clinical immunophenotyping by flow cytometry

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CLINICAL IMMUNOPHENOTYPING BY FLOW CYTOMETRY

Carleton C. Stewart, PhD and Sigrid J. Stewart

Laboratory of Flow Cytometry,

Roswell Park Cancer Institute, Buffalo, NY

..At a glance .1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Data Acquisition . . . . . . . . . . . . . . . . . . . . . . 5

Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . 6

Three Color Immunophenotyping: . . . . . . . 10

The Clinical Immunophenotyping Report . . 12

Introduction

Immunophenotyping is the term appliedto the identification of cells using antibodies to

antigens expressed by these cells. Theseantigens are actually functional membraneproteins involved in cell communication,adhesion or metabolism. Antibodies bind to three dimensional molecular structures onthese antigens called epitopes and eachantigen contains several hundred differentepitopes. A monoclonal antibody is specific fora single epitope, while polyclonal antibodies,are actually the natural pool of several hundred monoclonal antibodies producedwithin the animal, each one binding to itsunique epitope. Immunophenotyping usingflow cytometry has become the method ofchoice in identifying and sorting cells withincomplex populations. Applications of thistechnology have occurred in both basicresearch and clinical laboratories. TheNational Committee for Clinical LaboratoryStandards has prepared guidelines for FlowCytometry that describe in detail the currentrecommendations for processing clinicalsamples (H42 Landay, 1992 and H43,Borowitz, 1993).

Portions of this text are taken verbatim from: Methods in Cell1

Biology, Volume 41, Flow Cytometry, Second Edition Part A, edited byZbigniew Darzynkiewicz, J. Paul Robinson, and Harry A. Crissman, pp.40 -78, 1994 and from Methods in Molecular Biology, Volume 45,Monoclonal Antibody Protocols, edited by William C. Davis. pp. 129 -147, Humana Press, 1995. These references may be consulted formore information not presented here.

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Clinical Immunophenotyping by Flow Cytometry C. Stewart & S. Stewart


Methods

There are three basic methods forstaining cells. These three methods can becombined to stain cells with multiple antibodiesusing different colored fluorochromes. Werecommend receiving all samples at roomtemperature and processing them on ice in dimambient light until fixed.

Basic Method 1: Primary antibody followedby fluorochrome conjugated secondantibody.

1. Add 50 µl of cells (5-20 x10 /ml) to a126

x 75 mm tube.

2. Add 10 µl IgG fraction (2mg/ml) derivedfrom the same species as the secondantibody to the cells and incubate 10min. This IgG will block Fc receptorsand non-specific binding.

3. Add appropriate amount of primaryantibody to cells and incubate 15 min.

4. Add 3.5 ml PBS, centrifuge cells at 1500x g for 15 min., decant supernatant, blotexcess buffer on an absorbent toweland resuspend cells in residual buffer. This process will subsequently be calleda wash.

5 If erythrocyte lysis is desired, substitutethe lysis reagent for PBS in step 4.

6. Add the appropriate amount offluorochrome conjugated secondantibody, incubate 15 min. and wash.

7. Resuspend cells in 0.5 ml of 2%ultrapure formaldehyde. [If it is desiredto analyze viable cells, substitute thedesired resuspension medium].

Basic Method 2: Biotinylated primary

antibody followed by fluorochromeconjugated Avidin.

1. Add 50 µl of cells (5-20x10 /ml) to a12 x6

75 mm tube.

2. Add 10 µl IgG fraction (2mg/ml) derivedfrom the same species as the primarybiotinylated antibody to the cells andincubate 10 min. This IgG will block Fcreceptors and non-specific binding.

3. Add appropriate amount of biotinylatedprimary antibody to cells and incubate15 min. and wash. If erythrocyte lysis isdesired, substitute the lysis reagent forPBS.

4. Add the appropriate amount offluorochrome conjugated streptavidin,incubate 15 min. and wash.

5. Resuspend cells in 0.5 ml 2% ultrapureformaldehyde. [If it is desired to analyzeviable cells, substitute the desiredresuspension medium].

Basic Method 3: Directly ConjugatedPrimary Antibody

1. Add 50 µl of cells (5-20 x 10 /ml) to a126

x 75 mm tube.

2. Add 10 µl IgG fraction (2 mg/ml) derivedfrom the same species as theconjugated antibody to the cells andincubate 10 min. This IgG will block Fcreceptors and non-specific binding.

3. Add appropriate amount of conjugatedantibody to cells and incubate 15 min.and wash. If erythrocyte lysis isdesired, substitute the lysis reagent forPBS then wash again with PBS.

4. Resuspend cells in 0.5 ml 2% ultrapure



formaldehyde. [If it is desired to analyze It is possible to evaluate the extent ofviable cells, substitute the desired the inappropriate binding component using anresuspension medium]. isotype control. Each antibody should be

Multicolor Immunophenotyping For Up ToFour Antibodies Simultaneouslyusing Basic Method 3.

1. Add 50 µl of cells (5-20 x 10 /ml) to a126

x 75 mm tube.

2. Add 10 µl IgG fraction (2 mg/ml) derivedfrom the same species as the antibodyto the cells and incubate 10 min. ThisIgG will block Fc receptors and non-specific binding.

3. Add appropriate amount of each directlyconjugated, properly titered antibody tocells and incubate 15 min. and wash. Iferythrocyte lysis is desired, substitutethe lysis reagent for PBS then washagain with PBS.

4. Resuspend cells in 0.5 ml 2% ultrapureformaldehyde. [If it is desired to analyzeviable cells, substitute the desiredresuspension medium].

Precautions:

Antibodies interact with cells in threeways: first by binding specifically to theirepitope, secondly by binding to Fc receptorsand thirdly by binding non-specifically. OftenFc receptor binding and nonspecific bindingare referred to as nonspecific binding todistinguish between specific binding of theF(ab) portion of the antibody to its epitope. Actually Fc receptor binding of IgG’s to cell Fcreceptors is very specific and it, like epitopebinding, is saturable. In contrast, nonspecificbinding exhibits lower affinity binding and it isnot saturable.

titered prior to use so that the correct amountto optimally stain the cells is used. Ourgeneral practice is to have the optimal amountin 10 µl. Commercially available antibodiesthat have already been titered may be diluteddifferently by the supplier so that more or lessthan 10 µl is required. In these cases, use thesuppliers recommended amount (unless it isfound to be incorrect by titering). This controlconsists of the same cells, incubated with amyeloma protein having no epitope specificity, but having the same isotype and subclass asthe primary antibody. This myeloma protein ispresumed to bind to cells in the same wayimmunoglobulins bind excluding epitopebinding. The percentage of positive cellsrevealed by the isotype control is then used toset markers for analysis (described in detailbelow).

The autofluorescence control shouldalso be analyzed. It contains no antibodiesand is otherwise processed the same way asthe other samples. This control provides abaseline to determine the minimumfluorescence. Ideally, the isotype control andautofluorescence control would give identicalresults and to the extent they differ is indicativeof a problem.

All second antibodies should be F(ab’)2

fragments to eliminate Fc binding. Thesereagents are currently available againstvirtually all mammalian species andconjugated with all the common fluorochromes.A whole blood (or bone marrow) lysing methodshould be used to remove erythrocytes andthere are several reagents commerciallyavailable. While all of them work with blood,some are better than others for lysing bonemarrow erythroid cells. Cells obtained fromother sources such as lymph nodes or pluralfluid generally do not need to be lysed.



Lysing reagent: staining protocol, it is equally important to stain

4.13 g Ammonium Chloride (NH Cl) -- a directly conjugated isotype control4

Sigma A-5666 immunoglobulin. If the isotype control binds to

0.5 g Potassium Bicarbonate (KHCO ) - primary/secondary combination, the block has3

- Sigma P-4913 not been effective and the data will be

0.0185 g Tetra Sodium EDTA -- SigmaED4SS Dead cells can bind antibodies

Measure carefully the above chemicals. percentage of positive cells for somePour into a flask (must be at least a 500 ml antibodies. It is important to account for theseflask) to which a stir bar has been added. Add dead cells because antibodies bind to them500 ml double distilled water. Place on stir nonspecifically.plate and stir until the powder is dissolved.Do not pH. Use at room temperature. EMA will photolabel the DNA of

This reagent must be prepared daily (white light). These cells can then be fixedbecause the dissolved CO in water combines and the EMA, if incorporated, will remain2

with NH Cl to form the carbonate (NH ) CO bound to the DNA. The procedure follows:4 4 3 2

that is ineffective in lysing erythrocytes. Werecommend weighing the reagents and storing 1. Add 50 µl of cells to a12 x 75 mm tube,them as packets. The dry reagents are wash. If erythrocyte lysis is desired, lysedissolved in water as required. Quantities for cells with lysing reagent centrifuge,daily use can be made by scaling the listed decant and blot tube.reagents up or down.

Paraformaldehyde when dissolved in 18 inches beneath a fluorescent light foraqueous solution becomes equivalent to 10 mins. (Lay flat in white pan). Wash.ultrapure formaldehyde. This product isavailable as a 10% solution from Polysciences 3. Resuspend cells in 0.5 ml of 2% ultrapureInc. Do not use any other kind of formaldehyde. [If it is desired toformaldehyde for fixing cells because their analyze viable cells, substitute theautofluorescence can markedly increase. desired resuspension medium].

Any of the three basic methods can be EMA may stain viable cells slightlycombined to stain cells with antibodies using causing some interference with setting thedifferent colored fluorochromes. There are two marker if combined with other antibodies. Werules that must be followed to prevent recommend using a separate tube for viabilityartefactual data. Method 1, if used, mustalways be performed first. The secondantibody must be blocked by IgG from thesame species as the first antibody used sothat it will not bind any subsequent primaryantibodies. When method 1 is included in the

cells using method 1 followed by the block and

any of the cells stained with the

artifactual.

nonspecifically leading to an erroneous

nonviable cells when exposed to visible light

2. Add 8 µl of EMA (5 µg/ml) and put the tube

assessment and backgating to locate the liveand dead cells in the FSC vs SSC view. Thegate on viable cells is then applied to theanalysis of subsequent antibody stainedsamples.

Reference: Reidy, M. C., Muirhead, K. A.,



Figure 1: Color Compensation for Three Color Data Acquisition

Jensen, C. P., and Stewart, C. C. The Use Of Up to four fluorescence antibodies canA Photolabeling Technique To Identify be combined because the colors can beNonviable Cells In Fixed Homogenous Or separately measured, but we will consider onlyHeterologous Cell Populations. Cytometry three colors. To accomplish color separation,12:133-139, 1991 spectral filters and dichroic mirrors are used

Data Acquisition

We first need to define parameters. This is a generic term used to describe theentities we will measure. There are threecommon kinds of parameters: forward scatter(FSC) and side scatter, (SSC), which is alsocalled orthogonal or 90 scatter, because it iso

measured at 90 degrees to the laser beam andfluorescence. The FSC is proportional to thecross sectional area of the particle andprovides a measurement of size, while SSC issensitive to cell granularity. The intensity offluorescence is also measured orthogonal tothe cell stream.

The five commonly used fluorochromesthat are conjugated to second antibodies,avidin or to monoclonal antibodies arefluorescein isothiocyanate (FITC),phycoerythrin (PE), aphycoerythrin-texasred tandemcomplex(PETR), aphycoerythrin-cyanin5 tandemcomplex(PECy5)andperdinin chlorophil-P (PerCP). Thesefluorochromes can each be excited at 488 nMand the fluorescence emission from FITC =530 nM, from PE = 575 nM, from PETR = 613nM and from both PECy5 and PerCP = 670nM.

that reflect or transmit specific colors. Because the fluorochrome's emissionwavelengths can be very broad, it is alsonecessary to remove unwanted overlappingcolors by electronic compensation. This isaccomplished by appropriately adjusting theinstrument's compensation circuits. Toperform this task it is necessary to usecompensation standards. These standardscan be purchased if only FITC or PEfluorochromes are to be used, but if the othercolors are also desired, compensationstandards must be prepared by the user.

To prepare these standards for up tothree separate colors, 50 µl of blood is placedin four separate tubes. Only cells are added tothe first tube, and this is used to adjust thehigh voltage. To the second tube add PE-CD4, to the third tube add FITC-CD45, and tothe last tube add PECy5-CD45 (a PETR orPerCP antibody could be used instread). After

15 minutesincubation,on ice,wash, lyseerythrocytes and fixthe cells. Only cellsstainedwith theantibodiesfor thedesired

fluorochromes need to be prepared and usedfor compensation.

A properly compensated instrument forfour color data acquisition is shown in Figure1. All five tubes are superimposed in this



TABLE 1: CORRELATED LIST MODE FILEValues for each parameter

Cell P1 P2 P3 PnC1 122 84 20 440C2 424 92 20 460C3 86 83 562 22C4 101 98 487 33Cn 330 79 30 452

Figure 2: Setting Markers in a UnivariateHistogram

single figure as are plottedif the cells had against thebeen mixed measuredtogether in one values (on thetube after x axis orstaining them abscissa).with the We need anantibodies. objective wayThe FSC vs to tell positiveSSC is from negativedisplayed and and there area lymphocyte two populargate (R1) is methods. Theproduced. first is toUsing the high voltage adjustments, the acquire data from cells that have beenunstained cells in tube one are centered in the procedurally treated the same as thoselower left corner of each bivariate plot. Taking receiving antibody but do not actually receiveeach tube beginning with the first, eachcompensation is adjusted so that cells labeledwith the appropriate fluorochrome/antibody arein the correct quadrant. The instrument is nowready to acquire data derived from patientspecimens.

Data Analysis

All flow cytometers collect data in thesame way using a process called LIST MODE. A standard list mode file includes a headerfollowed by a list of data in the order collected. The values that are stored in a list mode fileare the voltages from the detectors for eachparameter (P1, P2, P3,... Pn) after they havebeen electronically processed. As shown inTable 1, the data in a list mode file, consists ofthe voltage for each parameter for cell #1followed by cell #2 and so forth until the lastcell, n, is measured. If six parameters aremeasured for 10,000 cells, the file will contain60,000 numbers in the order they wereacquired.

The univariate histogram shown inFigure 2 is the simplest of all ways to displaydata. It is a frequency distribution where thenumber of events (on the y axis or ordinate)

any antibody. As shown in Figure 2A, thesecells provide a minimum fluorescence valueand a marker can be created above whichcells are called positive. This is called anautofluorescence distribution and the marker isset so that usually, no more than 0.5 to 2% of



Figure 3: Gating Histogram

Figure 4: Bivariate Histogram

cells are above it. The second method is to we restrict the values of interest to a regionuse an isotype control immunoglobulin asdiscussed above. As shown in Figure 2B the

cells may be somewhat brighter than theautofluorescence because of non-specific andFc receptor binding. Blocking can reduce thiscomponent significantly.

Again, the user must decide whatmarker position will be used to distinguishpositive from negative cells and a positionbetween 0.5% to 2% of cells above the markeris customary.

Using the univariate histogram, we cananalyze a list mode data file. In Figure 3A, wehave defined two regions: R1 for smallparticles and R2 for large particles in the FSChistogram. We could find out the percentageof cells that are positive in R1 or R2 becausethe data analysis program would sequencethrough every FSC value in the list betweenthe selected values. We could then displaythem in the fluorescence histograms for allcells (B) or for those in R1 (C) or for those inR2 (D). We term this process gating because

that we wish to know about. Only cells thatmeet the region’s criteria are evaluated.

As shown in Figure 3C, there aretwo distinct frequency distributionssimilar in position to the same negativecells and all the bright cells in Figure3B.. The ones on the left of the markerexhibit low fluorescence while the oneson the right exhibit high fluorescence soit is easy to tell the cells that arepositive from those that are negative. InD, some of the negative cells are alsofound, but so is the dimmer positivepopulation.

When one univariate histogram isplotted on the x axis and a secondunivariate histogram is plotted on the yaxis as shown in Figure 4, a bivariatehistogram results. If the two univariatehistograms from Figure 2 are eachprojected into the area of overlap, the

values in common of FSC and FL1 produceclusters of the cells represented by the circles.



Figure 6: Gating a Selected Population

Figure 5: Setting Quadrant Markers

These clusters can be of any size and shapedepending upon the relationship between thetwo properties that were measured.

In order to make analysis easier whenbivariate plots of data are used, quadrantmarkers are used. These markers are setalong the x and y axis, as shown in Figure 5,to produce quadrants so that all combinationsof + and - cells can be displayed. For twoantibodies, there are four possiblecombinations, +-, ++, -+ and --.

We cannow combinegating andquadrantanalysis toanalyze 4parameter flowcytometry dataconsisting ofFSC, SSC andtwo colors offluorescence.

As shown in Figure 6, a region (R1) can bedrawn in the FSC vs SSC display (Figure 6A)to include only the small cells with low SSC. Inthis example blood was stained with CD4 andCD8 and the small cells are lymphocytes. Thisregion is used as a gate to display theirfluorescence in a second bivariate display. Using an isotype control, the quadrant markersare set in Figure 6B so that 1% of cells areabove them. In Figure 6C, CD4 and CD8 areshown and the percentage of positive events ineach category can be obtained.

One problem associated with gatingcells on the basis of size is an assumption thatthere are no cells of interest outside theselected region. In the above example,lymphocytes were selected but are all thelymphocytes in the region selected andsecondly are all the cells in the region selectedlymphocytes? For example, the cells coloredblue (CD4+) that are below the major bluecluster are not lymphocytes; they aremonocytes in the lymphogated region. It ispossible to answer the two questions quiteobjectively by using CD45 that labels allleukocytes and CD14 that labels onlymonocytes.

The purity of the region in the FSC vsSSC display for lymphocytes and theproportion of them within the gate can beevaluated. To accomplish this, all cells are



Figure 7: Quality of Gate

acquired and theirCD45 vs CD14distributionsdisplayed as shownin Figure 7 forblood. A region R2containing all viablelymphocytes isdrawn around theCD45 bright CD14-

cells. This region isapplied as a gate tothe FSC vs SSCdisplay and thelocation of alllymphocytes isfound. A secondregion, R1, is drawnaround thelymphocytes andtheir percentagerecorded.

If this region

is drawn largeenough, it willcontain undesiredcells so lymphocytepurity is reduced. To evaluate thepurity, this gate isapplied to the CD45CD14 bivariate display and the percentage ofcells in R2 representing the lymphocytes isrecorded. As shown in the lower right display,green dots are lymphocytes and red dots arecontaminating cells, so the purity is only92.5%. If the purity of the lymphogate is low, itmay be improved by reducing the size of Rl.This will cause a loss of lymphocytes whileimproving purity. As long as the loss of only asmall proportion of lymphocytes occursconcomitantly with a significant improvement inpurity, the procedure can be used to optimizethe lymphogate. This gate, once established,is then applied to the analysis of allsubsequent samples. This same strategy can

be applied to the monocyte and granulocyteregions.



Figure 9: Combining Quadrant Markers

Figure 8: Displaying Three Parameters

Three ColorImmunophenotyping:

When a third color is introduced, thereare three views for displaying all thefluorescence data, shown in Figure 8. Theyare FL1 vs FL2, FL1 vs FL3, and FL2 vs FL3. Since there are three plots, we need to be ableto visualize the position of cells in one viewwith the same cells in another. This can beaccomplished by using color.

A population of cells in FL1 vs FL2 hasbeen colored red. These cells could beanywhere along the projection of FL2 onto theFL3 axis or FL1 on to the FL3 axis.

As shown in Figure 8A, a singlepopulation is seen, but in Figure 8B, twodistinct populations, one that is FL3 and one-

that is FL3 are found. Since we colored the+

cells red, both populations resolved in FL3would also be red, even though one of them isFL2 FL3 and the other is FL2 FL3 . This+ - + +

situation can be corrected by coloring theFL2 FL3 population green. In Figure 8C we+ +

are able to see that both populations are alsoFL1 .+

For the automatic analysis of largenumbers of specimens, this method of coloringis time consuming. A more streamlinedstrategy can be developed using the quadrantmarkers shown in Figure 9. Each quadrant isassigned a region, R1-R8. Only two of the

three bivariate displays need to havequadrants because that will produce all thepossible combinations for the three fluorescentantibodies. Any two can be used and in thisexample FL1 vs FL2 and FL3 vs FL2 waschosen. Based on whether any given cell ispositive or negative for any given antibody,there are eight possible binary combinations,

shown in Table 2.

Cells with any givenphenotype can, therefore, beexplicitly defined by combining theregions using boolean algebra. Cells that are not stained can onlybe found in R3 and R7. We cannotuse R3 alone because they couldalso be found in either R7 or R8. Similarly, we cannot use R7 alonebecause they could be found in

either R3 or R4. Thus, R3 and R7 explicitlydefines the only location cells negative for allthree antibodies can be found. This samelogic is used for all the other seven possiblecombinations. The next step is to assign each of thesecombinations specific colors. Each color willcorrespond to a specific positive and negative



Figure 10: Normal T Cells

TABLE 2:BINARY COMBINATIONS OF QUADRANT MARKERS.

FL1 FL2 FL3- - - R3 and R7+ - - R4 and R7- + - R1 and R5+ + - R2 and R5- - + R3 and R8+ - + R4 and R8- + + R1 and R6+ + + R2 and R6

combination of antibodies it was assigned molecules (cellular differentiation antigens) onregardless of the antibody combinations used. the cell’s membrane that bind the specificEach combination of different antibodies will antibodies for them are highly regulated. Thisproduce its own unique colored pattern as is not true for malignant cells which exhibitshown for the alteredpanel FITC-CD3, expression ofPE-CD4, and TC- some, but notCD8 in Figure 10.

In Figure10, a lymphogatewas firstestablished usingthe method shownin Figure 7 andusing the binarycombinationsshown in Table 2. FITC-CD3 is FL1,PE-CD4 is FL2 andPerCP-CD8 is FL3. Compare this withFigure 6 where theCD4 dim cells (blue in Figure 6), are actuallyCD3 negative (green in Figure 10).

For non-malignant cells, these patternswill always be the same because the

all, antigens. Each person’sleukemia orlymphoma isunique and notwo are exactlyalike. This doesnot mean,however, thatthere aren’tgeneralcategories intowhich differenthematopoieticmalignanciesfall. These

categories fall into lineage assignments thatshare some properties in common with normalcells of the same differentiation stage. Anexample for a T-lineage acute lymphocyticleukemia is shown in Figure 11. Most T-ALL



Figure 11: T Cell Acute Lymphocytic Leukemia

cells are colored red but in comparison with processing, data acquisition and analysis andFigure 10, the normal T-cell pattern can alsobe seen.

The Clinical ImmunophenotypingReport

There are two kinds of clinical tests currentlyrecognized by the medical reimbursementcommunity. The first is called a clinicallaboratory test (CLT). The second is called aclinical anatomical test (CAT). The majordistinction between these two tests is thesecond requires an interpretation by aprofessional, while the first provides theclinician with numerical values only. FlowCytometry tests are considered clinicalanatomical tests because an interpretation isoften, but not always, required. This test canbe divided into two additional components: atechnical component that consists of sample

the professional component which consists ofa written interpretation of the data. Theprofessional interpretation is performed by acertified individual that does not have to be aphysician. The degree of interpretationdepends on the test. For example, an HIV

screen that consists only of a CD3 CD4+ +

count requires no interpretation and is like aclinical laboratory test. In contrast, a leukemiascreen will require the use of severalantibodies in combination to produce patternsthat need to be interpreted. In this case theuse of percentages in the report is of lessvalue for the clinician who would rather knowthe diagnosis and classification of theleukemia or lymphoma, i.e. the interpretation. Suggested antibody combinations are shownin Table 3.



TABLE 3. SUGGESTED ANTIBODY COMBINATIONS FOR IMMUNOPHENOTYPING

Immunodeficiency Lymphocytic Malignancy Myeloid MalignancyFL-CD45 FL-CD45 FL-CD45PE-CD14 PE-CD14 PE-CD14PP-HLADr PP-HLADr PP-HLADr

FL-CD3 FL-CD3 FL-CD3PE-CD4 PE-CD4 PE-CD4PP-CD8 PP-CD8 PP-CD8FL-CD16 FL-CD5 FL-CD33PE-CD56 PE-CD19 PE-CD13PP-CD3 TC-CD10 PP-HLADr

FL-CD20 FL-CD16PE-CD11c PE-CD32TC-CD22 TC-CD64

FL-KAPPA FL-CD11bPE-LAMBDA PE-CD13

TC-CD19 TC-CD33FL-IgD FL-CD38PE-IgM PE-CD34

TC-CD19 PP-HLADrFL-CD5 FL-CD15

PE-CD34 PE-CD34TC-CD19 TC-CD56

FL = FITC, PE = phycoerythrin, TC = PE-Cy5 tandem, PP = PerCP

Isotype Control containing FL-IgG1, PE-IgG2b and TC-IgG2a, autofluorescence sample and EMAsample included in every screen

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clinical immunophenotyping by flow cytometry

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