytometryassays, NIST has further developed a new stan-dard reference material, SRM 1934, to supportthe calibration of microspheres in the unitsof ERF. The SRM 1934 includes four fluo-rophore solutions or suspension, Fluorescein,Nile Red, Coumarin 30, and Allophycocyaninfor ERF value assignment performed with thethree laser excitations most commonly usedin commercial flow cytometers, 405 nm, 488nm, and 633 nm. A standard operating pro-cedure for ERF value assignment using thisSRM 1934 has been documented recently byNIST (Wang et al., 2016a). Uniformed ERFvalue assignment to calibration microspheresensures the traceability of the value assign-ment and enables the standardization of thefluorescence intensity scale of flow cytome-ters in quantitative ERF unit.

INSTRUMENT PERFORMANCECHARACTERIZATION,STANDARDIZATION ANDCALIBRATION

A flow cytometer may be characterizedor standardized by running samples, suchas manufactured particles, that have someknown properties. Controlling the parametersand characteristics provides consistent results


1.3.12


over time from one instrument but, unless theinstrument was calibrated, does not necessar-ily allow results to be quantitatively comparedwith those from other instruments. Perfor-mance characterization gives detailed quan-titative information about the analytical capa-bilities of a flow cytometer such as linearity ofthe measurement scale, sensitivity to measuredim fluorescence, resolution of small differ-ences in DNA content, or an estimate of thesmallest size of particles that can be detectedby light scatter.

Flow cytometers have many parametersthat can be standardized and controlled (e.g.,see Optical Alignment; see Light Scatter andParticle Sizing; see Fluorescence and LightScatter Resolution; see Measurement Re-sponse and Logarithmic Calibration; see Sen-sitivity or Signal/Noise for Dim Fluorescence;and see Spectral Overlap Compensation). Thissection also discusses aspects of standardiza-tion relating to specific applications (see DNAMeasurements; see Sorting Purity and Recov-ery; see Standardization with a Particle in theAnalysis Sample; and see Particle Concentra-tion). The final subsection provides a summary(see What Measurements Can Be Calibrated,and How Frequently Is Calibration Neces-sary?). Actual procedures and guidelines forcharacterizing beads follow (see Characteriz-ing Particles for Calibration and Control of aFlow Cytometer).

Optical AlignmentOptical alignment is most critically as-

sessed and most easily optimized using par-ticles with very uniform scatter and fluores-cence. The objective of optical alignment isto center the sample stream in the light beamand simultaneously image the intersection ofsample stream and light beam through the de-tection optics. At optimal alignment, signalpulses will have maximum amplitude and min-imum width, and be most reproducible. In ahistogram of the fluorescence or scatter, thedistribution will be at its narrowest with high-est amplitude. The most uniform particles aregenerally smaller (1 to 3 μm) than typical cells.Although much more difficult to make, largeruniform particles are available. FluorescenceCVs <2% can be expected for alignment par-ticles (see Fig. 1.3.6). Other than cell sorters,most modern flow cytometers have a fixedalignment that is not intended to be adjustedby the user. The objective then is to evaluatethe alignment using the CV of the uniform flu-orescent particles. Instrument manufacturersprovide expected and maximum CV’s from de-

fined alignment particles. There will also typ-ically be expected CVs for light scatter. Somemanufacturers give specifications for low sam-ple flow rate (minimum sample stream diame-ter) as well as for high sample flow rate (maxi-mum sample stream diameter) which will havea larger CV due to wider range of particle po-sition within the excitation laser beam.

Light Scatter and Particle Sizing

ScatterLight scatter is a difficult parameter to stan-

dardize because it depends critically on thescatter angles measured and the geometry ofthe collection optics. The scattering of parti-cles has strong nonlinear dependence on theangles measured and the refractive index ofthe particle, as well as particle size, internalstructure, and content (Salzman et al., 1990;Doornbos et al., 1994; Shapiro, 2003). It iseven possible at certain scatter angles to havea smaller signal from larger particles. The re-fractive index of most manufactured particlesis much larger than that of cells, and beads andcells of the same size have different scatter in-tensities. For these reasons, it is not advisableto use scatter for quantitative cell sizing. Scat-ter is primarily useful for discriminating cellsbased on relative scatter properties (e.g., dis-crimination of lymphocytes, monocytes, andgranulocytes is a common application).

Although it is difficult to standardize lightscatter across instrument types using manu-factured particles, it is possible to standardizewithin a particular instrument type and to mon-itor the relative performance of an instrument.The relative and absolute scatter intensities oftwo different-sized particles provide one wayto standardize scatter. The ellipsoidal shapeof fixed chicken red blood cells (CRBC) pro-duces a characteristic light scatter pattern withtwo peaks in the forward scatter distribution.Region R3 in Figure 1.3.5A shows an exampleof the light scatter distribution for CRBC. Al-though the scatter pattern for CRBC may varyamong different instrument designs, it may beuseful for monitoring a particular instrument(Horan and Loken, 1985; Horan et al., 1990).At the present time, however, the most reliableapproach is to use a biological sample of thesame or similar type that is to be used for theflow cytometric analysis (Owens and Loken,1995; CLSI, 2008).

Pulse widthPulse widths of fluorescence or scatter sig-

nals depend on both the height of the laser


1.3.13


beam (in the direction of flow) and the size ofthe particle passing through the beam. Whenthe particle diameter is at least as large as thebeam height, it is possible to measure particlediameter accurately (Sharpless and Melamed,1976; Sharpless et al., 1977; Eisert and Nezel,1978; Leary et al., 1979; Shapiro, 2003). Amixture of two or more sizes of particles ofknown, calibrated diameter can be used to cal-ibrate this measurement. For a detailed proto-col UNIT 1.23 (Hoffman, 2009).

Electronic cell volumeElectronic cell volume (Schwartz et al.,

1983; Kachel, 1990; Shapiro, 2003) is pro-vided as a parameter in some commerciallyavailable flow cytometers. Microbeads arean excellent material for standardization andcalibration of electronic cell volume. Accu-rate measurement of particle volume requiresthe particles to be electrically nonconductive.Thus, caution should be used when comparingresults with fixed cells in which the plasmamembrane has become permeable and electri-cally conductive.

Fluorescence and Light ScatterResolution

Resolution is usually estimated by runninga sample of uniform particles and measuringthe CV. For fluorescence measurements, CVis a good estimate of resolution because flu-orescence signals are generally proportionalto the amount of fluorochrome on the particle[i.e., the measurement and the characteristicof the particle are linearly related; see UNIT

1.4 (Wood, 2009) on system linearity]. Reso-lution of dimly stained particles is considereda special case (see Sensitivity or Signal/Noisefor Dim Fluorescence). Resolution of uniformparticles characterizes the alignment of the op-tics and should not be greater than the instru-ment manufacturer specifications for the spec-ified beads.

For light-scatter measurements, the CV ob-tained with one particle may not be a goodmeasure of how well the cytometer may beable to resolve particles of different sizes, sincethe relationship between light-scatter signaland particle size is usually not linear.

Measurement Response andLogarithmic Calibration

The measurement response of a flow cy-tometer can be determined in relative or ab-solute terms. Evaluating the relative responsecan be as simple as determining whether twoidentical particles stuck together give twice

the signal as one. Alternatively, it may be ascomplex as determining the response over arange of four or more decades. Absolute re-sponse calibrates the measurement (e.g., chan-nel number in a histogram) in units such asmolecules of equivalent soluble fluorochrome(MESF), equivalent number of reference fluo-rophores (ERF), or antibody binding capacity(ABC).

Knowing how well the displayed data ac-tually follow a linear response (or logarithmicresponse for instruments that use log ampli-fiers) is particularly important when the dataare used to compute compensation. Small de-viations in linearity can cause large absoluteerrors when a fraction of one number is sub-tracted from another. With the exception ofthe BD FACSCalibur, which does fluorescencecompensation using analog amplifiers, com-pensation is done using the linear data (or lin-earized data from log amplifiers) by mathe-matical calculation that assumes the data areperfectly linear. It is easy to demonstrate sit-uations in which a deviation from linearityof 3% in a large number (say in the lastdecade of a histogram) causes a truly negativepopulation to be displayed and then assumedto be positive following correctly computedcompensation.

Linear and logarithmic responseTesting the relative linearity of a measure-

ment on a limited range of a linear scale isconveniently done using small beads or otherstained particles such as cell nuclei that con-tain aggregates. Measurement of the sampleshould then give histogram distribution meanchannels that are multiples of the mean chan-nel of a single particle.

Measuring the response of a nominally log-arithmic scale can be done in two ways. Thefirst uses a known linear scale as reference andcompares measurements on the linear and log-arithmic scales (Muirhead et al., 1983; Horanet al., 1990). The second approach uses a mix-ture of particles with known, different intensi-ties. The log response is determined by mea-suring the separation of the peaks on the logscale as the signal level is varied by changingthe PMT voltage (Schmid et al., 1988). A moreconvenient version of the second approachuses a bead mixture containing a wide rangeof fluorescence levels whose relative intensi-ties are known (e.g., see Fig. 1.3.5C). If thebeads in the mixture are calibrated in terms ofMESF or ERF, then the absolute log responsemay be determined (Schwartz and Fernandez-Repollet, 1993; Schwartz et al., 1996).


1.3.14


Another standard method for measuring thelinearity (or logarithmic) response of an opti-cal detector is to use pulses from a light emit-ting diode (LED) that alternate at two differentintensities, and vary the amount of light emit-ted into the detector without changing the ratioof the input intensities. If the detection systemis linear, the ratio of the measured signals willbe constant and independent of the absolutemagnitudes of the two LED pulses. A simpleway to change the intensity of the LED lightwithout changing the inputs to the LED is tomove the LED further from the detector or in-troduce neutral density filters or apertures thatreduce the amount of light reaching the detec-tor. By measuring the output of the system asmeans of populations in a flow cytometer, onecan determine any deviation from linearity bynoting where the ratio of the measured meansdeviates from the expected ratio. A somewhatless rigorous method can use the intensitiesof two fluorescent particles and vary the in-put to the detection electronics by changingthe voltage on a PMT while measuring the ra-tio of the mean signals from the two beads. Ahigh degree of linearity (e.g., no more than 2%deviation from linearity) is required in instru-ments that calculate fluorescence compensa-tion rather than use analog electronics to per-form compensation. Most currently availableflow cytometers use calculated compensation,with the BD FACSCalibur being the exception.A seemingly small (e.g., 5%) percentage devi-ations from linearity can cause large (order ofmagnitude) errors in the absolute calculatedcompensation value. Thorough discussion ofestablishing and maintaining system linearityappears in UNIT 1.4 (Wood, 2009).

Secondary calibratorsCross-calibration of gCRBC or hard-dyed

particles to surface-labeled beads calibratedin MESF or ERF can generally be done forany one instrument (Schwartz et al., 1996; seeCharacterizing Particles for Calibration andControl of a Flow Cytometer). To ensure thatthe secondary calibrator on an instrument isreliable, however, the emission filters and thewavelength and intensity of the excitation lightmust remain unchanged. See Figures 1.3.1 and1.3.2 and cautions given by Schwartz et al.(1996). (Also see Types of Particles and dis-cussion of comparison of spectrally matchedand unmatched particles.)

Antibody binding capacity (ABC)A further step in immunofluorescence stan-

dardization and calibration is to express

measurement results in terms of antibody bind-ing capacity or antibodies bound per cell (seeDefinitions). Three approaches have been usedto estimate ABC. Each approach has differentcritical technical requirements and potentialsources of error. Although not, strictly speak-ing, a source of error, it must be kept in mindthat different antibody clones with the samecluster designation (CD) can have differentbinding affinity and capacity. Particular ex-amples of clone variability have been notedfor CD4 (Davis et al., 1998) and CD34 (Serkeet al., 1998). Therefore, if the three approachesto quantitative ABC are to be compared, theyshould be compared with the same clone orwith clones that are demonstrated to give thesame ABC. In addition, the sample prepara-tion method can affect the antibody bindingand must be taken into consideration (Islamet al., 1995; Serke et al., 1998).

The earliest approach (quantitative indi-rect immunofluorescence, or QIFI) uses acalibrated anti-mouse fluorescent second-stepreagent (Poncelet and Carayon, 1985; Bikoueet al., 1996). The anti-mouse reagent can becalibrated using particles coated with knownamounts of mouse immunoglobulin G (IgG).Polyvalent reagents are used in this method,however, and their reactivity with certain mon-oclonal mouse antibodies may produce arti-facts. Bikoue et al. (1996) observed that differ-ent monoclonal antibodies to a molecule (e.g.,CD8) can give different ABC values on thesame cells. Whether this is due to differencesin binding of the primary antibody to cells orto differences in binding of the secondary an-tibody to the primary antibody is not clear.Altered reactivity of the second-step reagentwith fluorochrome-conjugated mouse mono-clonal antibody is another possible variable.

A second approach (Quantum Simply Cel-lular or QSC) uses particles coated withknown amounts of polyvalent anti-mouseantibody (Schwartz and Fernandez-Repollet,1993; Schwartz et al., 1996). The QSCmethod is designed to capture quantitativelyany mouse monoclonal antibody independentof fluorochrome conjugation or IgG isotype.However, conjugates of the same monoclonalantibody with different fluorophores give dif-ferent ABC values when calibrated with QSCbeads, and different antibody clones directedagainst the same molecule can give differingresults with QSC beads (Lenkei and Anders-son, 1995). The QSC beads could, however,be calibrated to a specific monoclonal anti-body reagent whose production is carefully Flow Cytometry

Instrumentation

1.3.15


controlled (Lenkei and Andersson, 1995; A.Schwartz, pers. comm.).

A third method uses antibody conju-gates that have been prepared with a knownMESF/antibody ratio and a flow cytometerthat has been calibrated in MESF. Phycoery-thrin is an attractive fluorochrome for thisapproach since antibody conjugates can beprepared with exactly one PE molecule perantibody. Initial experiments with this ap-proach were promising (Davis et al., 1998; Iyeret al., 1998). In fact, it was this system thatled to the development of the QuantiBRITEproducts of purified 1:1 PE-antibody conju-gates and freeze-dried beads surface-stainedwith known numbers of PE molecules perbead.

The QIFI and QuantiBRITE methodshave been found to be generally compara-ble (Lenkei et al., 1998; Serke et al., 1998)for ABC quantitation, but the QSC methodfrequently gives significantly different resultsfrom the other methods (Lenkei et al., 1998;Serke et al., 1998). Since the QuantiBRITEmethod uses direct fluorescence staining, it canbe easily used in a multicolor staining proto-col. The general recommendation for compar-ing ABC across laboratories or over time is touse a single method along with the manufac-turers’ recommended reagents.

Certain cell-surface markers may be usefulas biological calibrators with a relatively smallvariability and uncertainty. Although not asreproducible as the amount of DNA per cell,the amount of CD4, CD45, and many othermolecules on normal human lymphocytes isgenerally reproducible (Brown et al., 1986;Poncelet et al., 1991; Bikoue et al., 1996); thus,CD4 content may be a useful biological cal-ibrator for immunofluorescence analogous tothe use of normal lymphocytes or other de-fined nucleated cells as biological calibratorsfor DNA quantitation (Hultin et al., 1998).

A protocol of quantitative flow cytome-try measurements in ABC based on humanCD4 reference marker has been recently de-veloped jointly by NIST and FDA (UNIT 1.29;Wang et al., 2016b). The reference marker,CD4 receptor protein on human T helper cells,can come from either whole blood of nor-mal healthy individuals or Cyto-Trol Con-trol Cells, a commercially available peripheralblood mononuclear cells (PBMC) prepara-tion, depending on the preference of users andthe accessibility of normal individual wholeblood samples. The CD4 expression levels inABC are approximately 45,000 for fixed nor-

mal whole blood samples and approximately40,000 for Cyto-Trol cells, respectively. TheseCD4 expression levels have been verified byorthogonal measurement methods, quantita-tive flow cytometry and mass cytometry, usinga well characterized anti-human CD4 mon-oclonal antibody (SK3 clone from BD Bio-sciences) as well as quantitative mass spec-trometry using an isotope-labeled, full-lengthrecombinant CD4 receptor protein as the in-ternal quantification standard. The known ref-erence CD4 expression enables the translationof a linear fluorescence intensity scale to theABC scale, which ultimately ensures quantita-tive measure of target antigen expression levelsindependent of flow cytometers used.

Separately, another method for estimat-ing how many bound antibodies are requiredto produce a particular measured signal andthereby antibodies bound per cell has been de-veloped by Kantor and colleagues (UNIT 1.30;Kantor et al., 2016). This method, so calledthe Test-Fill method, is based on complemen-tary binding of Test and Fill antibody reagentsto antibody capture microspheres. Quantita-tive values for the reference reagents areanchored using QuantiBRITE PE beads to cal-ibrate the PE channel scale and a 1:1 antibody-PE test reagent in complementation bead stainswith the Fill reagents. While replacing an-tibody capture microspheres with biologicalcells, and implementing the Test-Fill methodon the reference flow cytometer with knownstatistical photoelectron scales under the sameoperational conditions, estimation of ABC val-ues for the Test antibody reagents can be car-ried out in the same manner as on the antibodycapture microspheres.

Sensitivity or Signal/Noise for DimFluorescence

The practical notion of fluorescence sen-sitivity involves the ability to resolve pop-ulations of dimly fluorescent particles. Thefactors affecting the ability to reliably detectdim fluorescence include electronic noise, theamount of fluorescent light collected, the effi-ciency with which the fluorescent light is con-verted into electrons in the detector, and theamount of background light that is present.Characterization of an instrument’s detectioncapability should include the effect of thesefactors. An overly simplified method for char-acterizing fluorescence sensitivity can givemisleading results, as illustrated by the fol-lowing example.


1.3.16


Figure 1.3.7 Comparison of three methods for estimating green fluorescence sensitivity. His-tograms are four-decade log with 256 channels per decade. Each column of histograms representsa different measure of fluorescence sensitivity. (A-E) Histograms of autofluorescent unstained Cal-iBRITE beads (low peak) and gCRBC (high peak). (F-J) Histograms of background noise froma test pulse trigger (low peak) and gCRBC (high peak). (K-O) Histograms of autofluorescentunstained CaliBRITE beads and dim Rainbow beads. Each row of histograms shows results fora different condition or perturbation on the optical system of the instrument (see Sensitivity orSignal/Noise for Dim Fluorescence).

Resolution of dimly stained fromunstained particles

Three methods for assessing fluorescencesensitivity are illustrated in Figure 1.3.7.Data for each row of panels were obtainedwith exactly the same instrument condi-tions with a single sample that containedall the particle and test pulse events. Thefluorescence histograms were gated to con-tain the events of interest by setting regionsin a scatter dot plot, as in Figure 1.3.5A.The left column in Figure 1.3.7 (panels A-E) are histograms of unstained “autofluores-cence” beads and glutaraldehyde-fixed CRBC(gCRBC). The middle column (panels F-J)shows histograms of background light (fromtest pulse–triggered fluorescence) and thesame gCRBC. The right column (panels K-O)shows histograms of autofluorescence beadsand dimly stained beads. The instrument con-dition for each row of histograms was variedby adding background light or reducing theamount of fluorescent light that reached thePMT.

Delta channel and detection thresholdmethods: traditional and not verysensitive

The “delta channel” approach measuresthe mean or median channel of autofluores-cent or nonfluorescent particles (or test pulse–triggered background) and that of relativelybright particles. This approach is illustrated inFigure 1.3.7A-E, where the negative popula-tion is a bead with about the same autofluores-cence as unstained lymphocytes and the brightpopulation is gCRBC. With autofluorescenceas the negative reference point, there is almostno difference in mean channel or delta chan-nel for the different instrument conditions.There are considerable differences, however,in the ability of the instrument to resolve auto-fluorescent beads from dimly stained beads(Fig. 1.3.7K-O). For very dim populations, thedelta channel method does not critically as-sess the ability to resolve unstained from dimlystained particles. This is primarily because themethod does not take into account the broad-ness of the fluorescence distributions; rather, it


1.3.17


considers only the mean or median channels.Resolution of the dimly stained population,however, is critically dependent on the broad-ness of the distributions, which is determinedby background fluorescence and the amountof fluorescence signal reaching the PMT.

If nonfluorescent particles or test pulse–triggered fluorescence is used as the neg-ative population, one obtains additional in-formation about sensitivity. For Figure 1.3.7F-J, the instrument was configured to have thesame response for bright particles, but differ-ent amounts of background signal. In the ter-minology used by Schwartz et al. (1996), theinstrument had the same “window of analy-sis” for data in Figure 1.3.7F-J. Comparingpanels F-J with panels K-O in Figure 1.3.7,one sees an improved ability of this method topredict whether dimly stained particles will beresolved from unstained particles. The methodis not perfect, however. Panels L and M havethe same amount of background signal or, asexpressed by Schwartz et al. (1996), the same“detection threshold,” but they differ notice-ably in resolution of the autofluorescent anddimly stained particles. The same considera-tions hold for panels N and O of Figure 1.3.7,which have the same, intermediate amount ofbackground signal. So, the detection thresholdmethod also fails to reliably measure differ-ences in the ability to resolve dim populationsfrom the unstained population.

Resolution of dimly stained fromunstained particles

The most direct measure of the ability to re-solve unstained from dimly stained particles issimply to run a mixture of the particles and seeif they are resolved. Panels K-O in Figure 1.3.7illustrate this approach. This method at leastguarantees unambiguously that the instrumentis able to resolve a certain low level of flu-orescence. Making this approach quantitativerequires not only attention to the relative num-bers of unstained and dimly stained particles,but also some limits on the inherent particlefluorescence CV. By calibrating the unstainedand dimly stained particles in MESF or ERF,one could have a method that would standard-ize and calibrate sensitivity.

The Q and B method to characterizeinstrument sensitivity

To address the quantitative characteriza-tion of fluorescence sensitivity, the underlyingphysics of optical detectors needs to be takeninto account (Wood and Hoffman, 1998). Atheoretical model for the CV of dimly fluo-

rescent particles (Gaucher et al., 1988; Steen,1992) can be developed based on optical detec-tion efficiency, Q (number of photoelectronsper fluorescence intensity of fluorochromemolecule analyzed), and background light, B.B can be expressed in units of the equiva-lent number of fluorochrome molecules thatwould produce that background. The studiesby Gaucher et al. (1988) and Steen (1992) usedflashes from a light-emitting diode (LED) toproduce dim signals to a flow cytometer detec-tor and used the CVs of the resulting signals todetermine Q. The contribution to the CV frombackground light was determined by compar-ing CVs that were obtained from LED flasheswith the laser shining on the sample stream tothose obtained with the laser blocked. A fluo-rescence intensity standard bead was used tocalibrate the flow cytometer in MESF and ERFunits. Interest in using LED pulses as the idealsignal for determining Q and B has encour-aged development of at least one commercialLED test unit specifically for use on flow cy-tometers, e.g., quantiFlash from AngewandtePhysik & Elektronik GmbH in Germany.

As a generally practical approach, it hasbeen shown that sets of beads with uniformbut dim fluorescence could be used instead ofLED flashes to determine Q and B (Chase andHoffman, 1998; Wood, 1998). The intrinsicCVs of the dim beads could be determined bycomparing their measured bead CVs with CVsof LED light flashes and CVs of identical butbrightly stained beads (Chase and Hoffman,1998). Thus, a set of beads stained at varyinglevels from dim to bright could be character-ized for intrinsic properties and then used tomeasure Q and B in a flow cytometer. Q andB can then be determined by using a fluores-cence intensity standard to calibrate the flowcytometer in terms of MESF/ERF and measur-ing the CVs (or SDs) of the dimly and brightlyfluorescent beads. There are several ways toanalyze the resulting bead data to determineQ and B (Chase and Hoffman, 1998; Wood,1998).

A robust method for determining Q is tocorrect the standard deviations of the beadsfor the illumination uniformity contribution toSD (this is determined from the CV of a brightbead). A plot of standard deviation squared(SD2) versus the mean bead intensity in MESFunits gives a straight line whose slope is 1/Q.An estimate of B can be determined from theintercept of the line with the SD2 axis. Theintercept is B/Q. Alternatively, and probablymore accurately, B can be determined from


1.3.18


the SD of a blank bead or noise distribution(Chase and Hoffman, 1998).

UNIT 1.20 (Hoffman and Wood, 2007) pro-vides a detailed protocol for determining Qand B using the SD2 approach and simple lin-ear data fitting. The degree to which the datafit a straight line at every point is an indi-cation of how well assumptions used in themethod are met—particularly the assumptionthat all beads in the set used have the sameintrinsic CV and same uniformity of illumina-tion, which is accounted for by the brightestbead in the set. An alternative approach usesa quadratic fitting function that estimates thecontribution of intrinsic and illumination uni-formity from the fit parameters rather than di-rectly measured by the brightest bead in theset. Another alternative is used in BD’s auto-mated CS&T system, which has beads withonly three different brightness levels, but in-cludes data for the important intrinsic CV dif-ferences. All these approaches to measuring Qgenerally give somewhat similar results.

An important reason for characterizing sen-sitivity in terms of Q and B is that these val-ues can be used to predict the resolution ofdimly fluorescent populations of cells (Chaseand Hoffman, 1998). Q and B can also be usedto determine the effect of background con-tributions from unbound fluorescent antibodyor from spectral overlap on the resolution ofpopulations.

Spectral Overlap CompensationMeasurement of fluorescence in a partic-

ular spectral range is not the same as mea-suring the fluorescence from a fluorochrome.One usually measures fluorescence in a spec-tral range that contains the emission peak ofthe fluorochrome, but emission from other flu-orochromes used simultaneously to stain thecells may also overlap into the desired spec-tral region. In Figures 1.3.1 and 1.3.2, the spec-tral overlap of FITC fluorescence extends intothe range where PE has maximum emissionat about 575 nm. Similarly, PE has a smallamount of fluorescence in the range whereFITC has peak emission. The effect of thespectral overlap is illustrated in Figure 1.3.8A,which shows a dot plot of yellow and green flu-orescence from a mixture of unstained, FITC-stained, and PE-stained beads.

To make the dot-plot axes read in units ofFITC and PE fluorescence rather than greenand yellow fluorescence, the amount of spec-tral overlap from each fluorochrome can besubtracted (Bagwell and Adams, 1993). Themethod used to accomplish the subtraction is

instrument dependent; ask the manufacturerfor details. Since the percentage of FITC flu-orescence in the yellow detector is always aconstant fraction of the amount of FITC fluo-rescence in the green detector, the same per-centage of green FITC fluorescence can besubtracted from the yellow signal no matterwhat the FITC signal is. For example, PE flu-orescence = yellow fluorescence – f × greenfluorescence, where the fraction f is a constant.

Figure 1.3.8B shows the same sample asin Figure 1.3.8A but with spectral compen-sation applied. Spectral overlap compensationsimply transforms the readout of the fluores-cence from green and yellow to somethingmore directly related to the analysis results:the amount of FITC and PE fluorescence. Cor-rectly adjusting the fraction of compensationrequires care, however, since small differencesin the emission spectrum of the particle canhave a large effect on the amount of compensa-tion (Schwartz and Fernandez-Repollet, 1993;Schwartz et al., 1996). Manufactured particlesstained with fluorochromes such as FITC orPE may not have exactly the same emissionspectrum as cells labeled with the same fluo-rochromes. If beads are used to adjust compen-sation, it is always advisable to check compen-sation with labeled cells at least once for eachnew batch of beads (Schwartz and Fernandez-Repollet, 1993; Owens and Loken, 1995).

Antibody capture beads are an alternativeto using fluorochrome-stained beads or stainedcells for compensation. It is advisable to stainthe antibody capture beads with the same an-tibody conjugates that will be used to stain thecells. Antibody capture beads are especiallyuseful as compensation control samples formulticolor analysis, since the unstained refer-ence particle is identical for all fluorochromes,and all fluorochromes are controlled with astained population that is relatively uniformand bright.

When more than two fluorochromes areused simultaneously to stain cells, compen-sation becomes significantly more complex. Ifthe third and fourth fluorochromes in the sam-ple have little or no spectral overlap, compen-sation can be set manually. However, if three ormore fluorochromes all have significant spec-tral overlap among one another, correct com-pensation requires a matrix calculation thatis practical only using software (Bagwell andAdams, 1993; Roederer, 2001). In instrumentsthat use log amplifiers, a combination of hard-ware and software compensation may give themost accurate results (Baumgarth and Roed-erer, 2000). With compensation performed by


1.3.19


Figure 1.3.8 Example of spectral overlap with FITC- and PE-stained beads. Three populationsare shown in each dot plot: unstained beads, FITC-stained beads, and PE-stained beads, withunstained beads appearing in the lower left quadrant. (A) Uncompensated data with FITC-stainedbeads in the upper right quadrant with about 300 units of green fluorescence and 80 units ofyellow fluorescence. PE-stained beads are in the upper left quadrant with about 8 units of greenfluorescence and 800 units of yellow fluorescence. Unstained beads have about 6 units of greenand 5 units of yellow fluorescence. (B) Same data after compensating for spectral overlap tomake the dot-plot axes represent fluorescence from a specific fluorochrome rather than a color offluorescence signal. Note that the FITC-stained beads in the lower right quadrant of B have thesame mean “PE” fluorescence as the unstained beads, and the PE-stained beads in the upper leftquadrant have the same amount of “FITC” fluorescence as unstained beads. FITC beads have nomore PE signal than unstained beads, and PE beads have no more FITC signal than unstainedbeads.

software and with proper controls, accuratelycompensated results are possible and practicalwith ten or more fluorochromes (Baumgarthand Roederer, 2000).

DNA MeasurementsFor DNA measurements, fluorescence lin-

earity and resolution must be assured. Sam-ple preparation and data analysis must also becarefully controlled. Reviews of standardiza-tion issues for DNA analysis include those byDressler (1990), Bauer (1993), Darzynkiewicz(1993), and Wheeless (1993); also see Chap-ter 7 for nucleic acid analysis. Approaches toperforming instrument standardizations werediscussed earlier (see Optical Alignment, seeFluorescence, and see Light Scatter Resolu-tion, and Measurement Response and Loga-rithmic Calibration: Linear and LogarithmicResponse). Either fluorescent beads (see Fig.1.3.6) or stained cells or nuclei can be used.For determination of abnormal DNA content itis important to use an internal staining controlin the sample. Chicken erythrocytes, trout ery-throcytes, and normal human cells have beenused for internal controls. Calibrated measure-ment of the amount of DNA per cell has beenreported (see Shapiro, 2003,for a brief review),but sensitivity of the staining to chromatin

structure should caution against overinterpre-tation of the results (Darzynkiewicz, 1993).

Sorting Purity and RecoveryVarious manufactured particles can be used

to determine purity and recovery for sorting. Amixture of beads with different fluorochromesis typically used, but using only two types ofstained particles can give results that are overlyoptimistic. This is because a mixture of twodifferently stained beads has “built-in” doubletdetection. For example, if two different stainedbeads with primary fluorescence in fluores-cence channels 1 and 2, respectively, are mea-sured together (coincident in time), the eventis easily discriminated in a dot plot of channel1 versus channel 2 fluorescence. This coinci-dent event appears as doubly stained with sig-nal in both fluorescence channels 1 and 2. Asort gate that was set only for a singly stainedbead (i.e., in channel 1 only) would excludethese coincidences. If the real sample to besorted has unstained cells that produce unde-tectable coincidences with stained cells, thesort electronics will either (1) detect the coin-cidence, abort the event, and reduce yield; or(2) miss the coincidence, sort the coincidence,and give lower purity. If one of the beads is un-stained, however, it is not possible to detect a


1.3.20


coincidence in a dot plot of channel 1 versuschannel 2.

Standardization with a Particle in theAnalysis Sample

Adding the particle used for standardiza-tion or control to the sample to be analyzedgives an extra level of confidence to a flow cy-tometric analysis. Fluorescent beads are gener-ally used for “standardization in tube” methods(Horan and Loken, 1985; Horan et al., 1990).The standardization or control particle must besufficiently different in some measurement pa-rameters to be distinguished from the cells inthe sample. For example, the particles can bechosen to have lower forward scatter but muchhigher fluorescence than the cells in the sam-ple. If a known number of particles is addedto a known volume of sample, one can alsouse the particles to measure the concentrationof cells (cells per volume) in the sample (seeParticle Concentration). One commercial andclinical assay kit, Leuko64, for instance, usesan internal FITC bead control for assay valueassignment of activated granulocytes.

Particle ConcentrationParticle concentration (i.e., the number of

particles per unit volume) is becoming a morewidely used measurement. This is largely mo-tivated by the clinical need to measure theconcentration of CD4 cells in HIV-positiveindividuals and in AIDS patients. In this con-text, the concept of “absolute count” has beenused instead of cell concentration. The countis “absolute” in numbers per microliter of theoriginal blood sample, as calculated from theconcentration of cells (e.g., CD4 T cells) forthe sample analyzed and the known dilution ofblood used in preparing the sample. Referencesamples of known particle concentration canbe used to standardize particle concentrationmeasurements on a flow cytometer. For stan-dardizing and calibrating the absolute count, acontrolled or calibrated dilution of the originalsample must be made.

Some instruments are capable of measuringparticle concentration directly, as they mea-sure a fixed volume of sample. An alternativeapproach uses a known number, N, of refer-ence particles added to a sample. The ratio, r,of sample particle events (e.g., CD4 T cells)to reference particle events is measured in theflow cytometer. Then, the number of sampleparticles is computed from the product r ×N (Stewart and Steinkamp, 2005; Stebbingset al., 2015).

What Measurements Can BeCalibrated, and How Frequently IsCalibration Necessary?

Flow cytometry measurements that cur-rently can be calibrated in at least some com-mercially available instruments are particle di-ameter or volume, numbers of fluorochromes(or MESF, ERF or ABD) per particle, anti-body binding capacity/antibodies bound percell (ABC), and particle concentration (e.g.,particles per microliter). Calibration need notbe a daily practice, but the instrument mustbe quality controlled daily prior to calibration.Monitoring measurements with control mate-rial ensure that the instrument is still in cal-ibration as long as the measurements do notexceed acceptance limits determined by theapplication. If controls are out of range, it willbe necessary to recalibrate. If the instrumentis changed or serviced, it is usually advisableto recalibrate.

CHARACTERIZING PARTICLESFOR CALIBRATION ANDCONTROL OF A FLOWCYTOMETER

This section gives suggestions for how toassign fluorescence values to test beads (seeCalibrating Particle Fluorescence in MESFand ERF) and for assignment and use of in-herent fluorescence CV of a particle (see De-termining Inherent Fluorescence CV of a DimParticle Sample and see Measuring Signal toNoise from Dim Particles).

Calibrating Particle Fluorescencein MESF/ERF

Assigning intensity values to fluorescentparticles

Relatively bright beads or other particlesof known concentration can be measuredin terms of molecules of equivalent solublefluorochrome (MESF) and equivalent num-ber of reference fluorophores (ERF) with aspectrofluorometer using a solution of fluo-rochrome as reference (Brown et al., 1986;Schwartz and Fernandez-Repollet, 1993). Thespectrofluorometer is adjusted for the excita-tion and emission of the fluorochrome to bemeasured. Depending on how close the fluo-rescence excitation and emission wavelengthsare, it may be necessary to use a band-pass fil-ter in the excitation light path and a high-passor band-pass filter in the emission path to ade-quately reduce light scatter from the particles.The signal from a reference concentration of


1.3.21


fluorochrome is measured to calibrate the re-sponses from a spectrofluorometer, and then asuspension of the particles to be calibrated ismeasured under exactly the same conditions asthe fluorochrome solution. The fluorescence ofthe particle suspension is expressed in terms ofequivalent molecules of fluorochrome by com-paring it with the reference fluorochrome solu-tion. The particle concentration (particles/ml)is determined, correcting for doublets or ag-gregates if necessary. The MESF or ERF perparticle is calculated as the fluorochrome con-centration equivalent of the bead suspensiondivided by the particle concentration.

The U.S. National Institute of Standardsand Technology (NIST) has published a se-ries of papers (Gaigalas et al., 2001; Schwartzet al., 2002; Wang et al., 2002; Wang andGaigalas, 2011; Wang et al., 2016a) detail-ing the fundamental scientific basis and refer-ence methods for assigning MESF or ERF val-ues to particles. NIST has recently developeda standard fluorescein solution kit, StandardReference Material 1934, that includes flu-orescein, Nile Red, coumarin 30, and al-lophycocyanin for ERF value assignment.Compared to the use of a commercial spec-trofluorometer for fluorescence value assign-ment, a spectral response–calibrated fluorome-ter equipped with laser excitations such as 405nm, 488 nm, and 633 nm, commonly used inflow cytometers, and a CCD detector, is usedby NIST to perform ERF value assignment ofcalibration particles. The use of SRM 1934establishes the traceability of the ERF valueassignment and ultimately enables the stan-dardization of the fluorescence intensity scaleof flow cytometers in quantitative ERF units.

At low-particle MESF or ERF, the spec-trofluorometer may become inaccurate or lacksensitivity because of the measurement per-formed in suspension. If the log response forthe fluorescence channel of the flow cytome-ter has been carefully calibrated [see Mea-surement Response and Logarithmic Calibra-tion; also see UNIT 1.4 (Wood 2009)], then thebrighter particles can be used to calibrate theupper range of the fluorescence channel inMESF per linear fluorescence unit on the his-togram. Alternatively, the same process can bedone using linear amplification. Dimmer parti-cles can now be assayed on the calibrated flowcytometer and assigned MESF or ERF values.

A different approach to establishing inten-sity values is essentially arbitrary assignmentof values to a reference lot of beads, and us-ing the reference lot to assign values to future

lots of beads. BD Biosciences has used thisapproach for Assigned BD units (ABD units)of fluorescence intensity to the CS&T beadsused in their instrument performance charac-terization and tracking system. The creationof ABD units was necessary at the time dueto the absence of traceable fluorescence stan-dards for the many fluorophores that are in rou-tine use. Hopefully, the concept of ERF willallow a universal, traceable fluorescence inten-sity unit. When ERF standards are available,alternative fluorescence intensity units such asMESF and ABD can be cross calibrated toERF.

Assigning relative intensity valuesto hard-dyed beads

A flow cytometer can also be used to mea-sure and assign accurate relative fluorescenceintensity values to hard-dyed particles suchas Rainbow beads from Spherotech. For ex-ample, a flow cytometer with calibrated log-arithmic or linear amplifier could be used tomeasure fluorescence from each peak in a flu-orescence histogram for a mixture of particles.Figure 1.3.5C shows data for Spherotech Rain-bow beads.

Using hard-dyed particles as asecondary calibrator on one flowcytometer

A mixture of stable, hard-dyed particleswith a range of intensity levels and a sampleof fluorochrome-labeled particles with knownMESF values are used to calibrate a flow cy-tometer as follows:

1. Calibrate or verify calibration of theelectronic response of the data acquisi-tion electronics. Alternatively, a mixture offluorochrome-labeled beads (e.g., labeled withFITC) of varying, known MESF can be usedto calibrate the intensity scale (Schwartz et al.,1996).

2. Adjust the PMT voltage so thefluorochrome-labeled bead(s) of known MESFis in an appropriate histogram channel(s). Thiscalibrates the histogram scale in MESF.

3. Run the hard-dyed bead mixture atthe same PMT voltage as the fluorochrome-labeled beads.

4. Use the histogram calibrated in MESFto assign MESF values to each of the hard-dyed bead populations. The hard-dyed beadsare now a calibrator or “secondary standard”for this instrument and only this instrument.On subsequent days and at other PMT voltagesettings the hard-dyed beads can be used tocalibrate the fluorescence histogram in MESF.


1.3.22


Cautions using hard-dyed particles asMESF calibrators

Because hard-dyed particles do not have ex-actly the same excitation and emission spectraas a fluorochrome used to stain cells, they willnot necessarily have the same fluorescence in-tensity relative to that fluorochrome on anotherinstrument. Caution and skepticism should beused in trying to assign to the hard-dyed beadsa “global” MESF value that is valid for cali-bration in terms of MESF on all instruments(Schwartz et al., 1996). For further informa-tion, see Figures 1.3.1, 1.3.2, and 1.3.4, as wellas the discussion in the section above on Com-parison of Spectrally Matched and UnmatchedFluorescent Particles.

Determining Inherent FluorescenceCV of a Dim Particle Sample

The inherent fluorescence coefficient ofvariation (CV; see Definitions, “Coefficientof variation”) of a particle sample should bedue only to variation in the amount of fluo-rochrome in the particles. Measurement limi-tations or noise in the flow cytometer broadenthe measured CV (see Steen, 1992 and Chaseet al., 1998, for more detailed discussion).The contribution to the CV from variation inillumination can be estimated by running avery bright, uniform “alignment” particle. Fordimly fluorescent particles, background noiseand photoelectron statistics become dominantcontributors to the CV. To determine the in-herent CV of a dim particle, where the to-tal CV may be �10%, one must measure thebackground noise and photoelectron statistics.This can be done by using dim light flashesfrom a light-emitting diode (LED). Dim sig-nals are created by using filters to attenuate thelight from the LED or by simply holding theLED far from the detector. The inherent CVof the dim particles is found by subtracting (inquadrature) the noise CV from the total CV(Steen, 1992).

The inherent CV of dim, hard-dyed beadscan be determined in this way, effectively cal-ibrating the particles in terms of populationCV. When the particles are subsequently an-alyzed on any flow cytometer, the noise con-tribution from the measurement can be deter-mined by the broadening of the CV. Essentiallyone works in reverse from what was explainedabove: the noise CV is determined by subtract-ing (in quadrature) the inherent particle CVfrom the total measured CV. For dim signals,this gives a measure of the fluorescence sensi-tivity in terms of conventional signal/noise.

Measuring Signal to Noise from DimParticles

In engineering, a standard definition of min-imum resolvable signal is that for which thesignal (S) and noise (N) are equal, that is, S/N= 1. In flow cytometry, it is customary to useCV rather than S/N, but the simple relationCV = N/S can be used to translate betweenthe two measures.

If the inherent CV of a dim particle is known(see Determining Inherent Fluorescence CVof a Dim Particle Sample), the system noisecan be determined. The fluorescence of thedim particle is measured on the flow cytome-ter, and the CV of the resulting distribution isdetermined. Although a range of particle in-tensities might be required to measure systemnoise accurately, a single particle can be usedto determine whether S/N at a particular MESFis above a required minimum. Noise can alsobe expressed in MESF, and the MESF level atwhich S/N = 1 or some other predeterminednumber is a measure of sensitivity.

The instrument noise determined with thedim particle sample correlates with the abilityto resolve dim particles from unstained parti-cles. The other important factor is how largea signal is produced by unstained particles orcells. A convenient measure of the response totruly nonfluorescent cells is provided by thetest pulse mode available on many flow cy-tometers. The pulse detection electronics canbe triggered by an electronically generated sig-nal to a nonfluorescence parameter such as for-ward scatter. The fluorescence channels thenmeasure the response only to background lightand other noise sources.

CONCLUDING REMARKSFlow cytometry has had rapid growth since

the mid 1980s. It has moved from a technologyplatform that only a few hundred “initiated”experts understood and could use to becomeboth a common laboratory tool and clinical di-agnostic system. To name a few applications,flow cytometry is essential for accurate mea-surement of CD4+ cell counts for ensuring thatpatients receive the appropriate antiretroviraltreatment for HIV/AIDS monitoring. Count-ing the number of viable CD34+ cells us-ing flow cytometry for reconstitution of thehematopoietic immune system of patients af-ter chemotherapy has become a gold-standardclinical practice. Multiplexed flow cytometryassays (�12 fluorescence parameters) are rou-tinely used in clinics for disease diagnosis andtherapies. This is clearly seen in the clinical


1.3.23


flow cytometric analysis of hematologic ma-lignancies. Moreover, it has also become anessential clearance tool for the production ofprotein and cell therapeutics. All these applica-tions essentially require that comparable andreproducible results can be generated usingdifferent flow cytometer platforms at differentlocations and times. The consistency of themeasurements can only be accomplished withthe use of multiple controls, e.g., particles forinstrument standardization and calibration andbiological cell reference materials in the mea-surement process. Without proper use of theseprocess controls, the value of this information-rich instrument will not be realized nor willfurther advancement be made into new bio-logical and clinical applications.

DISCLAIMERCertain commercial equipment, instru-

ments, and materials are identified in this paperto adequately specify the experimental proce-dure. In no case does such identification implyrecommendation or endorsement by the Na-tional Institute of Standards and Technology,nor does it imply that the materials or equip-ment are necessarily the best available for thepurpose. Definitions provided in this unit arewell accepted in the field of flow cytometry,and a few may be slightly different from thoseprovided by the International Vocabulary ofMetrology (VIM) under the International Bu-reau of Weights and Measures (BIPM).

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