(PDF) leu2012122a

来自 : 发布时间：2024-12-23

SPECIAL REPORTEuroFlow standardization of flow cytometer instrument settingsand immunophenotyping protocolsT Kalina1,11, J Flores-Montero2,11, VHJ van der Velden3, M Martin-Ayuso4,SBo¨ ttcher5, M Ritgen5, J Almeida2, L Lhermitte6, V Asnafi6,A Mendonc¸a7, R de Tute8, M Cullen8, L Sedek9, MB Vidriales10,JJPe´ rez10, JG te Marvelde3, E Mejstrikova1, O Hrusak1, T Szczepan´ski9,JJM van Dongen3and A Orfao2on behalf of the EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708)The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis andclassification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory proceduresand antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection ofoptimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures(SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for theevaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentallyversus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility basedon strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments andthe analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first timelaboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal andmalignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a singledatabase.Leukemia (2012) 26, 1986–2010; doi:10.1038/leu.2012.122Keywords: flow cytometry; standardization; compensation; software; fluorochromes; immunophenotypingINTRODUCTIONImmunophenotyping is currently one of the fundamental pillarsfor the diagnosis and classification of leukemia and lymphoma.1Inthe last two decades multiparameter flow cytometry has becomethe preferred method to assess the immunophenotypic featuresof cells present in peripheral blood (PB), bone marrow (BM), lymphnode (LN) biopsy specimens, cerebrospinal fluid (CSF) andother types of samples suspected of containing neoplastichematopoietic cells.1,2During the first part of this period, the listof clinically useful antibodies (Abs) has progressively increased,3–5leading to the definition of complex immunophenotypic profiles.In parallel, the number of antigens that can be assessed in a singlemeasurement has increased dramatically owing to the availabilityof new multicolor digital instruments and a greater number ofcompatible fluorochromes.6,7This has facilitated more preciseidentification and phenotypic characterization of specificpopulations of tumor cells in samples over the background ofthe coexisting residual normal leukocyte subsets.8However, thehigher complexity of the immunophenotypic approaches andpanels of reagents involved in such characterization demandedincreasing expertise for correct interpretation of the dataobtained. As a consequence, disturbing levels of subjectivityhave been introduced, depending on the experience andknowledge of individual experts and the variable panels ofreagents applied in different clinical diagnostic laboratories.In order to decrease such variability and subjectivity, consensusrecommendations and guidelines have been produced by severalexpert groups.3,5,9–14These documents have had a wide impactand they have been followed by many centers around the world,but they have been only partially successful for several reasons.First, they focus on lists of Markers without specificrecommendations about reagent clones, fluorochromeconjugates or optimally designed antibody combinations in thepanel. Second, they fail to provide robust protocols for theselection of the most appropriate (i) combinations offluorochromes and fluorochrome-conjugated reagents in apanel, (ii) sample preparation techniques, (iii) standard operatingprocedures (SOPs) to establish instrument settings prior to themeasurements and (iv) the most adequate strategies for dataanalysis. Most importantly, the so far proposed sets of markershave never been prospectively evaluated.In 2006 the EU-supported EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708) started a project aimed at the prospectivedesign and evaluation of panels of antibodies for the diagnosis1Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University (DPH/O), Prague, Czech Republic;2Cancer Research Center (IBMCC-CSIC),Department of Medicine and Cytometry Service, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain;3Department ofImmunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands;4Cytognos SL, Salamanca, Spain;5Second Department of Medicine, UniversityHospital of Schleswig Holstein, Campus Kiel (UNIKIEL), Kiel, Germany;6Department of Hematology, Hoˆpital Necker and UMR CNRS 8147, University of Paris Descartes (AP-HP),Paris, France;7Department of Hematology, Portuguese Institute of Oncology (IPOLFG), Lisbon, Portugal;8Haematological Malignancy Diagnostic Service (HMDS), University ofLeeds (UNIVLEEDS), Leeds, UK;9Department of Pediatric Hematology and Oncology, Medical University of Silesia (SUM), Zabrze, Poland and10Department of Hematology,University Hospital Salamanca (HUS) and IBSAL, Salamanca, Spain. Correspondence: Professor JJM van Dongen, Department of Immunology, Erasmus MC, University MedicalCenter Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.E-mail: j.j.m.vandongen@erasmusmc.nl or b.vanbodegom@erasmusmc.nl11Shared first authorship, because TK and JFM have equally contributed to this manuscript.Received 12 January 2012; accepted 14 February 2012Leukemia (2012) 26, 1986– 2010 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12www.nature.com/leu and classification of the most frequent subtypes of leukemias andlymphomas in which immunophenotyping has proven to berelevant. The major objectives were (i) to provide comprehensivemulticolor combinations of fluorochrome-conjugated antibodiesaimed at answering those medical questions for which multicolorflow cytometry immunophenotyping is indicated, (ii) to prospec-tively evaluate their performance in multiple diagnostic labora-tories and (iii) to optimize the reagent panel whenever required.For this purpose, proven reproducibility in multiple diagnosticlaboratories was mandatory. Therefore, the definition of optimalantibody panels also required an effort in the selection of the mostappropriate combination of compatible fluorochromes, the designand evaluation of adequate SOPs for instrument setup, fluores-cence compensation and sample preparation and elaboration ofadequate software tools for the overall evaluation of thephenotypic profiles obtained.In the first five sections of this paper, we provide detailedinformation about the selection of the most appropriatecombination of fluorochromes for 8-color panels, the protocolsrecommended for instrument settings, fluorochrome compensa-tion and sample preparation, together with the data analysisstrategies adopted to evaluate the tested antibody reagents andpanels. In the last section, results of multicentric evaluation of thelevel of reproducibility that can be achieved by implementation ofall standardization efforts are provided. In each of the sections, weindicate what has been specifically evaluated versus adoptedbased on existing data.SECTION 1. FLUOROCHROME SELECTION FOR 8-COLORPANELSJ Flores-Montero1, T Kalina2,JJPe´rez3,SBo¨ttcher4,VHJ van der Velden5, J Almeida1, L Lhermitte6, A Mendonc¸a7,R de Tute8, M Cullen8, L Sedek9, E Mejstrikova2,JJM van Dongen5and A Orfao11USAL, Salamanca, Spain;2DPH/O, Prague, Czech Republic;3HUS, Salamanca,Spain;4UNIKIEL, Kiel, Germany;5Erasmus MC, Rotterdam, The Netherlands;6AP-HP, Paris, France;7IPOLFG, Lisbon, Portugal;8UNIVLEEDS, Leeds, UK and9SUM, Zabrze, PolandBACKGROUNDSelection of the most appropriate combination of fluorochromesis a key step in designing a multicolor immunophenotypic panel.15Usage of the new digital flow cytometers capable ofsimultaneously measuring multiple (for example, X6) differentfluorescence emissions has only recently become possible indiagnostic laboratories because of the increasing availability ofcompatible fluorochromes.16–21However, the varying spectraloverlap of such fluorochromes has also led to a higher complexityof fluorescence compensation matrices.6,22,23Fluorochrome selection largely depends on the intrinsiccharacteristics of each individual fluorescent compound, particu-larly its excitation and emission profile, its relative brightness, thespillover into other fluorescence detectors and its stability.24Theselection of the most adequate fluorochrome combination alsodepends on the specific optical configuration of the flowcytometer, that is, the number and type of laser lines itcontains, the number of detectors available for each laser andthe specific set of filters for each individual laser.7Furthermore,the aim of an antibody panel, the type of samples to be stained(that is, PB, BM versus small cell samples) and the cells containedin it also contribute to the decision on the minimum number ofreagents to be simultaneously assessed in individual tubes.25,26Finally, the availability of optimal clones of fluorochrome-conjugated antibodies also determines the selection of specificcombinations of reagents in a panel.27,28On the basis of the innovative immunophenotyping strategydesigned by the EuroFlow group in which new data merge andcalculation tools are combined for improved diagnosis andclassification of hematological malignancies, a minimum require-ment of 8-color panels for cost-effective immunophenotypingwas foreseen. Such panels should allow simultaneous usageof (i) backbone markers aimed at specific identification of thecell populations of interest and (ii) additional antibody markersdevoted to a more detailed characterization of the said cellpopulations.29In this section we review the selection of flow cytometerinstruments, their optical configuration and the set of compatiblefluorochromes, as performed during the construction and evalua-tion of the EuroFlow 8-color panels.Selection of flow cytometry instruments and their opticalconfigurationsAt the time the EuroFlow project started in March 2006, fourX8-color flow cytometry instruments from two different manu-facturers were available, with flexible and compatible opticalconfigurations (Table 1), which could potentially be used indiagnostic laboratories. The four instruments were taken intoconsideration in selecting the combinations of fluorochromes tobe used in the EuroFlow panels. All four instruments have a threelaser-line configuration, with blue (488 nm), red (633 or 635 nm)and violet (405 or 407 nm) lasers.Selection of fluorochromesA two-step approach was used by the EuroFlow group forselection of fluorochromes: (i) some fluorochromes were pre-defined without further specific testing based on previousexperience, whereas (ii) others were evaluated prior to theirselection. Accordingly, the first two positions for the blue laser line(emission at 488 nm) were pre-selected as fluorescein isothiocya-nate (FITC) and phycoerythrin (PE) because of the extensiveexperience available with both fluorochromes, the large numberof high-quality commercially available reagents and their compat-ibility with the optical configuration of all the four X8-colorinstruments listed in Table 1. The same selection criteria wereapplied for Allophycocyanin (APC) as the first fluorochrome for thered laser line (emission at 633/635 nm). Similarly, either peridinin–chlorophyll–protein complex (PerCP) or PerCP–Cyanin5.5(PerCPCy5.5) and PE–Cyanin7 (PECy7) were left as the mostsuitable fluorochrome choices for the third and fourth detectors ofthe blue laser line, respectively. In contrast, APC-Cyanin7 (APCCy7),Alexa Fluor 700 (AF700) and APC-Hilite7 (APCH7) were comparedfor the second detector of the red laser line, and Pacific Blue(PacB) versus Horizon V450 (HV450) and Pacific Orange (PacO)versus Anemonia Majano cyan fluorescent protein (AmCyan)17versus Horizon V500 (HV500) were evaluated for the first andsecond detector of the violet laser line (emission at 405/407 nm),respectively.For these evaluations several fluorochrome-conjugated anti-body reagents were compared: PacB-conjugated CD2(TS1/8),CD3(UCHT1), CD4(RPA-T4), CD20(2H7), CD45(T29/33) andHLADR(L243) versus HV450-conjugated CD2(S5.2), CD3(UCHT1),CD4(RPA-T4), CD20(L27), CD45(HI30) and HLADR(L243); AmCyan-conjugated CD45(2D1) versus PacO-conjugated CD45(HI30) versusHV500-conjugated CD45(HI30); and APCCy7-conjugated CD4(RPA-T4) versus AF700-conjugated CD4(RPA-T4) versus APCH7-conju-gated CD4(RPA-T4) antibody(clone) reagents. Antigen expressionwas evaluated as both mean fluorescence intensity (MFI) and stainindex (SI; defined as the difference between the MFI of positiveand negative cells divided by 2 s.d.’s of the MFI observed for thenegative cell population).24In all cases, staining of X5 PB sampleswas used to evaluate the staining patterns of each pair/group ofreagents to be compared. Sample preparation and instrumentEuroFlow standardization of flow cytometry protocolsT Kalina et al1987 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 settings were performed in the eight different EuroFlowlaboratories as described in Section 2 and Section 4 of thismanuscript.Comparison between the Pacific Blue (PacB) and Horizon V450(HV450) fluorochromesThe PacB and HV450 fluorochromes showed very similar fluorescenceprofiles that adequately fit with the optical configuration of the firstdetector for the violet laser of the four flow cytometry instruments.Detailed comparison of the needs for compensation for the spilloverinto other detectors of the fluorescence emissions of these twofluorochromes showed that thesewereslightlyhigher(P40.05;Mann–Whitney Utest) for PacB versus HV450; nonetheless, bothfluorochromes showed no spillover into any detector except for thesecond detector of the violet laser (Table 2).Regarding MFI and SI values, similar results with o10%differences were found when the same clone and manufacturerwere compared. Conversely, when either the clones or themanufacturers were not the same, differences between reagentswere higher (Table 3). We have chosen PacB for the EuroFlowpanels, based on broader availability of PacB conjugates at thetime of testing.Comparison among the Anemonia Majano cyan fluorescentprotein (AmCyan), Pacific Orange (PacO) and Horizon V500(HV500) fluorochromesSpecific comparisons for the second detector of the violet laserline were made for the AmCyan, PacO and HV500 fluorochromedyes. These fluorochromes showed clearly different fluorescenceprofiles. Accordingly, in terms of needs for fluorescence compen-sation, a higher spillover into other channels was observed forAmCyan, particularly in the first detector of the violet laser line(Po0.01 versus both PacO and HV500; paired Student’s T-test) andin the first detector of the blue laser (Po0.01 versus both PacOand HV500; paired Student’s T-test), where either PacB or HV450,and FITC, respectively, are typically measured. Table 2 summarizesthe compensation matrix values obtained for these three dyes. Ingeneral, the MFI obtained for monoclonal Ab reagents conjugatedwith these fluorochromes directed against the same antigen wasalso higher for AmCyan, although different clones were comparedand fluorescence differences may not be solely related to thefluorochrome (Table 3). AmCyan showed a higher resolutionpower, but the higher fluorescence intensity represented adisadvantage when a strong AmCyan signal for a marker wascombined with a dim signal of FITC-conjugated reagents in thesame cell populations, because of its relatively higher overlap withthe first detector of the blue laser (data not shown). In turn, PacOshowed low spillover into other channels (Table 2), together withclearly dimmer MFI values (Table 3); nonetheless, its resolutionpower, as reflected by the observed SI, was comparable to that ofAmCyan (Table 3). HV500 showed an intermediate profile betweenAmCyan and PacO in terms of both needs for compensation andfluorescence intensity of positive cells (higher than PacO but lowerthan AmCyan), associated with a comparable resolution power (SI)between different cell populations (Table 3).Comparisons among the Allophycocyanin–Cyanin7 (APCCy7),Alexa Fluor 700 (AF700) and Allophycocyanin–Hilite7 (APCH7)fluorochromesComparison of APCCy7, AF700 and APCH7 was performed insequential steps. First, the performance of each individualfluorochrome was assessed. Accordingly, APCCy7 showed arelatively high intensity (Table 3), while its main disadvantagewas the over-time instability, especially in the presence offormaldehyde-based fixatives. This instability resulted in arelatively high and variable degradation-associated ‘spillover’ intothe first channel of the red laser and the appearance in thisTable 1. Typical default optical configuration and most common fluorochromes available for each detector of three lasers, X8-color flow cytometry instruments available in March 2006Channel FACSCanto II (BD Biosciences) FACSAria (BD Biosciences) LSR II (BD Biosciences) CyAn ADP (Dako/Beckman Coulter) Most commonly availablefluorochromesLaser DM EF Laser DM EF Laser DM EF Laser DM EF1 30 mW Violet(405 nm)450/50 10 mW Violet(407 nm)450/50 25 mW Violet(405 nm)450/50 100mW Violet(405 nm)450/50 PacB/HV4502 30 mW Violet(405 nm)502 510/50 10 mW Violet(407 nm)502 530/30 25 mW Violet(405 nm)502 525/50 100mW Violet(405 nm)502 530/40 AmCyan/PacO/HV5003 20 mW Blue(488 nm)502 530/30 13 mW Blue(488 nm)502 530/30 20 mW Blue(488 nm)502 530/30 25 mW Blue(488 nm)502 530/40 FITC/AF4884 20 mW Blue(488 nm)556 585/42 13 mW Blue(488 nm)556 585/42 20 mW Blue(488 nm)556 575/26 25 mW Blue(488 nm)556 585/42 PE5 13 mW Blue(488 nm)610 616/23 25 mW Blue(488 nm)613/20 PE-TR6 20 mW Blue(488 nm)655 670LP 13 mW Blue(488 nm)655 695/40 20 mW Blue(488 nm)655 695/40 25 mW Blue(488 nm)655 680/30 PerCP/PerCPCy5.57 20 mW Blue(488 nm)735 780/60 13 mW Blue(488 nm)735 780/60 20 mW Blue(488 nm)735 780/60 25 mW Blue(488 nm)735 750LP PECy78 17 mW Red(633 nm)660/20 11 mW Red(633 nm)660/20 35mW Red(633 nm)660/20 60 mW Red(635 nm)665/20 APC/AF6479 17 mW Red(633 nm)735 780/60 11 mW Red(633 nm)735 780/60 35mW Red(633 nm)735 780/60 60 mW Red(635 nm)735 750LP APCCy7/APCH7/AF700aAbbreviations: AF, alexa fluor; AmCyan, Anemonia Majano cyan fluorescent protein; APC, allophycocyanin; Cy5.5, cyanin5.5; Cy7, cyanin7; DM, dichroic mirror; EF, emission filter; FITC, fluorescein isothiocyanate;H7, hilite7; HV450, Horizon V450; HV500, Horizon V500; LP, long pass; PacB, pacific blue; PacO, pacific orange; PE, phycoerythrin; PerCP, peridinin–chlorophyll–protein; TR, Texas Red.aAF700 requires a 710/50emission filter.EuroFlow standardization of flow cytometry protocolsT Kalina et al1988Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited channel of false-positive events (data not shown), in line withprevious observations.24More recently, such instability has alsobeen related to a cell-dependent degradation phenomenon.30Inaddition, APCCy7 showed great lot-to-lot differences in brightnessand compensation needs (data not shown). AF700 showed littlespillover into this latter channel (Table 2), but this dye required theuse of a different mirror and filter –680 nm long pass (LP) and 710/50 nm band pass (BP), respectively– than those available bydefault in all four flow cytometers evaluated. In addition, thefluorescence intensity of AF700 translated into suboptimaldiscrimination of some antigens expressed at relatively low levels,particularly when they were expressed on cells that had a brightAPC signal (decreased SI due to compensation-induced dataspread; data not shown). Finally, the APCH7 dye, a more stableAPC-based tandem dye with a long Stoke’s shift, was tested.It showed a lower SI and MFI than its equivalent APCCy7-antibodyTable 2. Mean values of compensation matrices (n¼5) obtained at different time points in up to five different EuroFlow flow cytometer instrumentsfor fluorochromes compared for the same fluorescence channelLaser channel Compensation requirements inother fluorescence channelsPacB HV450 PacO AmCyan HV500 APCCy7 AF700 APCH7Violet-1 NA NA 2.2±0.3* 11.5±1.5 7.5±1.6 0.2±0.3 0.1±0.1 0.1±0.3Violet-2 27.9±2.8 23.8±2.3 NA NA NA 0.3±0.4 0.1±0.1 NRBlue-1 0.1±0.1 0.1±0.1 0.8±0.4 17.1±2.6 2.8±1.2 0.4±0.4 0.2±0.1 0.2±0.4Blue-2 NR 0.1±0.1 0.4±0.2 1.4±0.2 0.5±0.2 0.2±0.2 NR 0.1±0.2Blue-3 0.1±0.1 0.1±0.1 0.5±0.2 0.4±0.1 0.3±0.2 1.2±1.0 3.6±0.7 0.6±1.2Blue-4 NR NR 0.1±0.1 NR NR 3.3±1.9 1.3±0.3 1.7±0.8Red-1 NR NR 0.3±0.4 NR NR 4.8±2.5 0.8±0.2 2.0±1.1Red-2 0.1±0.1 NR 0.1±0.2 NR NR NA NA NAAbbreviations: AF700, alexa fluor 700; AmCyan, Anemonia Majano cyan fluorescent protein; APC, allophycocyanin; Cy7, cyanin7; H7, hilite7; HV450, HorizonV450; HV500, Horizon V500; NA, not applicable; NR, not required; PacB, pacific blue; PacO, pacific orange. Results are expressed as percentage values±s.d.*Po0.01 versus both AmCyan and HV500 (paired Student’s T-test).Table 3. Mean fluorescence intensity (MFI) and stain index (SI) values obtained for different sets of reagents evaluated in normal PB samples (n¼5)Marker PacB HV450 P-valueaCD2 Clone(manufacturer)TS1/8(BioLegend)S5.2 (BD B)MFI±s.d. ofCD2þT and NK-cells5741±755.7 8259±1792.8 0.02SIc37.0 41.4CD3 Clone(manufacturer)UCHT1 (BD Ph) UCHT1 (BD B)MFI±s.d. ofT-cells15 774±1503.7 17 246±814.2 0.08 APCCy7 AF700 APCH7 P-valuebSId117.2 130.5CD4 Clone(manufacturer)RPA-T4 (BD Ph) RPA-T4 (BD B) RPA-T4 (BD B) RPA-T4 (BD B) RPA-T4 (BD B)MFI±s.d. ofCD4þT-cells9474±710.2 9195±408.2 0.46 13 596±686.5 4307±174.1 9910±414.3 o0.001SIe61.8 66.6 42.6 41.4 35.0CD20 Clone(manufacturer)2H7(eBiosciences)L27 (BD B)MFI±s.d. ofB-cells30 073±3783.5 38 152±2857.4 0.005 PacO AmCyan HV500 P-valuebSIf219.61 222.8CD45 Clone(manufacturer)T29/33 (Dako) HI30 (BD B) HI30(Invitrogen)2D1 (BD B) HI30 (BD B)MFI±s.d. oflymphocytes30 742±824.8 53 709±2062.2 o0.001 5521±150.6 30 681±2838.3 19 157±686.3 o0.001SIg3.9 5.2 3.8 4.8 3.8HLADR Clone(manufacturer)L243(BioLegend)L243 (BD B)MFI±s.d. ofmonocytes11 509±1721.4 16 874±1934.3 0.002SIh47.7 64.8Abbreviations: AF700, alexa fluor 700; AmCyan, Anemonia Majano cyan fluorescent protein; APCCy7, allophycocyanin–cyanin7; APCH7, allophycocyanin–hilite7; BD Ph, BD Pharmingen; BD B, BD Biosciences; HV450, Horizon V450; HV500, Horizon V500; PacB, pacific blue; PacO, pacific orange.aPaired Student’sT-test.bPo0.001 for the following comparisons: APCH7 versus AF700, AF700 versus APCCy7, APCCy7 versus APCH7, PacO versus AmCyan, PacO versus HV500and AmCyan versus HV500 (paired Student’s T-test).cPositive reference population (PRP): CD2þT- and NK-cells and negative reference population (NRP),CD2lymphocytes.dPRP, T-cells; NRP, B- and NK-cells.ePRP, CD4þT-cells; NRP, CD4T-cells.fPRP, B-cells; NRP, T- and NK-cells.gPRP, lymphocytes; NRP,neutrophils.hPRP, monocytes; NRP, lymphocytes.EuroFlow standardization of flow cytometry protocolsT Kalina et al1989 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 conjugates (Table 3), but the major advantages of APCH7conjugates included (i) improved stability and (ii) better compen-sation profile, while (iii) keeping the default optical configurationof the instrument unchanged. These results are illustrated bydirect comparison of APCCy7, APCH7 and AF700 conjugates of thesame CD4 monoclonal Ab clone from the same manufacturer afterstaining of normal PB samples (n¼5) (Table 3).CONCLUSIONSelection of appropriate fluorochromes to be combined was a keyand pre-requisite step in developing the 8-color EuroFlow panels.On the basis of existing knowledge, experience and provenquality of evaluated reagents, several fluorochromes werepre-selected. For other fluorochrome positions, extensive compar-isons were required. Finally, we selected the combination of PacB(or HV450), PacO (or HV500), FITC, PE, PerCPCy5.5, PECy7, APC andAPCH7. However, it should be noted that some of thesefluorochromes performed at the desirable conditions, but others(for example, APCH7) still leave room for improvement. Substitu-tion of PacO by HV500 and PacB by HV450 might be feasible,provided that identical clones are used, that the new reagents areextensively compared to the reference reagents, and that newcompensation matrices are applied, which are adequate for theselected fluorochromes.SECTION 2. EUROFLOW STANDARD OPERATING PROCEDURE(SOP) TO ESTABLISH STANDARDIZED INSTRUMENT SETTINGSAT MULTIPLE SITEST Kalina1, JG te Marvelde2, VHJ van der Velden2, J Flores-Montero3,D Thu+rner1,SBo¨ttcher4, M Cullen5, L Lhermitte6, AS Bedin6,L Sedek7, A Mendonc¸a8, O Hrusak1, JJM van Dongen2and A Orfao31DPH/O, Prague, Czech Republic;2Erasmus MC, Rotterdam, The Netherlands;3IBMCC-CSIC-USAL, USAL, Salamanca, Spain;4UNIKIEL, Kiel, Germany;5UNIVLEEDS, Leeds, UK;6AP-HP, Paris, France;7SUM, Zabrze, Poland and8IPOLFG, Lisbon, PortugalBACKGROUNDFlow cytometers are relatively flexible instruments that allowsimultaneous measurement of the light scatter propertiesof different types of cells and the fluorescence emissions ofdistinct fluorophores attached to them.31Because of theirflexibility, adequate setting of instrument conditions, includingfine tuning of the light scatter and fluorescence detectors, isrequired prior to a specific measurement, in order to establish theoptimal window of analysis. An additional goal within theEuroFlow project was to define SOPs to establish standardizedinstrument settings that would allow reproducible (identical or atleast highly comparable) measurements at different times in thesame instrument or in different instruments at the same or atdistinct sites through the application of predefined scatter andMFI values for specific reference particles. In general, with suchSOPs, all particles that will be measured should fall in thepreviously defined window of analysis for the light scatter andeach fluorescence detector.The EuroFlow light scatter settings aim at reaching two goals: (i)all populations of interest (from small erythroblasts to eosinophilsand plasma cells) fall centered within the scale limits and (ii)adequate scatter resolution between individual cell populations isobtained, for both cell surface and intracellular staining proce-dures. Lymphocytes were chosen as an internal biologicalreference population to control for adequate placement ofinstrument light scatter settings.The EuroFlow setting of photomultiplier tube (PMT) voltages fora fluorescence detector is established at a voltage above theelectronic noise in such a way that the least autofluorescent celltype to be measured is placed at the left side of the scale, as‘negative’ events clearly distinguishable from debris in themultidimensional space generated, dim fluorescent events canbe discriminated from the negative, and no cell- or bead-associated fluorescence measurement reaches the upper limit ofthe scale.32Each PMT is characterized by a response of accuracy toPMT voltage measured, as the robust coefficient of variation (rCV)of a dim particle. Optimal PMT voltage is set at the beginning ofthe plateau of a rCV versus PMT voltage curve.32In this way, theelectronic noise contribution to the signal is minimal whereasmaximal dynamic range is left for the measurement of fluore-scence. At the time of writing, Cytometer Setup and Tracking (CS T)beads (BD Biosciences, San Jose, CA, USA) and Cyto-Cal MultifluorPlus Violet Intensity Calibrator (Thermo Scientific, Freemont, CA,USA) are being evaluated by EuroFlow as potentially suitableadditional calibrators for long-term, multi-center studies.In this section we summarize the most critical and relevantsteps included in the EuroFlow SOPs developed for optimalplacement of instrument settings.Instruments and reagentsFACSCanto II (BD Biosciences) flow cytometers were used in sevencenters and both an LSR II (BD Biosciences) and a CyAn ADP (Dako,Glostrup, Denmark/Beckman Coulter, Brea, CA, USA) were used inanother center. All cytometers were equipped with three lasersemitting at 405/407, 488 and 633/635 nm. Optical filter config-urations were identical, with the exceptions described in Table 1.Eight-peak Rainbow bead calibration particles (Spherotech, LakeForest, IL, USA) were used throughout the study for initial PMTcharacterization and for setting target MFI values, as well as fordaily checks; the same master lot of beads (RCP-30-5A master lotX02) was used throughout the study.Placement of PMT voltages for fluorescence measurementsTo place PMT voltages, the following sequential steps were used:the Rainbow 8-peak bead population showing the seconddimmest fluorescence was gated and the rCV of that peak wascalculated in each fluorescence channel for PMT voltages rangingfrom 300 to 999 mV at increments of 50 mV.33The optimal voltagefor each channel was first determined on one instrument (LSR II)and set at the beginning of the plateau phase of the curvegenerated. Using the PMT value obtained in this way, the brightestpeak was gated and its fluorescence intensity recorded in allchannels and then used as preliminary ‘Target MFIs’ for all otherinstruments. Subsequently, verification of PMT settings wasperformed on each individual EuroFlow instrument. Forverification of the lower boundary, PMT settings were checkedon the rCV versus PMT voltage curve, as described above for thereference instrument. For the ‘Target MFI’ to be accepted, PMTvoltage on each instrument had to be at the plateau of the curvefor all nine instruments. Additionally, all bright markers fromthe EuroFlow antibody panels29were tested in the correspondingchannels of all instruments; if the target MFI setting resulted insuboptimal PMT setting on any instrument, the target MFI valueswere adjusted accordingly till consensus target MFI valuesassuring optimal PMT settings for each instrument were reached.Placement of instrument settings for light scattermeasurementsFine tuning of scatter settings was based on usage of normalhuman PB lymphocytes. For this purpose, 50 ml of PB samplesobtained from healthy donors (after informed consent was given)and measured within the first 24 h after venipuncture were usedat each site. Prior to measurement, non-nucleated red cells werelysed (10 min) using 2 ml of 10X FACS Lysing Solution (BDBiosciences) and diluted 1/10 (vol/vol) in distilled water (dH2O),EuroFlow standardization of flow cytometry protocolsT Kalina et al1990Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited according to the recommendations of the manufacturer. Then, thesample was centrifuged (5 min at 540 g), the cell pellet waswashed with 2 ml of phosphate buffer saline (PBS; pH ¼7.4)containing 0.5% (w/v) bovine serum albumin (BSA; SIGMA-ALDRICH, St Louis, MO, USA) and 0.09% of sodium azide (NaN3;SIGMA-ALDRICH), centrifuged again under the same conditionsand finally resuspended in 250 ml of PBS with 0.5% BSA þ0.09%NaN3, and measured in the flow cytometer at a ‘low’ flow ratemode within the first hour after sample preparation. PMT voltageswere adjusted so that forward scatter (FSC)/sideward scatter (SSC)-gated lymphocyte singlets reached mean SSC and FSC values of55 000±5000 and 13 000±2000, respectively.EuroFlow instrument settingsFinal PMT voltages for each fluorescence channel were set foreach instrument to reach target MFI values using the brightestpeak of Rainbow 8-peak beads of the same lot. Subsequentrainbow bead lots were assigned new target MFI values by cross-calibration using the previous lot for an instrument in a singlelaboratory (DPH/O, Prague, Czech Republic) (Table 4, see alsowww.euroflow.org for the updated target MFI of other Rainbowbead lots). In turn, light scatter settings were placed as describedabove. Inclusion of the FSC-H parameter will allow discriminationof doublets in a FSC-Area (FSC-A) versus FSC-Height (FSC-H)bivariate plot, contributing further to the accuracy of the results.34The final instrument settings for both light scatter andfluorescence-associated PMT voltages are further referred asEuroFlow settings. The detailed EuroFlow SOP for instrumentsetup is available at the EuroFlow website (www.euroflow.org).Monitoring of instrument performanceMonitoring of instrument performance was done daily (at eachcold start) after laser stabilization was allowed for 30 min. Rainbow8-peak beads were acquired under EuroFlow settings (under‘disabled compensation’ conditions) and the MFI of the brightestpeak in each fluorescence channel was compared with thecorresponding target MFI value. The following criteria had to bereached for the instrument to pass the check: (i) MFI values withinthe target MFI±15%, and (ii) coefficient of variation (CV) of thebrightest peak o4% for the blue and violet laser channels, buto6% for the red laser channels and the PECy7 channel. Wheneverinstrument performance failed, measures such as thoroughcleaning, de-gassing flow cell and laser delay verification weretaken. When the performance was not restored to pass themonitoring criteria, a service visit was requested. After a servicevisit, PMT settings were adjusted as described above and a newcompensation experiment was performed as described in Section3 of this manuscript.MFI values of the brightest Rainbow bead peak weredaily reported for each individual flow cytometer. As the scalingof axes is different on FACSCanto II and LSR II (262 144 channels)as compared to CyAn ADP (4096 channels), the Rainbow beadsTable 4. Target mean fluorescence intensity (MFI) values obtainedafter optimal PMT adjustments for each fluorescence channel for thebrightest peak of Rainbow 8-peak calibration beads in the LSR IIinstrumentFluorochrome channel MFI values Rainbow lot no.X02, Y02 Z02 EAB01PacB 195 572 194 818 215 352PacO 231 265 216 293 217 908FITC 59 574 58 372 65 283PE 101 900 98 520 84 847PerCPCy5.5 216 064 223940 228 818PECy7 27 462 27 185 29 865APC 176 780 226 435 252 000APCH7 56 437 81 371 102 099Abbreviations: APC, allophycocyanin; Cy7, cyanin7; FITC, fluoresceinisothiocyanate; H7, hilite7; PacB, pacific blue; PacO, pacific orange;PE, phycoerythrin; PerCPCy5.5, peridinin–chlorophyll–protein–cyanin5.5.Figure 1. Comparison of cytometer setting tracking (CS T)module and EuroFlow baseline settings obtained for the fluoresceinisothiocyanate (FITC) channel (blue laser line) in one representativeinstrument. CS T (mid-fluorescence peak in a) and EuroFlow(Rainbow beads brightest, eighth peak in b) baseline settings arecompared in c(gray and red vertical lines, respectively) for therobust coefficient of variation (CV) and robust electronic noise(SDEN). Note that although EuroFlow settings used lower PMTvoltages, the robust CV values (orange line) and robust SDENvalues(green line) are still in their plateau phases.EuroFlow standardization of flow cytometry protocolsT Kalina et al1991 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 data file was first converted to FCS 2.0 format and then read with theCyAn ADP’s Summit software (Dako) to calculate the correspondingnumerical values with the same distribution over the scale.Automated baseline settings and instrument monitoringWhen FACSDiVa V6.0 software with the CS T module and BD CS Tbeads (BD Biosciences) were introduced in 2008, baseline PMTsettings were placed according to the manufacturers’ instructions forthe FACSCanto II and the LSR II instruments. Subsequently, PMTvoltage settings were adjusted manually in the CS T module, tocreate EuroFlow baseline settings. Electronic noise (SDEN)andrCVofthe dimmest CS T bead values obtained with the two baselinesettings were compared for eight instruments (data of onerepresentative instrument is shown in Figure 1).Instrument monitoring with the CS T module was performed inparallel to the EuroFlow instrument performance-monitoring SOPon three different instruments (two FACSCanto II and one LSR II),for a 3-month period. To evaluate instrument performance, wecalculated the CV of MFI values obtained for the brightest peak of8-peak Rainbow particles.Reproducibility of fluorescence intensity measurements withEuroFlow settingsThe level of standardization of the EuroFlow settings wasevaluated at two different time points, before standardizationevaluation experiments were performed as described in Section 6.Results of such evaluation showed nearly identical MFI values forindividual PMTs when their voltage was set to match the targetMFI fluorescence channels listed in Table 4. In all eightinstruments, the CV for the MFI values obtained for the brightestpeak of Rainbow beads was systematically lower than 5.5%(Table 5).Long-term evaluation of the MFI signal fluctuation withfixed PMT voltages revealed that in each of the eight instrumentsevaluated, changes of up to ±15% of the mean target MFI mighttransiently occur, whereas significant maintenance or hardwareissues were highlighted by not meeting the above-describedmonitoring criteria, with deviations in these values (Figure 2).Electronic noise level with EuroFlow settingsThe SDENlevel obtained with individual flow cytometers usingEuroFlow settings was highly comparable to that obtained throughthe CS T module (Figure 1), except for the PerCPCy5.5 channel(Table 6). Thus, it could be concluded that the EuroFlow approachfor PMT settings yields high-quality data with no impairment of thequality of the results obtained, due to higher electronic noise overindividual CS T module baseline. On average, the EuroFlowapproach set PMT voltages at lower levels (Table 6), which allowsfor slightly larger dynamic ranges for measurements on thedetectors. Of note, the significantly higher SDENvalue obtainedTable 5. Variation of mean fluorescence intensity (MFI) valuesobtained for the brightest bead population of the Rainbow 8-peakbeads in individual instruments placed in eight different EuroFlowcenters (seven FACSCanto II and one LSR II flow cytometers)Fluorochrome-associatedPMT detectorTargetMFIMean MFIaof individualmeasurements (n¼12)CVPMT 1—PacB 195572 193 109 5.40%PMT 2—PacO 231 265 225 152 4.63%PMT 3—FITC 59 574 59 003 2.08%PMT 4—PE 101 900 100 763 2.38%PMT 5—PerCPCy5.5 216 064 215 596 2.11%PMT 6—PECy7 27 462 27 639 3.13%PMT 7—APC 176 780 176 190 1.68%PMT 8—APCH7 56 437 56 610 2.16%Abbreviations: APC, allophycocyanin; CV, coefficient of variation; Cy7,cyanin7; FITC, fluorescein isothiocyanate; H7, hilite7; PacB, pacific blue;PacO, pacific orange; PE, phycoerythrin; PerCPCy5.5, peridinin–chlorophyll–protein–cyanin5.5; PMT, photomultiplier tube.aResults are expressed asarbitrary MFI channel values scaled from 0 to 262 144.Figure 2. Overtime stability of Rainbow 8-peak bead meanfluorescence intensity (MFI) profile, illustrating the results obtained forthree fluorescence channels: pacific blue (PacB) channel of the violetlaser (blue dots); phycoerythrin (PE) channel of the red laser (yellowdots); and fluorescein isothiocyanate (FITC) channel of the green laser(green dots), for the same flow cytometer instrument upon long-termmonitoring of MFI measurements for the brightest peak of the Rainbow8-peak beads. As shown, faulty violet laser was recognized as a sourcefor the decreased MFI values falling below 15% of the target MFI (boxesAandC).Acceptable±15% range for each channel are depicted bygray lines and a colored background. After a service visit and laseralignment, MFI values above 15% of the target MFI were detected (boxB); thus, photomultiplier tube (PMT) voltages were adjusted at this timepoint manually (closed circles). Please note that by placement ofinstrument settings as per the cytometer setting tracking (CS T)module the PMT could be adjusted to correct for the violet laser failure(open circles) until the laser failed completely and was replaced.Table 6. PMT voltages and electronic noise (SDEN) obtained with the EuroFlow settings versus the CS T moduleFluorochrome-associated PMT detector PMT voltage SDENP-valueaCS T module settings EuroFlow settings CS T module settings EuroFlow settingsPMT 1—PacB 431 (357–490) 412 (360–460) 24.1 (20–29.8) 24 (20.6–29.1) 0.92PMT 2—PacO 509 (414–633) 466 (395–581) 25.2 (21.3–28.1) 24.5 (20.2–27.3) 0.08PMT 3—FITC 483 (399–555) 438 (375–518) 28.2 (25.4–31.2) 28.9 (26.2–29.7) 0.98PMT 4—PE 462 (411–501) 395 (370–445) 30.9 (18.1–33.6) 31.1 (18.3–32.4) 0.46PMT 5—PerCPCy5.5 543 (456–610) 522 (440–591) 28.1 (18.1–31.3) 29.1 (18.2–32.9) 0.03PMT 6—PECy7 624 (589–757) 552 (539–707) 29 (22.1–32.6) 29.5 (20.7–31.8) 0.49PMT 7—APC 614 (543–687) 576 (501–629) 26 (16.8–28.9) 25.9 (12.8–28.9) 0.95PMT 8—APCH7 489 (435–662) 524 (481–687) 25.1 (17.5–36) 26 (14.1–36.6) 0.50Abbreviations: APC, allophycocyanin; CS T, Cytometer Setting Tracking; Cy7, cyanin7; FITC, fluorescein isothiocyanate; H7, hilite7; PacB, pacific blue; PacO,pacific orange; PE, phycoerythrin; PerCPCy5.5, peridinin–chlorophyll–protein–cyanin5.5; PMT, photomultiplier tube.aTwo-tailed Student’s T-test. Results areexpressed as mean (min–max) values.EuroFlow standardization of flow cytometry protocolsT Kalina et al1992Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited for the PerCPCy5.5 channel was still well-fitted in the plateau phaseof the voltage versus SDENcurve.CONCLUSIONThe EuroFlow SOP was designed to establish and daily monitorstandard instrument settings for a common bright signal placed atthe same level in different flow cytometer instruments. Overall, ourresults show optimal performance at different sites and instruments(even from different manufacturers), with early alarms for changes inhardware components that may impact the results. At the sametime, the EuroFlow SOP avoids performing full calibration of theinstrument (including compensation) on a daily basis.SECTION 3. DESIGN AND EVALUATION OF EUROFLOWSTANDARD OPERATING PROCEDURE FOR ESTABLISHINGOPTIMAL COMPENSATION SETTINGST Kalina1, JG te Marvelde2, VHJ van der Velden2, J Flores-Montero3,D Thu+rner1,SBo¨ttcher4, M Cullen5, L Lhermitte6, L Sedek7,A Mendonc¸a8, O Hrusak1, JJM van Dongen2and A Orfao31DPH/O, Prague, Czech Republic;2Erasmus MC, Rotterdam, The Netherlands;3USAL, Salamanca, Spain;4UNIKIEL, Kiel, Germany;5UNIVLEEDS, Leeds,UK;6AP-HP, Paris, France;7SUM, Zabrze, Poland and8IPOLFG, Lisbon,PortugalBACKGROUNDMost fluorochromes used in multicolor flow cytometry haverelatively broad fluorescence emission spectra.7,35Therefore,measurement of their fluorescence emissions is typically notrestricted to a single fluorescence channel but the emissions arealso measured in detectors other than the primary channel for aparticular fluorochrome (secondary fluorescence channels).7Spectral overlap of light into secondary channels might lead tofalse-positive signals. However, the proportion of light spilloverfrom the total fluorescence emission is constant for eachfluorochrome, implying that this spillover can be mathematicallycalculated and subtracted.7The term ‘fluorescence compensation’is typically used to describe this calculation and subtractionprocess. In general, the specific compensation values requireddepend on the spectral characteristics of the dyes, the opticalbandpass filters and dichroic mirrors mounted in the flowcytometer, the intensity of the measured signal and the specificvoltage used for the PMT where it is detected.7In digital flowcytometers, fluorescence compensation is applied after dataacquisition.36Accurate calculation of the compensation valuesfor a set of fluorochromes across multiple detectors is provided bythe compensation tools available in conventional flow cytometrysoftware once applied to data derived from the flow cytometricmeasurement of one or more sets of single fluorochrome-stainedstandards/controls.36A full compensation matrix is calculated bythe software based on each standard/control, and then it isapplied to the measured data. A prerequisite to establishappropriate compensation settings is that the spectralcharacteristics of light emissions collected in individual channelsfor the standards/controls exactly match those of the dye(s) usedin the experiment. Despite this, special attention should be paid tothe fact that several currently used dyes are compound tandemdyes, where one fluorochrome serves as an acceptor of laser lightenergy and transfers this energy to the second dye of the tandemby fluorescence resonance energy transfer (FRET).7Tandem dyesgreatly enhance the Stoke’s shift of the compound fluorochrome,but their manufacturing process may lead to non-uniform spectralcharacteristics of the tandems.7Thus, tandem dyes (that is, PECy7,APCH7) present with variable spillover light to the donor dyechannel depending on the proximity and amount of FRETacceptor dyes used;7this frequently translates into the need forspecific compensation controls/standards and settings forindividual 8-color combinations containing different reagentsconjugated to the same tandem dye.7A second prerequisite foroptimal compensation settings is that standards/controls mustcontain bright signals, so that the distance between the positiveTable 7. List of fluorochrome-conjugated antibodies used to set up fluorescence compensation matrices at individual centersGeneric fluorochromes and tandem fluorochromesGeneric fluorochromes Tandem fluorochromesGeneric targets Positive target(bead or cell)populationaPECy7 targets Positive target (bead orcell) populationaAPCH7 targets Positive target (bead orcell) populationaCD20-PacB B-cells CD2-PECy7 CD2þT/NK-cells CD3-APCH7 T-cellsCD45-PacO Lymphocytes CD8-PECy7 CD8hiT-cells CD4-APCH7 CD4þT-cellsCD8-FITC CD8hiT-cells CD10-PECy7bCompBead CD8-APCH7 CD8hiT-cellsCD8-PEcCD8hiT-cells CD16-PECy7 NK-cells CD9-APCH7bCompBeadCD5-PerCPCy5.5dCD5þT-cells CD19-PECy7 B-cells CD10-APCH7bCompBeadCD8-APCcCD8hiT-cells CD45RA-PECy7 CD45RAþT-cells CD14-APCH7eMonocytesCD45RO-PECy7 CD45ROþT-cells CD19-APCH7 B-cellsCD56-PECy7 NK- and CD56þT-cells CD24-APCH7 B-cellsCD117-PECy7bCompBead CD38-APCH7 CD38hiLymphocytesHLADR-PECy7 B- and HLADRhiT-cells CD43-APCH7 T-cellsCD49d-APCH7 T-cellsCD71-APCH7bCompBeadCD81-APCH7 B-cellsanti-lAPCH7bCompBeadAbbreviations: APC, allophycocyanin; Cy7, cyanin7; FITC, fluorescein isothiocyanate; H7, hilite7; PacB, pacific blue; PacO, pacific orange; PE, phycoerythrin;PerCPCy5.5, peridinin–chlorophyll–protein–cyanin5.5.aUnless otherwise indicated, the negative reference population used for each reagent was thelymphocytes from the ‘unstained’ control tube. For more information about the specific clones used, please see van Dongen et al.29 b‘Negative’ CompBeadused as negative reference population.cThe CD8-PE and CD8-APC antibodies are not part of the EuroFlow antibody panels and might be used from anyreliable source.dThis tandem dye requires generic compensation;eArtificially CD14monocyte population created by ‘appending’ 5000 events from theunstained tube to this single antibody-stained tubes (SAbST) acquisition.EuroFlow standardization of flow cytometry protocolsT Kalina et al1993 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 and negative subsets of events used to calculate fluorescencecompensation values is as high as the maximum distance in theexperimental samples to be measured. In practice, single reagent-stained cells or mouse immunoglobulin (Ig)-capture beads areused as compensation standards.37It should be noted thatcompensation settings must be defined only after the PMTvoltage is set for the experiment, because of its impact onfluorescence intensity and spillover into secondary channels.37In this section we describe the procedures used to design andevaluate the compensation matrix required for routine use of theEuroFlow panels proposed for the different 8-color combinationsof fluorochrome-conjugated antibodies, defined in the EuroFlow8-color panels.29Fluorescence compensation standards and controlsSpecific subsets of PB leukocytes stained with fluorochrome-conjugated antibody reagents in single antibody-stained tubes(SAbST) were used as standards (Table 7) to establish thefluorescence compensation matrices to be applied to flowcytometric data measured using the 8-color EuroFlow panels forthe diagnosis and classification of leukemias and lymphomas.SAbST were prepared as described in Section 4 for multiple single-stained aliquots of a normal PB sample showing negative to verybright expression of the stained reagents. In addition, reagent-specific SAbSTs for molecules not present on normal PB cells (forexample, CD117 PECy7) were created using Ig-capture beads(CompBead, BD Biosciences) as specific standards for thesespecific reagents in the panel. Furthermore, normal and patientsamples stained with the preliminary and final versions of theEuroFlow panels were used to confirm the utility of the calculatedcompensation matrices. The specific set of reagents used forfluorescence compensation purposes varied depending on theselected fluorochrome-conjugated antibodies at each round ofevaluation of the EuroFlow panels, as described in van Dongenet al.29Table 7 displays the set of markers used for the finalversion of the EuroFlow panels.29Fluorescence compensation setupCompensation standards and controls were acquired withFACSDiVa software or Summit software using the softwarecompensation tools. The setup containing the PMT voltage foreach fluorescence channel and the compensation matrix calcu-lated by the software was saved as ‘EuroFlow’ Setup into theFACSDiVa Setup Catalog, or as ‘EuroFlow Protocol’ in Summit.Templates were prepared for experiments and tubes labeled withthe reagents’ names beforehand, linked to the EuroFlow settings.Thus, reagent-specific compensation was applied accurately to thematching reagent labels, even when the compensation matrix wasrecalculated. In every center, compensation setup experimentswere performed by default once a month. Whenever instrumentmonitoring failed and PMT voltages were reset to match targetMFI values, the compensation setup experiment was repeated.Comparison of fluorescence compensation matrices obtained atdifferent days and at distinct centersCompensation setup experiments showed that generic compen-sation matrices could be used for all antibody reagents in theEuroFlow panels conjugated with the PacB, PacO, FITC, PE andAPC fluorochromes, as well as for the PerCPCy5.5 tandemfluorochrome (data not shown). In contrast, different values wererequired for both the PECy7 and APCH7 tandem fluorochromes,depending on the specific reagent conjugates used(Supplementary Table 1).To evaluate and compare the fluorescence compensationsettings established at different times in each center, compensa-tion matrices were evaluated from 14 listmode data files in FCS 3.0format, measured in seven centers (two per center); each of theTable 8. Fluorescence compensation matrix values obtained from listmode data files (n¼14) generated in 7 centers at two different time points fora total of 7 different flow cytometry instrumentsaSecondary fluorescence channelPacB PacO FITC PE PerCPCy5.5 PECy7 APC APCH7Primary fluorescence channel PacB MIN 24.3 0.0 0.0 0.0MEDIAN NA 27.7 0.0 NR 0.0 0.0 NR NRMAX 31.0 0.2 0.6 0.1PacO MIN 1.9 0.2 0.0 0.0 0.0 0.0 0.0MEDIAN 2.4 NA 0.4 0.2 0.3 0.0 0.0 0.0MAX 2.9 0.5 0.3 0.5 0.1 0.3 0.5FITC MIN 0.0 4.8 10.0 3.0 0.2 0.0 0.0MEDIAN 0.0 5.6 NA 12.0 3.5 0.3 0.0 0.0MAX 0.1 6.4 16.0 4.0 0.5 0.2 0.2PE MIN 0.0 0.0 0.2 30.1 2.2 0.0MEDIAN 0.0 0.1 1.3 NA 32.9 2.5 0.1 NRMAX 0.1 0.3 1.7 38.9 2.8 0.1PerCPCy5.5 MIN 0.0 0.0 0.0 0.0 12.5 1.6 1.0MEDIAN 0.0 0.0 0.0 0.0 NA 16.5 2.4 5.5MAX 0.9 0.9 0.2 0.1 18.8 3.7 8.0PECy7 MIN 0.0 0.0 0.0 0.2 0.6 0.0 3.2MEDIAN 0.0 0.0 0.1 0.7 2.9 NA 0.0 6.8MAX 0.4 0.6 0.5 13.1 5.7 0.9 9.1APC MIN 1.0 0.1 8.5MEDIAN NR NR NR NR 1.2 0.1 NA 9.6MAX 1.4 0.2 11.6APCH7 MIN 0.0 0.0 0.0 0.0 0.0 1.3 1.3MEDIAN 0.0 0.0 0.0 0.0 0.0 1.5 1.8 NAMAX 0.2 0.1 0.2 0.1 0.2 2.0 3.9Abbreviations: APC, allophycocyanin; Cy7, cyanin7; FITC, fluorescein isothiocyanate; H7, hilite7; NA, not applicable; NR, compensation was never required;PacB, pacific blue; PacO, pacific orange, PE, phycoerythrin; PerCPCy5.5, peridinin–chlorophyll–protein–cyanin5.5.aResults are expressed as median percentagevalues and range. Median values are highlighted in bold.EuroFlow standardization of flow cytometry protocolsT Kalina et al1994Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited two compensation matrices used per center had been establishedafter a new compensation experiment (Table 8).Overall, compensation matrices were shown to be similar in allseven instruments evaluated (Table 8) and their variability amonginstruments was similar to that observed with time within each of thelaboratories for individual instruments (P40.05, paired Student’sT-test). Although compensation requirements depend on the specificPMT voltage settings, overall, high spillover was detected for thePacB into the PacO channel and for PE into the PerCPCy5.5 channel.Furthermore, intermediate spillover was found between PerCPCy5.5and PECy7, between FITC and PE, PECy7 and APCH7, and betweenAPC and APCH7 detectors (Table 8). Compensation experimentsperformed 1 month apart yielded very similar compensation values(P40.05; paired Student’s T-test).CONCLUSIONFluorescence compensation setup procedures were designed toestablish fluorescence compensation matrices for every individual8-color combination of fluorochrome-conjugated reagents in the8-color EuroFlow panels.29The complexity of the procedure washigher than desired due to the need for different compensationvalues for reagents conjugated with the PECy7 and APCH7fluorochrome tandems. Fortunately, the frequency ofcompensation could be set to a time interval of 1 month,during which only minor deviations from target MFI values wererecorded on well-performing instruments, as assessed by routine(daily) monitoring of the standard instrument settings (see Section 2).Notably, highly stable compensation matrices were obtained atdifferent times among all different EuroFlow laboratories with theproposed fluorescence compensation setup SOP. This suggeststhat in the future, software solutions for automated establishmentof compensation matrices to experiments performed withadjusted PMT voltages to target MFI values may potentially bedeveloped and implemented.SECTION 4. SAMPLE PREPARATION AND STAININGVHJ van der Velden1, J Flores-Montero2, JG te Marvelde1,SBo¨ttcher3, L Lhermitte4, AS Bedin4, J Almeida2,JJPe´rez5,M Cullen6, P Lucio7, E Mejstrikova8, T Szczepan´ski9, T Kalina8,A Orfao2and JJM van Dongen11Erasmus MC, Rotterdam, The Netherlands;2USAL, Salamanca, Spain;3UNIKIEL, Kiel, Germany;4AP-HP, Paris, France;5HUS, Salamanca, Spain;6UNIVLEEDS, Leeds, UK;7IPOLFG, Lisbon, Portugal;8DPH/O, Prague, CzechRepublic and9SUM, Zabrze, PolandBACKGROUNDAt present multiple protocols and reagents are available forstaining leukocytes.5,26,38–42Most protocols include a stainingFigure 3. Illustrating example of the differences in the light scatter characteristics of the major subsets of peripheral blood leukocytesobserved for the distinct lysing solutions and staining protocols. Please note the significant reduction in the light scatter CV for the differentleukocyte populations observed with FACS Lysing Solution and a SLW protocol (red square). Events shown in the upper-left corner of each dotplot correspond to PerfectCOUNT beads (Cytognos SL) introduced for the evaluation of cell loss. SLW, stain-lyse-wash; SLWF, stain-lyse-wash-fix; SLNW, stain-lyse-no wash.EuroFlow standardization of flow cytometry protocolsT Kalina et al1995 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 step, one or more washing steps and an erythrocyte lysing step(whenever non-nucleated red cells are present in the sample), butfor enumeration of leukocytes the washing step is frequentlyomitted.5Erythrocytes can be lysed using ammonium chloride orother commercially available reagents, for example, FACS LysingSolution, QuickLysis (Cytognos SL, Salamanca, Spain) andVersaLyse (Beckman Coulter).5For staining of intracellularproteins (for example, cytoplasmic (Cy)CD3, CyMPO, nuclear(Nu)TdT) the leukocytes need to be fixed and permeabilized aswell.38,42For this purpose, several reagents, such as BD Perm/Washbuffer (BD Biosciences), Fix Perm (AN DER GRUB Bio ResearchGmbH, Vienna, Austria), IntraStain (Dako) and IntraPrep (BeckmanCoulter), are commercially available. Cell samples other than BMand PB, such as LN biopsies, CSF, pleural effusion fluid andvitreous humor, may need extra steps prior to the stainingprocedure.43For example, CSF samples need to be collected intubes with special medium in order to prevent substantial cellloss26and LN biopsies need to be cut into small pieces andhomogenized.41The choice of procedure and reagents applied to stainleukocytes depends on the aim of the experiment, but generallythe best procedure should fulfill the following criteria: (a) low CVson FSC and SSC; (b) large differences in mean channel values forFSC and SSC between major leukocyte populations; (c) minimalcell loss; (d) preservation of fluorochrome brightness; (e) noimpact on the stability of tandem fluorochromes; (f) lowbackground staining; (g) minimal inter-laboratory variation; and(h) easy and fast performance. Taking this into account, theEuroFlow Consortium has evaluated several procedures for thestaining of samples suspected of containing neoplastic hemato-poietic cells.Cell samplesThe EuroFlow antibody panels29are designed for diagnosis andclassification of all major hematological malignancies. Althoughmost EuroFlow antibody panels are primarily designed forevaluation of BM and/or PB samples, other samples, forexample, pleural effusions and fine-needle aspirates, can beused as well. The preferred patient materials for these panels arediscussed elsewhere.29Erythrocyte lysing and staining procedures evaluatedOverall, four different erythrocyte lysing solutions (ammoniumchloride, FACS Lysing Solution, QuickLysis and VersaLyse) wereevaluated to assess which best fulfilled the above-listed criteria.Reagents were evaluated in all eight EuroFlow centers on PBsamples obtained from 30 healthy donors, who gave theirinformed consent to participate in the study. Three differenttubes were stained for each lysing solution: (1) CD4-PacB,CD8-AmCyan, CD45-FITC, CD19-PE and CD14-APC (all from BDBiosciences); (2) CD4-PerCPCy5.5, CD19-PECy7 and CD8-APCH7 (allfrom BD Biosciences) and (3) CD19-PECy7 (from Beckman Coulter).Briefly, 50 ml of PB was incubated (15 min in darkness) with theantibodies in a final volume of 100 ml. Subsequently, the lysingsolution was added to the tube according to the instructions ofthe manufacturers and incubated for 10 min at room temperaturein darkness. After centrifugation (5 min at 540 g), the supernatantwas discarded and the cell pellet resuspended in 2 ml PBS þ0.5%BSA. After another centrifugation step (5 min at 540 g), thesupernatant was discarded and the cell pellet resuspended in250 ml PBS þ0.5% BSA. For tube 1, 50 ml of PerfectCOUNT beads(Cytognos SL) was added immediately prior to the acquisition inFigure 4. Comparison of the absolute cell counts of major leukocyte populations (a) and lymphocyte subsets (b) obtained with the fourdifferent lysing solutions (FACS Lysing Solution, Ammonium Chloride, QuickLysis and VersaLyse Lysing Solution) evaluated in combinationwith the three different staining procedures (SLNW, SLW, SLWF) tested. Results are shown as mean values (open circles) and 95% confidenceintervals (vertical lines). FACS Lyse, FACS Lysing Solution; NH4Cl, ammonium chloride; VersaLyse, VersaLyse Lysing Solution. SLW, stain-lyse-wash; SLWF, stain-lyse-wash-fix; SLNW, stain-lyse-no wash.EuroFlow standardization of flow cytometry protocolsT Kalina et al1996Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited the flow cytometer. All samples were acquired in a flow cytometerat four different time points (0, 1, 3 and 24 h after staining) anddata about 100 000 events per tube were recorded and stored.Stained samples were stored at 4 1C till acquisition at the 1-, 3- and24-h time points.Data recorded for tube 1 included: (a) qualitative comparisonof the separation obtained among major leukocyte populations;(b) mean FSC and SSC channel and CVs detected for eosinophils,neutrophils, monocytes and total lymphocytes; (c) absolutenumber of eosinophils, neutrophils, monocytes, CD19þB-cells,CD4þT-cells and CD8hiT-cells; (d) MFI and CV values observed forCD45 (for each cell population) and for CD19, CD4, CD8 and CD14for CD19þB-cells, CD4þT-cells, CD8hiT-cells and CD14himonocytes, respectively. Data recorded for the other twomonoclonal Ab combinations (tubes 2 and 3) included MFI andCVs of positive cells in the specific channel, MFI and CVs ofnegative cells in the same channel, and, for the tandemfluorochromes, the fluorescence signals (MFI values) in all otherchannels than the primary fluorochrome-specific one.Overall, three different staining procedures were evaluated:stain-lyse-wash (SLW), stain-lyse-wash-fix (SLWF) and stain-lyse-nowash (SLNW). The SLW procedure is described above; for the SLWFprocedure the final cell pellet was resuspended in PBS containing0.5% paraformaldehyde instead of PBS þ0.5% BSA. For the SLNWprocedure, sample preparation ended after incubation (10 min)with the lysing solution without any further washing step.Qualitative comparison of the scatter characteristics of the majorPB cell populations for the four erythrocyte lysing solutions evaluatedshowed that FACS Lysing Solution and ammonium chloride yieldedthe best discrimination among them, independently of the stainingprocedure used. Furthermore, comparison between the threestaining procedures tested showed that CVs for both FSC and SSCwere lower and more homogeneouswiththeSLNWmethod,exceptwhen the FACS Lysing Solution was used, which improved the FSCand SSC CVs with the washing step (Figure 3).In general, the SLNW resulted in the highest cell numbers,whereas specific loss of lymphocytes (Figure 4a) and lymphocytesubsets (Figure 4b) was observed with the SLW and SLWFprocedures. However, cell loss was significantly lower when FACSLysing Solution was used (versus all other lysing reagents)(Figure 4).Subsequently, we evaluated the effect of the different lysingsolutions and staining procedures on the fluorescence intensities.Both the washing step and the final fixation step induced somedecrease in the MFI of all antibodies evaluated. Overall, FACSLysing Solution generally resulted in the highest MFI values(Figure 5). There were no clear differences in MFI values orspillover of fluorescence emissions into secondary channels (MFIof ‘non-specific’ channels) between the four different lysingsolutions tested.Based on the data derived from the performance of the fourdifferent lysing reagents and the different sample preparationprotocols, it was decided to use a stain-lyse-wash procedure withFACS Lysing Solution for all cell surface membrane (Sm) labelings.The detailed protocols recommended are shown in Table 9. Asdisplayed there, due to the presence of Igs in plasma, membranestainings for Ig chains (for example, Igk,Igland Igm) requiredwashing steps prior to antibody incubation. Based on experience,practical feasibility and additional testing (data not shown), it wasagreed to include NaN3(at a concentration of 0.09%) in allwashing solutions and to ensure that all immunostainingsincluding SmIgs were preceded by two washing steps with10 ml PBS þ0.5% BSA (Table 9). The latter procedure resulted inmaximal SmIg staining intensities (data not shown).Figure 5. Comparison of the mean fluorescence intensity (MFI) values of six fluorochrome-conjugated antibodies obtained with the fourdifferent lysing solutions evaluated in combination with the three different staining procedures (SLNW, SLW, SLWF) tested. CD45-fluoresceinisothiocyanate (FITC) was evaluated on total peripheral blood (PB) lymphocytes, CD14-allophycocyanin (APC) was evaluated on PB monocytes,CD4-peridinin chlorophyll protein cyanin5.5 (PerCPCy5.5) was evaluated on PB CD4þT-lymphocytes, CD8-APC hilite7 (H7) on PB CD8hiT-lymphocytes and the two CD19-phycoerythrin cyanin 7 (PECy7) reagents were both evaluated on PB CD19þB-lymphocytes. Results areshown as mean values (open circles) and 95% confidence intervals (vertical lines). FACS Lyse, FACS Lysing Solution; NH4Cl, ammoniumchloride; VersaLyse, VersaLyse Lysing Solution. SLW, stain-lyse-wash; SLWF, stain-lyse-wash-fix; SLNW, stain-lyse-no wash.EuroFlow standardization of flow cytometry protocolsT Kalina et al1997 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 Table 9. Detailed EuroFlow Standard Operating Procedures (SOPs) for sample preparation and stainingA. Common initial procedure when the EuroFlow antibody panel includes SmIg stainingIf the EuroFlow antibody panel is going to be applied to a sample that includes SmIg staining, follow these initial steps; otherwise go directly to thebackbone, surface or intracellular staining protocols (sections B, C, D, respectively):1. Pipette 300 ml of sample into a 10-ml tube (see Note 1). Note 1: For small samples (i.e. CSF, vitreous aspirates) spin down the total volume(5 min at 540 g), discard the supernatant (see point 5) and resuspend in 300 ml of PBS þ0.5% of bovine serum albumin (BSA) þ0.09% sodiumazide (NaN3).2. Add 10 ml filtered PBS þ0.5% BSA þ0.09% NaN33. Mix well4. Centrifuge for 5 min at 540 g5. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet6. Add 10 ml PBS þ0.5% of BSA þ0.09% NaN3to the cell pellet7. Mix well8. Centrifuge for 5 min at 540 g9. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet10. Resuspend the cell pellet in 200 ml of PBS þ0.5% BSA þ0.09% NaN311. Continue with conventional EuroFlow SOPs for staining of cell surface or cell surface plus intracellular markers as described below inprocedures B, C and D, respectivelyB. Staining of backbone markers1. Calculate the total volume of surface membrane backbone antibodies based on the number of tubes in the panel (see Note 2).Note 2: Intracellular backbone markers should not be added here.2. Pipette these antibodies in one tube (backbone tube)3. Calculate the total volume of sample to be stained, also based on the number of tubes in the panel and a volume of 50 ml per tube4. Pipette this sample volume into the backbone tube5. Mix well6. Pipette equal amounts of the sample/backbone mix into the various tubes included in the applied EuroFlow panel (see Note 3).Note 3: Both the volume pipetted into each tube and the overall number of tubes depends on the specific EuroFlow panel that is applied.7. Continue with the steps described below in procedure CC. Staining of surface markers only (see Note 4):Note 4: PCD tube 2 is processed identically to PCD tube 1 as described in section D if CD138-PacO is used.1. Add the appropriate volume of antibodies directed against cell surface markers (except for the backbone markers), as recommended for eachspecific EuroFlow panel2. If necessary, use PBS þ0.5% BSA þ0.09% NaN3to reach a final volume of 100 ml per tube (see information on the EuroFlow panels)3. Mix well4. Incubate for 15 min at room temperature (RT ) protected from light5. Add 2 ml of 1x FACS Lysing Solution (10x FACS Lysing Solution diluted 1/10 vol/vol in distilled water (dH2O))6. Mix well7. Incubate for 10 min at RT protected from light8. Centrifuge for 5 min at 540 g9. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 ml residualvolume in each tube10. Add 2 ml of PBS þ0.5% BSA þ0.09% NaN3to the cell pellet11. Mix well12. Centrifuge for 5 min at 540 g13. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 ml residualvolume in each tube14. Resuspend the cell pellet in 200 ml PBS þ0.5% BSA þ0.09% NaN315. Acquire the cells after staining or (if not immediately acquired) store at 4 1C maximally for 3 h until measured in the flow cytometerD. Combined staining of intracellular and surface membrane markers (see Note 5):Note 5: Tube 4 of the AML/MDS panel should be stained/processed further as described in Procedure E1. Add the appropriate volumes of antibodies for cell surface markers, as recommended for each specific EuroFlow panel2. If necessary, use PBS þ0.5% BSA þ0.09% NaN3to reach a volume of 100 ml per tube (see information on the EuroFlow panels)3. Mix well4. Incubate for 15 min at RT protected from light5. Add 2 ml of PBS þ0.5% BSA þ0.09% NaN3to the cell pellet6. Mix well7. Centrifuge for 5 min at 540 g8. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 ml residualvolume in each tube9. Resuspend the cell pellet by mixing gently10. Add 100 ml of Reagent A (fixative; Fix Perm, An der Grub, Vienna, Austria)11. Incubate for 15 min at RT protected from light12. Add 2 ml of PBS þ0.5% BSA þ0.09% NaN3to the cell pellet13. Mix well14. Centrifuge for 5 min at 540 g15. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 ml residualvolume in each tube16. Resuspend the cell pellet by mixing gently17. Add 100 ml of Reagent B (permeabilizing solution; Fix Perm)18. Mix well19. Add the appropriate volume of the intracellular antibodies (see EuroFlow panels)EuroFlow standardization of flow cytometry protocolsT Kalina et al1998Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited Intracellular stainingsFor the staining of intracellular antigens, special procedures areneeded to permeabilize and fix the cells.38,42On the basis of theextensive experience of the EuroFlow laboratories, the Fix Permreagents were selected for this purpose; no additional comparisonwith other commercially available reagents was performed. Thedetailed protocols are shown in Table 9.Although the Fix Perm reagents work well for NuTdT staining,it was decided that within the acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) protocol, staining of NuTdT willbe done using FACS Lysing Solution, based on the performancepreviously reported,38because all tubes can then be treatedin a similar way and additional effects on the light scattercharacteristics of leukocytes (which could potentially hamper theiruse as common parameters to every stained aliquot) are avoided.This was not applied to staining of NuTdT in the BCP-ALL andT-ALL panels,29because in such cases additional stainings forother intracellular markers were required (that is, CyIgm, CyTCRband CyCD3), for which Fix Perm reagents already was shownto be of utility.38,42To ensure similar staining intensities of the backbone markers inall tubes (for both membrane and intracellular stainings), allantibodies were titrated for a total volume (antibodies andsample) of 100 ml in every tube. If this volume was not reached,PBS þ0.5% BSA þ0.09% NaN3was added to increase the volumeto 100 ml. In some EuroFlow tubes, the total volume exceeded100 ml. This was accepted as long as the total volume remainedbelow 115 ml, as such minor deviations had no impact on thestaining intensities of the backbone markers (data not shown).Processing of cell samples with low nucleated cell countsAs described above, the sample preparation protocols and thedifferent lysing solutions tested here were evaluated for thestaining of whole BM and PB samples. However, in some patientsthe cell count may be rather low. This occurs, for example, in asubstantial number of pediatric MDS patients and certainly willoccur in samples obtained during therapy. We therefore evaluatedwhether it was possible to perform bulk lysis of erythrocytes withammonium chloride prior to the EuroFlow protocol, to increaseconsiderably the concentration of nucleated cells in the sample.Initially, within the AML/MDS panel,29slight differences wereobserved for CD16, CD11b and CD15, but after titration ofantibodies, fluorescence emissions were highly comparableTable 9.(Continued)20. Mix well21. Incubate for 15 min at RT protected from light22. Add 2 ml of PBS þ0.5% BSA þ0.09% NaN3to the cell pellet23. Mix well24. Centrifuge for 5 min at 540 g25. Discard the supernatant using a Pasteur pipette or vacuum system without disturbing the cell pellet, leaving approximately 50 ml residualvolume in each tube26. Resuspend the cell pellet in 200 ml PBS þ0.5% BSA þ0.09% NaN327. Acquire the cells after staining or (if not immediately acquired) store at 4 1C maximally for 3 h until measured in the flow cytometer.E. Nuclear (Nu)TdT staining (Tube 4 AML/MDS EuroFlow panel):1. Continued from procedure C step 132. Add the appropriate amount of the TdT antibody to the cell pellet3. Mix well4. Incubate for 15 min at RT protected from light5. Add 2 ml of PBS þBSA 0.5% þ0.09% NaN3to the cell pellet6. Mix well7. Centrifuge for 5 min at 540 g8. Resuspend the cell pellet in 200 ml PBS þBSA 0.5% þ0.09% NaN39. Acquire the cells after staining or (if not immediately acquired) store at 4 1C maximally for 3 h until measured in the flow cytometer.Overview of protocol Sections for the various EuroFlow antibody panels and corresponding tubes.Antibody panel Tube(s) Protocol procedureABCDEALOT 1 XBCP-ALL 1,4 X X X2,3 X X XT-ALL 1–4 X XAML/MDS 1–3, 5–7 X X4XXXLST 1 X XSST 1 X XPCD 1–2 X XB-CLPD 1–4 X X XT-CLPD 1,2,4,6 X X3,5 X XNK-CLPD 1,2 X X3XXAbbreviations: ALOT, acute leukemia orientation tube; AML/MDS, acute myeloid leukemia and myelodysplastic syndrome; BCP-ALL, B-cell precursor acutelymphoblastic leukemia; CLPD, chronic lymphoproliferative disorder; LST, lymphoid screening tube; PCD, plasma cell disorders; SST, small sample tube; T-ALL,T-cell acute lymphoblastic leukemia.EuroFlow standardization of flow cytometry protocolsT Kalina et al1999 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 between both procedures (Figure 6). Therefore, bulk lysis may beused prior to antibody staining when nucleated cell concentrationneeds to be increased, such as for the AML/MDS EuroFlow panel.29As low cell counts less likely occur in other hematological diseasesat diagnosis, prior bulk lysis was not specifically tested for theseprotocols.Sample acquisition in the flow cytometerAs the time between staining of the samples and data acquisitionin the flow cytometer may have an impact on the MFI of individualmarkers (particularly of those detected by reagents containingtandem fluorochromes), we acquired the samples immediatelyafter staining, as well as 1, 3 and 24 h after sample preparation wascompleted. Our results show that MFI generally decreased overtime, particularly when lysing solutions that did not containfixative (that is, ammonium chloride) were used (Figure 7a). Themost stable results were obtained with FACS Lysing Solutioncombined with either the SLNW or the SLW procedures(Figure 7b). Data became somewhat more variable when acquired3 h and particularly 24 h after staining (Figures 7a and b).On the basis of the results reported above, it was agreed that allsamples should preferably be acquired within 1 h after completingthe staining procedure. If not measured immediately, they shouldbe stored at 4 1C in the darkness. Samples should be acquired onflow cytometers that have been set up according to the EuroFlowSOPs as described in Sections 2 and 3. For the EuroFlow screeningand orientation tubes (acute leukemia orientation tube (ALOT),lymphoid screening tube (LST), small sample tube (SST) andplasma cell dyscrasia (PCD)),29a minimum of 50 000 cells (typically100 000) should be acquired in order to reach sufficient sensitivityfor recognition of abnormal populations.CONCLUSIONThe EuroFlow protocols for sample preparation and staining weredesigned based on previous experience and experimental dataavailable in the literature together with the results of specificexperiments performed by the EuroFlow Consortium. Based onthe combined results, the EuroFlow Consortium favors the use of aSLW procedure with FACS Lysing Solution for cell surface antigens,where measurements are performed shortly (o1 h) after samplepreparation is completed. Special situations were envisaged forthe staining of SmIgs, intracellular markers and samples with lownucleated cell counts, where introduction of additional washingsteps, a fixation/permeabilization step and bulk lysis prior tostaining, respectively, are recommended. The EuroFlow samplepreparation and staining protocols described here are designed tobe used together with EuroFlow SOPs for instrument setup(Section 2) and fluorescence compensation (Section 3) for theselected fluorochromes (Section 1). The proposed samplepreparation and staining protocols perfectly fit with the EuroFlowantibody panels designed for the diagnosis and classification ofhematological malignancies29when using the most commontypes of samples, such as PB and BM. Specific issues related toother types of samples that have peculiar features and requireunique sample preparation protocols (for example, CSF) areaddressed in the EuroFlow antibody panel report.29SECTION 5. EUROFLOW STRATEGIES AND TOOLS FOR DATAANALYSISM Martı´n-Ayuso1, ES Costa2, CE Pedreira3,QLe´crevisse4,J Herna´ndez1, L Lhermitte5,SBo¨ttcher6, JJM van Dongen7and A Orfao41Cytognos SL, Salamanca, Spain;2Universidade Federal do Rio de Janeiro(UFRJ), Rio de Janeiro, Brazil;3Engineering Graduate Program, ElectricalEngineering Program (COPPE PEE) and Faculty of Medicine (FM), UniversidadeFederal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil;4USAL, Salamanca,Spain;5AP-HP, Paris, France;6UNIKIEL, Kiel, Germany and7Erasmus MC,Rotterdam, The NetherlandsBACKGROUNDEven though we have seen considerable improvements of clinicalflow cytometry over the last years, the multicolor capabilities ofcurrently available flow cytometers are still far behind therequested needs in routine clinical diagnostic laboratories. Forexample, the current immunophenotypic diagnosis of distinctWHO categories of hematological malignancies frequentlyrequires the assessment of B30 different markers on neoplasticcells, which cannot be routinely studied on the same cell, owing totechnical limitations.44–47In order to overcome these technicallimitations, multiple aliquots of a sample are stained with differentcombinations of markers.47In this approach, a few markers aim atthe reproducible definition of the cell population(s) of interest; theso-called backbone markers are repeatedly used in every aliquotof the same sample and combined with other sets of markers,which together aim at the detailed immunophenotypiccharacterization of the cell population(s) of interest.47Despite their clear benefits, these advances in multiparameterflow cytometry have led to a significantly increased complexity ofdata analysis and data interpretation because of the higherFigure 6. Parameter band plot of all individual parameters evaluatedin a bone marrow sample from an MDS patient treated according tothe EuroFlow protocol with (light colors) or without (dark colors)prior bulk lysis. Colored circles represent median scatter andfluorescence intensity (MFI) values obtained for the lymphocytes(dark green/light green), monocytes (red/orange) and neutrophils(dark blue/light blue).EuroFlow standardization of flow cytometry protocolsT Kalina et al2000Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited number of parameters simultaneously assessed in greaternumbers of individual cells, and the expanded numberof variables that might have an impact on the quality of theresults.44–47Moreover, these technical improvements have notbeen paralleled (or followed) by innovations of data analysis andinterpretation tools in the software packages routinely used inhematology laboratories. This lack of innovation has furthercontributed to the increased complexity of immunophenotypingof hematological malignancies.44,45In recent years, the EuroFlowConsortium has proposed several new data analysis tools48–50aimed at decreasing such complexity through the development ofnew and more objective data analysis and interpretationstrategies.48–51These novel tools have been progressivelyincorporated into the Infinicyt software (Cytognos SL) developedby the EuroFlow Consortium.In this section we describe the new data analysis strategyproposed by the EuroFlow Consortium to be used in combinationwith the EuroFlow antibody panels and the EuroFlow SOPs formultiparameter immunophenotypic diagnosis and classificationof hematological disorders.Merge of flow cytometry data files and calculation of’missing values‘The EuroFlow antibody panels are composed of multiple 8-colorcombinations of antibodies that contain three or four fluoro-chrome-conjugated antibodies as common backbone markers,essential for gating the cells of interest in every aliquot of asample stained with a specific EuroFlow antibody panel.29TheMerge function (first step in Figure 8) was used to fuse differentdata files corresponding to distinct aliquots of the same sample,each stained with a unique combination of reagents from theEuroFlow antibody panels. This results in a new single mergeddata file that contains all information measured in the samesample.52,53Such data file consists of a data matrix (Figure 9) inwhich the information (the measured parameters) for eachdifferent cellular event evaluated is aligned in one column perFigure 7. Effect of time between completion of staining and data acquisition in the flow cytometer (0 h, 1 h, 3 h and 24 h) and the samplepreparation protocol on the mean fluorescence intensity (MFI) of CD19-phycoerythrin cyanin 7 (PECy7), CD4-peridinin chlorophyll proteincyanin5.5 (PerCPCy5.5) and CD8-allophycocyanin hilite7 (APCH7) on peripheral blood (PB) B-cells, CD4þT-cells and CD8hiT-cells, usingammonium chloride (a) or FACS Lysing Solution (b) as lysing reagents. Three different sample preparation protocols were evaluated: SLNW;SLW and SLWF. Results are shown as mean values (open circles) and 95% confidence intervals (vertical lines). FACS Lyse, FACS Lysing Solution;NH4Cl, ammonium chloride. SLW, stain-lyse-wash; SLWF, stain-lyse-wash-fix; SLNW, stain-lyse-no wash.Figure 8. Flow chart diagram illustrating the sequential steps usedduring data analysis for the evaluation of the performance of theEuroFlow antibody panels.EuroFlow standardization of flow cytometry protocolsT Kalina et al2001 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 tube, which includes light scatter- and fluorescence emission-associated parameters, placed in different rows of the data matrix.The data matrix contains filled and unfilled boxes, correspondingto parameters that were directly measured and parameters notevaluated directly (missing values) for an individual event in agiven aliquot of the sample, respectively48(Figure 9).A calculation function was used to fill in the ‘missing values’ in theabove-mentioned data matrix corresponding to the merged datafile. For this purpose, the common backbone parameters were usedfor identification of the neoplastic and/or normal cell populations ofinterest present in each of all tubes coming from a single sample(secondstepinFigure8).Afterwards,anewdatafilewascreated,which only contained information about those parameters mea-sured for individual events contained in the gated cell population.48Then, the ‘missing values’ in the data matrix corresponding to thegatedcellpopulationwerecalculated(thirdstepinFigure8).Forthislatter purpose, for each event to be calculated inside the selectedpopulation, the software searches for the ‘nearest neighbor’54,55event in each of the other aliquots of the sample, based only on itsunique position in the multidimensional space created by allcommon backbone parameters (Figure 10). Therefore, the ‘nearestneighbor’ of each event to be calculated in a merged data filemeasured in one sample aliquot will be the event showing theshortest distance from it in the n-dimensional space generated bythe same common parameters in another sample aliquot from themerged data file. Finally, the software applies the values obtainedfor the ‘nearest neighbor’ for all those parameters measured for theevent in the latter sample aliquot but not measured in the formersample aliquot. This calculation process is done for each individualevent in the merged data file till the data set is completed. At theend of the calculation process, the new data file contains both thedata that were actually measured in the flow cytometer for eachevent and the calculated data for those parameters not measured inthe same group of events in the other aliquots.48The calculationprocess requires optimal definition with maximum biologicalheterogeneity within the cell population to become apparent withthe common parameters (for example, backbone parameters) forthe cell population to be calculated; thus, backbone markerselection is crucial. In order to obtain a high accuracy of thecalculation process, each event in the cell population of interest isrequired for its definition in the EuroFlow antibody panels, based onfive or six backbone parameters (two scatter parameters and threeto four fluorescence markers).As previously described in this paper (see Section 4), EuroFlowantibody panels include both surface and intracellular stainings.Therefore, variations in the FSC/SSC values or in the fluorescencelevels of the backbone markers may occur because of the differentsample preparation procedures (Table 9). In order to allow thecalculation process when cells are treated with different stainingprotocols, a harmonization procedure was developed56andapplied to those cell populations of interest, for all parametersmeasured in common in the different sample aliquots, which areprepared differently (Figure 11). Such harmonization processconsists of the translation of a data matrix defined in a tube by agiven set of parameters for a given cell population into a datamatrix defined by the same parameters for the same cellpopulation measured in another tube under different conditions(for example, surface versus surface plus intracellular stainings).Of note, this harmonization tool did not affect the calculationprocess, as similar results were obtained when we compared thecalculated values in files that contained information about asample for which some aliquots/tubes were submitted tointracellular staining procedures and others were treated for Smstaining only.As an end result of the calculation procedure, all individualevents from each of the original data files corresponding todifferent aliquots of the same sample contain information abouteach reagent/parameter included in the whole antibody panel.The overall number of parameters for which values can beassigned to each individual cellular event included in the newdata file are virtually unlimited, and equals that of the number ofparameters measured in the whole set of merged data files for agiven number of stained aliquots of a sample. This allowsvisualization of previously ‘impossible’ bivariate dot plots forindividual events (for example, staining patterns for two reagentsconjugated with the same fluorochrome)48(Figure 10).Generation of reference data filesA reference data file is a data file constructed by merging two ormore data files, each corresponding to a cell population measuredin different samples with the same panel of reagents (fourth step inFigure 8). Hereby, the reference data file contains information aboutall parameters (measured or calculated) for each individual event ofthe targeted cell population.50Reference data files may containinformation about normal or neoplastic cell populations, which maybe homogeneous or heterogeneous with regard to differentparameters evaluated. The generation of the reference data filesaims at building libraries of reference cases to be comparedbetween each other or with a new case that has been stained withthe same panel of reagents (fifth step in Figure 8). On the basis ofthe existence of different patterns of protein expression in normalversus neoplastic cells, as well as among different WHO diseaseentities, a library can be built, which contains all normal andaberrant patterns that represent each of the different normal andpathological cell populations studied with the different EuroFlowFigure 9. Data matrix obtained from the EuroFlow B-CLPD (B-cellchronic lymphoproliferative disorders) antibody panel, showingmerging of five original data files into a single data file containingdata about 29 parameters (2 scatter parameters and 27 markers).Columns correspond to the different B-CLPD tubes (sample aliquots)measured and rows correspond to the different parametersevaluated. ‘C’ means ‘common’ marker defined as measured in allaliquots; ‘R’ means ‘real’ data measured in any of the tubes. Blankspaces represent the parameter information that was not measuredon an individual aliquot of the sample.EuroFlow standardization of flow cytometry protocolsT Kalina et al2002Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited antibody panels. Such a library can be used for (1) furtherevaluation of the utility and performance of antibody panels and(2) pattern-guided prospective classification of new cases diag-nosed in different individual laboratories, which use the sameEuroFlow antibody panels and laboratory procedures.50Evaluation of the EuroFlow antibody panels based on comparisonsof groups of reference data filesThe EuroFlow 8-color antibody panels for the diagnosis andclassification of hematological malignancies are designed toanswer specific clinical questions, which can be grouped intotwo general categories: (1) Is a given hematopoietic cellpopulation normal or reactive/regenerating or abnormal/neoplas-tic? (2) When an abnormal/neoplastic cell population is identified,which WHO disease category does it belong to? In order toevaluate the utility and performance of the EuroFlow antibodypanels, different groups of reference files that had been stainedwith the same antibody panels have been constructed. To answerthe first question, reference data files from a normal/reactive cellpopulation were compared with their neoplastic counterpart fromFigure 10. Schematic representation of the data calculation process with the Infinicyt software based on the ‘nearest neighbor’ principle. First,one event from a cell population (B-cells highlighted in red) in ais identified in a first data file (tube 2 of B-cell chronic lymphoproliferativedisorders (B-CLPD) panel) based on the backbone markers; then the event corresponding to the nearest neighbor of this event is identified inthe second data file (right; tube 5 of B-CLPD panel) as that event occupying the same (closest) position in a multidimensional space formed bythe same backbone parameters (b). Third, through the data calculation process the values for those parameters that were only measuredfor the later event in the second data file (d) but not for the former event in the first data file are assigned to the said event in the first data fileand vice versa (c). Finally, the calculation process is completed for all other events in the cell population of interest (red events). Through thisapproach, all events in the merged and calculated data file have information about each of the parameters measured in both tubes (e).EuroFlow standardization of flow cytometry protocolsT Kalina et al2003 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 one or multiple WHO disease entities in a multivariate 1 1 set ofcomparisons approach. To answer the second question, referencedata files corresponding to the neoplastic cell population frommultiple cases of a single WHO disease entity were comparedagainst single or multiple reference data files corresponding toone or more WHO disease entities.For such comparisons, multiple approaches such as principalcomponent analysis (PCA) can be used with the correspondingmultiple-dimensions (that is, bi- or tridimensional) graphicalrepresentations of, for example, Principal Component (PC) Xversus PC Y, and PC X versus PC Y versus PC Z, respectively, usingthe Automatic Population Separator (APS) graphical representa-tion of the Infinicyt software (Figure 12).On the basis of this APS representation, information about theseparation between the two groups of reference data files isobtained through definition of median and/or mean±s.d. borders(Figure 12) together with information about the most informative(versus redundant) parameters.57It also allows re-evaluation of apanel after excluding one or multiple markers to objectively evaluatethe contribution of each marker. A similar approach can then be usedto prospectively compare one new case against two different groupsof reference data files. Through such comparison, information isobtained about whether new cases belong to one of the referencegroups or whether they differ from the reference groups, for thosemarkers which are relevant in such comparison.Through such comparisons one can also easily and objectivelyidentify the phenotypic differences and similarities between the cellpopulations compared in the different reference groups and themarkers that account for them. In fact, it allows direct (multivariate)comparisons of one or more cell populations from a given samplewith other (for example, reference) cell populations from a pool ofX2 different samples (Figure 12). In a certain way, this mimics whatan expert follows in his mind when he compares the immunophe-notypic profiles obtained with a given antibody panel in a samplewith the profiles obtained for the same combinations of antibodiesin another sample (or group of samples) composed of normal,reactive, activated, aberrant or malignant cells. For example, theAPS comparison of normal with malignant B-cell precursors allowsidentification of the best combination of markers to distinguishbetween them and thereby define the most common aberrantFigure 11. Illustrating example of the impact of different sample preparation protocols on the immunophenotypic and light scatter features oflymphocytes from a normal peripheral blood (PB) sample (a) and blast cells from B-cell precursor acute lymphoblastic leukemia (BCP-ALL)(n¼9; c) and how the harmonization process reduces such impact (band d, respectively). In aand b, FSC versus SSC representation ofduplicates of a sample stained with two different protocols (permeabilized versus non-permeabilized lymphocytes) is shown without (a) andwith (b) data harmonization applied, respectively; in both aand b, green and violet populations correspond to non-permeabilized andpermeabilized aliquots, respectively. In cand d, different BCP-ALL blast cell populations from nine different BCP-ALL patients each stained infive different aliquots with the BCP-ALL EuroFlow panel are displayed. Each population is represented as median values in a principalcomponent (PC) 1 versus PC2 analysis diagram (automatic population separator (APS)1 view based on the discrimination obtained for thefollowing parameters: FSC, SSC, CD19, CD34 and CD45), where paired duplicated samples are colored identically. In csamples contain bothpermeabilized and non-permeabilized aliquots within the panel and the harmonization process was applied for five patient samples(duplicates colored dark yellow, light green, dark violet, red and cyan) for which duplicates show a very close position in the APS1 view;conversely for the other pairs of duplicates (light yellow, dark green, violet, dark blue show greater differences between paired samples). In d,one group of duplicates was processed by permeabilizing all aliquots within the panel, while in the other group each sample containedpermeabilized and non-permeabilized sample aliquots, with data harmonization being applied to the latter group; note that now all pairs ofsample duplicates overlap, confirming that with data harmonization blast cell populations processed differently (permeabilized versus non-permeabilized) are highly comparable to those who underwent a uniform sample preparation protocol.EuroFlow standardization of flow cytometry protocolsT Kalina et al2004Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited phenotypes. In addition, APS comparison between malignantB-cells from patients with different B-cell chronic lymphoproli-ferative disorders (B-CLPD) defined according to the WHO 2008classification1allows identification of the most informativeparameters for their differential diagnosis. Noteworthily, fullyobjective information is obtained through this approach about thespecific contribution of each marker in the panel.49CONCLUSIONDuring the last 6 years, the EuroFlow Consortium has built newapproaches for data analysis, which provide a new objectivestrategy for evaluation of the performance and utility of individualmarkers and antibody panels. The newly proposed strategy fordata analysis of samples stained with the EuroFlow antibodypanels includes a set of sequential steps (Figure 8): merge of datafiles corresponding to individual samples stained with theEuroFlow antibody panels, calculation of missing values for thecell populations of interest, merge of data files for the referencegroups and evaluation of antibody panels through multiplecomparisons between different sets of reference cases stainedwith the same panel of reagents. This new analytical strategy alsoprovides a pattern-guided approach for the immunophenotypicclassification of normal and malignant cell populations.49,50Thetools required to use the new analytical approach have beenimplemented into the Infinicyt software by the EuroFlowConsortium, which allows their usage in routine practice by anyother group around the world. Herewith a full set of flow cytometrydata analysis tools is provided to the flow cytometry field to helpexpert-based interpretation of highly complex multiparameter datasets. In combination with the reference data files generated, thenew software tools also provide a robust and reliable method fordata comparison between different diagnostic laboratories on asample-by-sample basis. The robustness and reliability of thisapproach is also based on the use of antibody panels with sufficientand adequate backbone markers that have been selected forcareful identification of the subset of cells of interest, which isessential for accurate calculation of missing data and their graphicalrepresentation. Accordingly, use of the EuroFlow antibody panelsand the EuroFlow SOPs for sample preparation allows for (1)building databases with reference groups of well-defined WHOcategories; (2) classification of a new case; (3) assuring internal andexternal quality control by any other user if the same tools andreference groups are used. Therefore, the new software toolscontribute significantly to the standardization of flow cytometrydata analysis.SECTION 6. RESULTS OF MULTICENTER MEASUREMENTST Kalina1, J Flores-Montero2, VHJ van der Velden3,SBo¨ttcher4,M Cullen5, L Lhermitte6, L Sedek7, A Mendonc¸a8, HK Wind3,JG te Marvelde3, E Mejstrikova1, O Hrusak1, JJM van Dongen3andA Orfao21DPH/O, Prague, Czech Republic;2USAL, Salamanca, Spain;3Erasmus MC,Rotterdam, The Netherlands;4UNIKIEL, Kiel, Germany;5UNIVLEEDS, Leeds, UK;6AP-HP, Paris, France;7SUM, Zabrze, Poland and8IPOLFG, Lisbon, PortugalBACKGROUNDIn order to design and apply the EuroFlow antibody panels for theimmunophenotypic diagnosis and classification of leukemias andlymphomas, SOPs were developed and evaluated as described inthe previous sections. However, multicentric implementation ofsuch antibody panels29and SOPs would require further evaluationof the protocols at the multicentric level. For this purpose twodifferent series of experiments could be envisaged: (1) staining ofcomparable samples with the same SOPs and antibody panels atFigure 12. Example of principal component (PC1 versus PC2) analysis (PCA; automatic population separator (APS)1 views) for comparison of anew sample—red circles (median values) and dots—with a library of cases (median values/case represented as circles) from three different referencegroups, each being colored differently (green, cyan and violet circles). In the upper panels the unknown case is compared to each pair of referencegroups and it only overlaps systematically with the dark blue cases (a–c). In the lower panels (d–f), the new sample is separately compared witheach individual reference group, showing again a high degree of overlap with the dark blue reference cases (f). Contour lines in each panelcorrespond to one (inner line) and two (outer line) s.d.’s of the mean value of the corresponding group of reference cases.EuroFlow standardization of flow cytometry protocolsT Kalina et al2005 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 multiple sites, and (2) staining of the same sample at different siteswith the EuroFlow antibody panels and SOPs.Results of the multi-step procedure to standardize EuroFlowsetup of all instruments were evaluated on a set of PB samplesfollowing the two approaches. The variation observed in multi-center experiments may be caused by multiple factors. Amongthem, the most relevant ones include (1) the pattern of expressionof the molecule investigated (that is, tight peaks for CD4 expressionversus non-homogeneous expression of CD27 in T-cells); (2) stabilityof the fluorochrome and its emission spectra (that is, stable FITCemitting in green versus relatively less stable APCH7 and PECy7tandem fluorochromes with emission in far red resulting invariation due to photon-counting statistics); and (3) affinity andthus titration profile of antibodies (that is, for some antibody clonespipetting errors may lead to changes in staining levels).The present experiment was chosen to allow analysis of distinctpopulations defined by positive markers in every fluorescencechannel and it was designed to mimic the performance ofthe antibody panels for the EuroFlow ‘small sample tube’.29A complex, 8-color tube was chosen as testing tube to includecorrect compensation in the end-point of the test. Allmeasurements were subjected to the previously describedEuroFlow SOPs, including analysis of merged data files using theInfinicyt software. The main question of the presented experimentwas whether biological differences between distinct cell subsetswill be resolved well when all setup procedures described so farare used in eight different EuroFlow laboratories and when themerged data are analyzed by the same software tools.Standardized instrument settings and SOP evaluationexperimentsThe PB of one donor was stabilized using TransFix reagent(Cytomark, Buckingham, UK) and distributed in 1-ml aliquots tothe eight EuroFlow centers; in addition, PB samples were obtained(after informed consent) from 30 different healthy volunteers—that is, one PB sample distributed to all eight centers and 30different PB samples analyzed at eight centers (three to foursamples per center). Instrument setup, compensation and samplepreparation were performed exactly as described in Sections 2, 3and 4, respectively. Reagents used for staining were modified fromone of the 8-color EuroFlow panels (that is, SST)29as follows:CD20-PacB (eBiosciences, San Diego, CA, USA), CD45-PacO(Invitrogen, Carlsbad, CA, USA), CD8-FITC, CD27-PE (ExBio,Prague, Czech Republic), CD4-PerCPCy5.5, CD14-APC and CD3-APCH7 (all from BD Biosciences) and CD19-PECy7 (BeckmanCoulter). After acquisition in the flow cytometers, data wereexported as FCS 3.0 data files. At each center, the following cellsubsets were gated: SSClo/CD45hitotal lymphocytes, CD14þ/CD45himonocytes, CD20hi/CD19þB-lymphocytes, and CD3þ/CD27þmemory T-lymphocytes with both CD3þ/CD4þT-cellsand CD3þ/CD8hiT-cells. Then the MFI values obtained forindividual markers were calculated and reported (Table 10 andFigure 13a). Subsequently, both MFI values and the originallistmode data files were sent to one center (DPH/O, Prague, CzechRepublic) for central analysis. Then, the CV of the MFI valuesobtained for each subset in each channel was calculated. Inaddition, listmode data files were merged with Infinicyt software(version 1.3), monocytes were gated as CD45hi/CD14þcellularevents and total lymphocytes were gated as FSClo/SSClo/CD45hievents and their subsets further defined as listed in Table 10. Next,the merged file was displayed in an APS view (PC1 versus PC2),where each subset was color-coded, and the median of eachsubset was depicted as a color-coded circle as illustrated inFigures 13b and c.Comparison of data obtained at each of the centers showed thatinstrument-related differences caused a CV of target MFI values ofo5.5% (see Section 2 and Table 10). When a stabilized PB sampleobtained at one center was stained, measured and analyzed manuallyat each of the eight centers, CVs for the MFI values of each cellpopulation evaluated were systematically o44%. Similarly, a maximalCV of 44% for CD3-APCH7 on T-cells was observed for normal PBsamples obtained, stained, measured and analyzed at each individualcenter. Notably, CVs below 17% were obtained for 4/8 fluorochrome-conjugated markers assessed in specific cell subsets. Merging alllistmode data files, followed by gating on the different subsets oflymphocytes and monocytes showed that we were able to clearlydistinguish clusters of PB events corresponding to the same cellsubsets from samples drawn from different donors, stained atdifferent centers and measured on different instruments (Figures 13aand b). This illustrates that biological differences are not hidden oraffected by the technical variability. To test the feasibility of mergeddata analysis across flow cytometry platforms, we acquired the sametube (except for CD14-APC) on LSR II and CyAn ADP instruments. Boththe conventional analysis of dot plots (Supplementary Figure 1A) andgraphical analysis of the APS view (Supplementary Figure 1B) showedseparation of major lymphocyte subsets.CONCLUSIONOur collective experiments showed that standardized instrumentsettings, compensation procedures, staining protocols and dataanalysis in multi-institutional collaboration programs are feasible.Data variation resulting from hardware differences (opticalTable 10. Overall results of synchronized experiments expressed in terms of variability obtained in the 8 EuroFlow laboratories for the measurementof antigen expression profiles in normal PB monocytes and lymphocytes (n¼30 different samples), stained, prepared and measured in all 8 centersin parallel versus a stabilized sample obtained in a single center, distributed and then stained, prepared and measured locally at each centerChannel PacB PacO FITC PE PerCPCy5.5 PECy7 APC APCH7Target MFI (Rainbow beads) 195 572 231 265 59 574 101900 216 064 27 462 176 780 56 437Mean ac tual MFI (Rainbow beads) 193 109 225 152 59 003 100 763 215 596 27 639 176 190 56 610CV of Rainbow MFI 5.4% 4.6% 2.1% 2.4% 2.1% 3.1% 1.7% 2.2%Antibody conjugate evaluated CD20 CD45 CD8 CD27 CD4 CD19 CD14 CD3Gating parameters and cell subset CD20hi/CD19þB-cellsCD45hitotallymphocytesCD3þ/CD8hiT-cellsCD3þ/CD27þmemoryT-cellsCD3þ/CD4þT-cellsCD20hi/CD19þB-cellsCD45hi/CD14þmonocytesCD3þT-cellsMFI CV for the cell subset (n¼8 for 1stabilized sample)15.2% 13.9% 11.4% 32.9% 24.7% 11.1% 43.8% 38.7%MFI CV of the cell subset (n¼30samples)16.9% 15.5% 16.9% 28.0% 28.4% 15.4% 22.7% 48.4%Abbreviations: APC, allophycocyanin; Cy7, cyanin7; CV, coefficient of variation; FITC, fluorescein isothiocyanate; MFI, mean fluorescence; H7, hilite7;PacB, pacific blue; PacO, pacific orange; PB, peripheral blood; PE, phycoerythrin; PerCPCy5.5, peridinin–chlorophyll–protein–cyanin5.5.EuroFlow standardization of flow cytometry protocolsT Kalina et al2006Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited elements might have different quality; in some channels differentfilters are used on LSR II and FACSCanto II instruments) or variationfrom other sources is negligible when compared to biologicaldifferences between cell types. However, initial training of localoperators in the applied SOPs is strongly recommended.DISCUSSIONIn constructing the EuroFlow antibody panels for the diagnosisand classification of leukemias and lymphomas using X8-colorflow cytometry, among all technical issues, selection of themost adequate and feasible combination of fluorochromes to beused in the available multicolor flow cytometers was a pre-requisite. Usage of an increasingly high number of fluorochromesis associated with an exponential increase in the amount ofinformation obtained from a single combination of fluorochrome-conjugated antibodies. However, such multicolor/multi-markerapproaches are associated with an increasing complexity and theneed to select the most appropriate/optimal combinations ofindividual reagents, including compatible fluorochromes for whichthe required high-quality antibodies are commercially available.As stated above, several fluorochromes were pre-selected becauseof the extensive experience and proven utility of a high number ofgood-quality antibody conjugates and their match with theFigure 13. Results of synchronized EuroFlow experiments performed on different centers and instruments. (a) Box plot representations ofmean fluorescence intensity (MFI) values observed for all antigens evaluated in the eight gated subsets of peripheral blood (PB) monocytesand lymphocytes from 30 healthy donor PB samples. Results corresponding to a total of 30 merged data files are displayed. (b) Principalcomponent (PC)1 versus PC2 view (automatic population separator (APS) 1 view) of individual cellular events of the cell populations depictedin a; the median values of each gated subset (circles) are color-coded as follows: B-cells, red; CD4þ/CD27þmemory T-cells, light blue; CD4þ/CD27T-cells, dark blue; CD8hi/CD27þmemory T-cells, dark green; CD8hi/CD27T-cells, light green; CD3þ/CD4and CD8T-cells, violet;NK-cells, yellow and monocytes, orange. (c) APS1 view of a single stabilized peripheral blood sample measured in 8 different EuroFlowlaboratories for illustration of intra-donor variability (color coding is the same as in b). In a, results are displayed as box plots, where the line inthe middle represents median values, the upper and lower limits of the box represent the 75th and 25th percentiles, respectively, and theupper and lower ends of vertical lines represent the 95% confidence interval. In b, each population is represented as a circle surrounded bydots corresponding to median values of median expression for all immunophenotypic parameters measured and to individual cells,respectively.EuroFlow standardization of flow cytometry protocolsT Kalina et al2007 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 default optical configuration of the available instruments. Incontrast, for other fluorochrome positions, extensive experi-mental comparisons among different fluorochrome conjugateswere required. Finally, the combination of PacB, PacO, FITC, PE,PerCPCy5.5, PECy7, APC and APCH7 was selected. However, whilethe majority of these fluorochromes performed satisfactorily well,others still require improvement (for example, APCH7). Preliminarytesting of several new alternative fluorochromes (e.g., HV450,HV500 and brilliant violet fluorochromes) show promising results,implying that they might be suited as replacements. However, forsome fluorochrome positions, alternative fluorochromes are notavailable (or became available very recently) or they are justconjugated with a restricted number of CD markers, which limitstheir current applicability but also points to the need for furtherimprovements.The technical EuroFlow approach was designed to establish andmonitor standard instrument settings to a common bright signalplaced at the same level in different flow cytometer instruments.This implies the possibility that some variation might occur in themeasurement of dim/negative signals owing to small differencesbetween individual instruments. However, it assures that indivi-dual flow cytometers work above their detector’s backgroundnoise. Slight differences in laser power output, sharpness of theoptical filters’ edges and other hardware-associated variablesmight account for such small deviations.32,33In fact, we detectedhigher MFI for unstained cells in the violet laser fluorescencechannels at one occasion in one instrument. The violet laser of thisinstrument had to be replaced by the manufacturer owing to alow laser power delivered to the flow cell. In 2008 the CS Tmodule was introduced in the 6.0 version of the FACSDiVasoftware.58Please note that this module is made for automatedinstrument characterization and automated calculation of optimalvoltage settings for single instruments and does not deal withstandardized multicenter setting approaches. With the CS Tmodule, PMT voltages are set at a value that is 10 times thestandard deviation of electronic noise. Small differences werefound between CS T and EuroFlow settings owing to the differentcriteria for optimal setting of PMT voltages. Whereas CS T favorshigher sensitivity for dim signals, the EuroFlow settings aretailored to measure both dim signals and signals generated bymolecules with very high levels of expression (for example, CD38expression on normal plasma cells). In practice, both methods (theEuroFlow settings and the CS T module) were associated withoptimal PMT settings and allow for an early detection ofinstrument failure (for example, laser failure). Unfortunately,automated adjustments at daily monitoring by the CS T modulesystematically require a full new compensation experiment, evenif the variation obtained for the new adjustment is as low as±1 mV with no real impact on the compensation matrix. Dailyperformance of a full compensation experiment is expensive,time-consuming and consequently inefficient in diagnosticlaboratories. In addition, it is not supported by the minorchanges observed in the compensation matrix. An additionaladvantage of the EuroFlow approach is its flexibility and itsapplicability for 8-color flow cytometers from differentmanufacturers. Based on the long-term stability of MFImeasurements, once-fixed PMT voltages were used. We adoptedacceptance criteria for deviations of up to 15% from the target MFIvalues, for instrument settings to pass during daily monitoring. Auser-friendly software tool and graphics were built into theInfinicyt software for a quick color-code assessment of anydeviation from the accepted criteria for optimal instrumentsettings. The stringency of such criteria should be driven by thepurpose of standardization. In immunophenotyping ofhematological malignancies, the biological intra- and inter-sample differences are quite high and they are not hidden oraffected by changes in fluorescence intensity values of up to 30%(EuroFlow data; not shown). As described in Section 5 with newautomated software-driven analytical approaches thatsimultaneously take into account all markers and theirintensities at the same time, the relative relevance of small MFIchanges in individual markers is significantly diminished. Inaddition, we also show that these criteria can be easily met bydifferent instruments at different sites.Usage of an optimal fluorescence compensation matrixis currently considered as a requirement for optimal identificationof single- versus double-positive cells in multicolor flowcytometry immunophenotyping.37The complexity of theprocedure designed to set up the optimal fluorescencecompensation matrix depends on the specific multicolorantibody panels. As could be predicted, single compounddyes were represented by one ‘generic’ SAbST (onerepresentative marker stained in a specific cell population),while tandem fluorochromes were represented by one tube foreach specific fluorochrome conjugate antibody. The onlyexception to this rule was the PerCPCy5.5 tandem fluorochrome.However, it should be noted that, in contrast to PECy7 and APCH7,PerCPCy5.5 is a tandem fluorochrome where both compounds ofthe tandem show maximum emission into the same bandpassfilter; this could explain why no fluorochrome-specificcompensation is needed. The similar spillover values fordifferent PerCPCy5.5 reagents were confirmed in a small-scaleexperiment (data not shown).Fluorescence compensation experiments consisting of a full setof compensation controls (n¼30 tubes) represent a challenge fortime-stressed laboratories as well as a burden for laboratorybudget. Thus, the frequency of compensation could be set to atime interval of 1 month, during which only minor deviations fromtarget MFI were recorded on well-performing instruments.However, gradual 405-nm laser power failures often resulted insignificant signal shifts that required new instrument setup andcompensation experiments, more frequently than initiallyplanned. Careful selection of reagents with sufficient life-span,especially with regard to tandem fluorochromes, and protection oflight-sensitive reagents is crucial for acquisition of high-qualitydata in the once-per-month compensation scheme. Based on thecomparison of the fluorescence compensation matrices obtainedover time for the same instrument, we concluded that it is notnecessary to repeat the compensation experiment whenever boththe reagents and the signal collection on the instrument arestable. However, the stability of tandem fluorochromes is notreliably constant for all manufacturers and it depends on thestorage and handling conditions. Our 1-month compensationapproach was feasible as judged by evaluation of X2000 mergedPB, BM, LN, CSF and vitreous humor samples of multiple diseasecategories acquired over the past 6 years (data not shown). In turn,a software solution that would allow automated and rapidestablishment of fluorescence compensation settings to experi-ments, after PMT voltages had been adjusted to ‘Target MFI’, couldbe of great help for clinical flow cytometry laboratories.Interestingly, our multicenter results indicate that such anapproach is apparently feasible, owing to the highly stablecompensation settings observed in our study at both the inter-and intra-laboratory level.The EuroFlow SOPs for sample preparation was developedbecause of its ability to provide robust and reliable data thatmeet all the criteria indicated above. In combination with thestandardized EuroFlow SOPs to define instrument settings andfluorescence compensation, it allows generation of highlycomparable and reproducible data for a single instrument andbetween different instruments within the same laboratory andbetween different laboratories. Such highly reproducible data arerequired not only for the comparison of data obtained within thedifferent EuroFlow laboratories, but also for the construction of adatabase with immunophenotypic data from large numbersof patients suffering from the various subtypes of relevantEuroFlow standardization of flow cytometry protocolsT Kalina et al2008Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers Limited hematological malignancies, which can be potentially used as areference by any laboratory worldwide.Until now, standardization of flow cytometry in hematologicaldiagnostic processes remains a challenge and it is rarely achieved ina multi-institutional setting. There have been some attempts tostandardize the analysis of minimal residual disease in multiplestudy groups9,59–61that restricted the standardization on theanalytical stage by exchanging the listmode data files. Kraan et al.62have presented general rules for cytometer setup in clinical settingsusing analog flow-cytometric systems with up to four colors,whereas Shankey et al.63presented complete standardization of4-color ZAP-70 investigation in three institutions. Our present studygoes further beyond the so-far-reported multi-center studies andaims at standardization of the data to the level at which listmodefiles measured in all centers can be meta-analyzed by software toolsspecifically designed for this purpose. The whole process ofcytometer settings, compensation settings, fluorochrome selectionand antibody panel selection was re-evaluated and fully controlled.The need for such extensive standardization arises from thepossibilities that are brought by three-laser X8-color digital flowcytometers to measure increasingly detailed subsets in complexcellular samples. Cell definitions using 44 colors are thought toenhance the accuracy of rare cell detection such as used forminimal residual disease studies. Analysis of surface/cytoplasmicexpression patterns on large cohorts of samples by computationaltools is possible only when the input data are supplied in a fullystandardized format. Sharing of knowledge and diagnosing rarediseases will be made possible by manual or computer-assistedanalysis of data files acquired in multi-institutional cooperation.Here again, appropriate interpretation of the data is possible onlywhen standardized instrument settings and controls are used, thequality of the data is ensured, and the performance of the antibodypanels is evaluated and taken into account during analysis.We conclude that the 6 years of extensive collaborativeexperiments and the analysis of hundreds of patients’ samplesin the EuroFlow centers have indeed provided innovativeprotocols, software tools and antibody panels for fully standar-dized diagnosis and classification of hematological malignancies.CONFLICT OF INTERESTThe EuroFlow Consortium is an independent scientific consortium which aims atinnovation and standardization of diagnostic flow cytometry. All acquired knowledgeand experience will be shared with the scientific and diagnostic community afterprotection of the relevant Intellectual Property, for example by filing of patents. Theinvolved patents are collectively owned by the EuroFlow Consortium and the revenuesof the patents are exclusively used for EuroFlow Consortium activities, such as forcovering (in part) the costs of the Consortium meetings, the EuroFlow EducationalWorkshops and the purchase of custom-made reagents for collective experiments.Cytognos is for-profit company developing the Infinicyt software.ACKNOWLEDGEMENTSWe are grateful to Dr Jean-Luc Sanne of the European Commission for his supportand monitoring of the EuroFlow project. We thank Marieke Comans-Bitter for graphicdesign of the figures and for her continuous support in the management of theEuroFlow Consortium, and Bibi van Bodegom, Caroline Linker, and Monique vanRossum for their secretarial support of the consortium activities. We are grateful toRia Bloemberg and Gellof van Steenis for support in the financial management of theproject funds. The research activities of the EuroFlow Consortium were supported bythe European Commission (grant STREP EU-FP6, LSHB-CT-2006-018708, entitled ‘Flowcytometry for fast and sensitive diagnosis and follow-up of hematologicalmalignancies’) and the following national grants: Spanish Network of CancerResearch Centers (ISCIII RTICC-RD06/0020/0035-FEDER), FIS 08/90881 from the ‘Fondode Investigacio´n Sanitaria’, Ministerio de Ciencia e Innovacio´n (Madrid, Spain), SA016-A-09 from the Consejerı´a de Educacio´n, Junta de Castilla y Leo´n, Valladolid, Spain,and PIB2010BZ-00565 from the Direccio´n General de Cooperacio´n Internacional yRelaciones Institucionales, Secretarı´a de Estado de Investigacio´n, Ministerio deCiencia e Innovacio´n (Madrid, Spain). T Kalina, E Mejstrikova and O Hrusak weresupported by the Czech Ministry of Education Grant No. MSM0021620813, CzechMinistry of Health grant NT/12425-4 and TK is supported as an ISAC Scholar by TheInternational Society for Advancement of Cytometry.REFERENCES1 Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H et al. WHO Classifi-cation of Tumours of Haematopoietic and Lymphoid Tissues. 4th edn. InternationalAgency for Research on Cancer: Lyon, 2008, 439 pp.2 Davis BH, Holden JT, Bene MC, Borowitz MJ, Braylan RC, Cornfield D et al.Bethesda International Consensus recommendations on the flow cytometricimmunophenotypic analysis of hematolymphoid neoplasia: medical indications.Cytometry B Clin Cytom 2007; 72(Suppl 1): S5–S13.3 Wood BL, Arroz M, Barnett D, DiGiuseppe J, Greig B, Kussick SJ et al. BethesdaInternational Consensus recommendations on the immunophenotypic analysis ofhematolymphoid neoplasia by flow cytometry: optimal reagents and reporting forthe flow cytometric diagnosis of hematopoietic neoplasia. Cytometry B Clin Cytom2007; 72(Suppl 1): S14–S22.4 Stewart CC, Behm FG, Carey JL, Cornbleet J, Duque RE, Hudnall SD et al.U.S.-Canadian Consensus recommendations on the immunophenotypic analysisof hematologic neoplasia by flow cytometry: selection of antibody combinations.Cytometry 1997; 30: 231–235.5 Stetler-Stevenson M, Ahmad E, Barnett D, Braylan R, DiGiuseppe J, Marti G et al.Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells. Approvedguideline. 2nd edn. CLSI document H43-A2 ed. Clinical and Laboratory StandardsInstitute: Wayne, PA, 2007.6 Perfetto SP, Chattopadhyay PK, Roederer M. Seventeen-colour flow cytometry:unravelling the immune system. Nat Rev Immunol 2004; 4: 648–655.7 Baumgarth N, Roederer M. A practical approach to multicolor flow cytometry forimmunophenotyping. J Immunol Methods 2000; 243: 77–97.8 Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neo-plasms. Blood 2008; 111: 3941–3967.9 van de Loosdrecht AA, Alhan C, Bene MC, Della Porta MG, Drager AM, Feuillard Jet al. Standardization of flow cytometry in myelodysplastic syndromes: reportfrom the first European LeukemiaNet working conference on flow cytometry inmyelodysplastic syndromes. Haematologica 2009; 94: 1124–1134.10 Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H et al.Report of the European Myeloma Network on multiparametric flow cytometry inmultiple myeloma and related disorders. Haematologica 2008; 93: 431–438.11 Ruiz-Arguelles A, Rivadeneyra-Espinoza L, Duque RE, Orfao A. Report on thesecond Latin American consensus conference for flow cytometric immunophe-notyping of hematological malignancies. Cytometry B Clin Cytom 2006; 70: 39–44.12 Owens MA, Vall HG, Hurley AA, Wormsley SB. Validation and quality control ofimmunophenotyping in clinical flow cytometry. J Immunol Methods 2000; 243: 33–50.13 Davis BH, Foucar K, Szczarkowski W, Ball E, Witzig T, Foon KA et al. U.S.-CanadianConsensus recommendations on the immunophenotypic analysis of hematologicneoplasia by flow cytometry: medical indications. Cytometry 1997; 30: 249–263.14 Bene MC, Nebe T, Bettelheim P, Buldini B, Bumbea H, Kern W et al. Immuno-phenotyping of acute leukemia and lymphoproliferative disorders: a consensusproposal of the European LeukemiaNet Work Package 10. Leukemia 2011; 25:567–574.15 Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay.Clin Lab Med 2007; 27: 469–485.16 Roederer M, Kantor AB, Parks DR, Herzenbergzzz LA. Cy7PE and Cy7APC: brightnew probes for immunofluorescence. Cytometry 1996; 24: 191–197.17 Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML et al.Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol1999; 17: 969–973.18 Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ et al. Quantitativecomparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of thedyes and their bioconjugates. J Histochem Cytochem 2003; 51: 1699–1712.19 Telford W, Kapoor V, Jackson J, Burgess W, Buller G, Hawley T et al. Violet laserdiodes in flow cytometry: an update. Cytometry A 2006; 69: 1153–1160.20 Abrams B, Diwu Z, Guryev O, Aleshkov S, Hingorani R, Edinger M et al. 3-Carboxy-6-chloro-7-hydroxycoumarin: a highly fluorescent, water-soluble violet-excitabledye for cell analysis. Anal Biochem 2009; 386: 262–269.21 Maecker H, Trotter J. Selecting Reagents for Multicolor Flow Cytometry. ApplicationNote. BD Biosciences: San Jose, CA, 2009.22 Stewart CC, Stewart SJ. Four color compensation. Cytometry 1999; 38: 161–175.23 Roederer M. Spectral compensation for flow cytometry: visualization artifacts,limitations, and caveats. Cytometry 2001; 45: 194–205.24 Maecker HT, Frey T, Nomura LE, Trotter J. Selecting fluorochrome conjugates formaximum sensitivity. Cytometry A 2004; 62: 169–173.25 Wood B. 9-color and 10-color flow cytometry in the clinical laboratory. Arch PatholLab Med 2006; 130: 680–690.EuroFlow standardization of flow cytometry protocolsT Kalina et al2009 2012 Macmillan Publishers Limited Leukemia (2012) 1986 – 2010 26 Kraan J, Gratama JW, Haioun C, Orfao A, Plonquet A, Porwit A et al. Flow cyto-metric immunophenotyping of cerebrospinal fluid. Curr Protoc Cytom 2008;Chapter 6: Unit 6 25.27 Basso G, Buldini B, De Zen L, Orfao A. New methodologic approaches forimmunophenotyping acute leukemias. Haematologica 2001; 86: 675–692.28 McLaughlin BE, Baumgarth N, Bigos M, Roederer M, De Rosa SC, Altman JD et al.Nine-color flow cytometry for accurate measurement of T cell subsets andcytokine responses. Part I: Panel design by an empiric approach. Cytometry A2008; 73: 400–410.29 van Dongen JJM, Lhermitte L, Bo¨ttcher S, Almeida J, van der Velden VHJ, Flores-Montero J et al. EuroFlow antibody panels for standardized n-dimensional flowcytometric immunophenotyping of normal, reactive and malignant leukocytes.Leukemia 2012; doi:10.1038/leu.2012.120.30 Le Roy C, Varin-Blank N, Ajchenbaum-Cymbalista F, Letestu R. Flow cytometryAPC-tandem dyes are degraded through a cell-dependent mechanism. CytometryA2009; 75: 882–890.31 Hulett HR, Bonner WA, Sweet RG, Herzenberg LA. Development and application ofa rapid cell sorter. Clin Chem 1973; 19: 813–816.32 Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and thedetermination of positivity. Cytometry A 2006; 69: 1037–1042.33 Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay P, Roederer M. Qualityassurance for polychromatic flow cytometry. Nat Protoc 2006; 1: 1522–1530.34 Hughes OR, Stewart R, Dimmick I, Jones EA. A critical appraisal of factors affectingthe accuracy of results obtained when using flow cytometry in stem cell inves-tigations: where do you put your gates? Cytometry A 2009; 75: 803–810.35 Shapiro HM. Excitation and emission spectra of common dyes. Curr Protoc Cytom2004; Chapter 1: Unit 1 19.36 Tung JW, Parks DR, Moore WA, Herzenberg LA. New approaches to fluorescencecompensation and visualization of FACS data. Clin Immunol 2004; 110: 277–283.37 Roederer M. Compensation in flow cytomet ry. Curr Protoc Cytom 2002; 1:1Chapter 1: Unit 1 14.38 Groeneveld K, te Marvelde JG, van den Beemd MW, Hooijkaas H, van Dongen JJ.Flow cytometric detection of intracellular antigens for immunophenotyping ofnormal and malignant leukocytes. Leukemia 1996; 10: 1383–1389.39 Macey MG, McCarthy DA, Milne T, Cavenagh JD, Newland AC. Comparative studyof five commercial reagents for preparing normal and leukaemic lymphocytes forimmunophenotypic analysis by flow cytometry. Cytometry 1999; 38: 153–160.40 Stewart CC, Stewart SJ. Immunophenotyping. Curr Protoc Cytom 2001; Chapter 6:Unit 6 2.41 Holmes K, Lantz LM, Fowlkes BJ, Schmid I, Giorgi JV. Preparation of cells andreagents for flow cytometry. Curr Protoc Immunol 2001; Chapter 5: Unit 5 3.42 Kappelmayer J, Gratama JW, Karaszi E, Menendez P, Ciudad J, Rivas R et al. Flowcytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interac-tion between monoclonal antibody clones, fluorochromes and sample prepara-tion protocols. J Immunol Methods 2000; 242: 53–65.43 Quijano S, Lopez A, Sancho JM, Panizo C, Deben G, Castilla C et al. Identificationof leptomeningeal disease in aggressive B-cell non-Hodgkin’s lymphoma:improved sensitivity of flow cytometry. J Clin Oncol 2009; 27: 1462–1469.44 Owens MA, Loken MR. Flow Cytometry Principles for Clinical Laboratory Practice:Quality Assurance for Quantitative Immunophenotyping. Wiley-Liss: New York;Chichester, 1995, xiii, 224 pp.45 Caraux A, Klein B, Paiva B, Bret C, Schmitz A, Fuhler GM et al. Circulating human Band plasma cells. Age-associated changes in counts and detailed characterizationof circulating normal CD1 38- and CD138 þplasma cells. Haematologica 2010; 95:1016–1020.46 Braylan RC, Orfao A, Borowitz MJ, Davis BH. Optimal number of reagents requiredto evaluate hematolymphoid neoplasias: results of an international consensusmeeting. Cytometry 2001; 46: 23–27.47 Sanchez ML, Almeida J, Vidriales B, Lopez-Berges MC, Garcia-Mar cos MA, Moro MJet al. Incidence of phenotypic aberrations in a series of 467 patients with Bchronic lymphoproliferative disorders: basis for the design of specific four-colorstainings to be used for minimal residual disease investigation. Leukemia 2002;16: 1460–1469.48 Pedreira CE, Costa ES, Barrena S, Lecrevisse Q, Almeida J, van Dongen JJ et al.Generation of flow cytometry data files with a potentially infinite number ofdimensions. Cytometry A 2008; 73: 834–846.49 Pedreira CE, Costa ES, Almeida J, Fernandez C, Quijano S, Flores J et al. A prob-abilistic approach for the evaluation of minimal residual disease by multi-parameter flow cytometry in leukemic B-cell chronic lymphoproliferativedisorders. Cytometry A 2008; 73A: 1141–1150.50 Costa ES, Pedreira CE, Barrena S, Lecrevisse Q, Flores J, Quijano S et al. Automatedpattern-guided principal component analysis vs expert-based immunopheno-typic classification of B-cell chronic lymphoproliferative disorders: a step forwardin the standardization of clinical immunophenotyping. Leukemia 2010; 24:1927–1933.51 Costa ES, Arroyo ME, Pedreira CE, Garcia-Marcos MA, Tabernero MD, Almeida Jet al. A new automated flow cytometry data analysis approach for the diagnosticscreening of neoplastic B-cell disorders in peripheral blood samples with absolutelymphocytosis. Leukemia 2006; 20: 1221–1230.52 Robinson JP, Durack G, Kelley S. An innovation in flow cytometry data collectionand analysis producing a correlated multiple sample analysis in a single file.Cytometry 1991; 12: 82–90.53 Robinson JP, Ragheb K, Lawler G, Kelley S, Durack G. Rapid multivariate analysisand display of cross-reacting antibodies on human leukocytes. Cytometry 1992;13: 75–82.54 Cover TM, Hart PE. Nearest neighbour pattern clasification. IEEE Trans Inf Theory1967; IT-13: 21–27.55 Duda RO, Hart PE, Stork DG, RO Duda. PC, scene a. In: Duda RO, Hart PE, Stork DG(eds) Pattern Classification. 2nd edn Wiley: New York; Chichester, 2001; 654 pp.56 Costa ES, Peres RT, Almeida J, Lecrevisse Q, Arroyo ME, Teodosio C et al. Har-monization of light scatter and fluorescence flow cytometry profilesobtained after staining peripheral blood leucocytes for cell surface-only versusintracellular antigens with the Fix Perm reagent. Cytometry B Clin Cytom 2010;78: 11–20.57 Lugli E, Roederer M, Cossarizza A. Data analysis in flow cytometry: the future juststarted. Cytometry A 2010; 77: 705–713.58 Stall A. Qr and Br in BD FACSDiva v6 Software: Parameters for CharacterizingDetector Performance. Application Note 23-10516-00. BD Biosciences: San Diego,CA, 2008.59 Bjorklund E, Matinlauri I, Tierens A, Axelsson S, Forestier E, Jacobsson S et al.Quality control of flow cytometry data analysis for evaluation of minimal residualdisease in bone marrow from acute leukemia patients during treatment. J PediatrHematol Oncol 2009; 31: 406–415.60 Dworzak MN, Gaipa G, Ratei R, Veltroni M, Schumich A, Maglia O et al. Standar-dization of flow cytometric minimal residual disease evaluation in acute lym-phoblastic leukemia: multicentric assessment is feasible. Cytometry B Clin Cytom2008; 74: 331–340.61 Irving J, Jesson J, Virgo P, Case M, Minto L, Eyre L et al. Establishment andvalidation of a standard protocol for the detection of minimal residual disease inB lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting. Haematologica 2009; 94: 870–874.62 Kraan J, Gratama JW, Keeney M, D’Hautcourt JL. Setting up and calibration of aflow cytometer for multicolor immunophenotyping. J Biol Regul Homeost Agents2003; 17: 223–233.63 Shankey TV, Forman M, Scibelli P, Cobb J, Smith CM, Mills R et al. An optimizedwhole blood method for flow cytometric measurement of ZAP-70 proteinexpression in chronic lymphocytic leukemia. Cytometry B Clin Cytom 2006; 70:259–269.This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view acopy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)EuroFlow standardization of flow cytometry protocolsT Kalina et al2010Leukemia (2012) 1986 – 2010 2012 Macmillan Publishers LimitedCitations (0)References (44)ResearchGate has not been able to resolve any citations for this publication.EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytesArticleFull-text availableMay 2012 J.J.M. van Dongen Ludovic LhermitteSebastian Böttcher Alberto OrfaoMost consensus leukemia lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.ViewShow abstractWHO Classification of Tumours of the Haematopoietic and Lymphoid TissuesBookFull-text availableJan 2008 Elaine S JaffeS. Swerdlow Elias CampoJ.W. WardimanViewCirculating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138- and CD138+ plasma cellsArticleFull-text availableJun 2010HaematologicaAnouk CarauxBernard Klein Bruno Paiva Martin Perez-AndresGeneration of B and plasma cells involves several organs with a necessary cell trafficking between them. A detailed phenotypic characterization of four circulating B-cell subsets (immature-, naïve-, memory- B-lymphocytes and plasma cells) of 106 healthy adults was realized by multiparametric flow cytometry. We show that CD10, CD27 and CD38 is the minimal combination of subsetting markers allowing unequivocal identification of immature (CD10(+)CD27(-)CD38(+), 6+/-6 cells/microL), naïve (CD10(-)CD27(-)CD38(-), 125+/-90 cells/microL), memory B lymphocytes (CD10(-)CD27(+)CD38(-), 58+/-42 cells/microL), and plasma cells (CD10(-)CD27(++)CD38(++), 2.1+/-2.1 cells/microL) within circulating CD19(+) cells. From these four subsets, only memory B lymphocytes and plasma cells decreased with age, both in relative and absolute counts. Circulating plasma cells split into CD138(-) (57+/-12%) and CD138(+) (43+/-12%) cells, the latter displaying a more mature phenotypic profile: absence of surface immunoglobulin, lower CD45 positivity and higher amounts of cytoplasmic immunoglobulin, CD38 and CD27. Unlike B lymphocytes, both populations of plasma cells are KI-67(+) and show weak CXCR4 expression.ViewShow abstractCy7PE and Cy7APC: Bright new probes for immunofluorescenceArticleJul 1996CytometryMario Roederer Aaron B. KantorDavid R. ParksLeonard A. HerzenbergWe demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a \"brightness” (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a \"brightness” comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC. © 1996 Wiley-Liss, Inc.ViewShow abstract3-Carboxy-6-chloro-7-hydroxycoumarin: A highly fluorescent, water-soluble violet-excitable dye for cell analysisArticleMar 2009ANAL BIOCHEMBarny Abrams Zhenjun Diwu Oleg Guryev Tim DubrovskyIn our search for new violet-excitable dyes with improved photophysical and photochemical properties, we examined several halogen-substituted hydroxycoumarins and found that chlorinated derivatives are at least as bright as their fluorinated analogs. A monochlorinated hydroxycoumarin was found to have a high quantum yield (∼0.98), and human leucocyte-specific monoclonal antibodies (CD3, CD4, and CD45) conjugated with this dye exhibited reliable performance in flow cytometry assays. Additional studies were performed, with BD Horizon V450–antibody conjugates being included in eight-color cocktails aimed at subsetting lymphocytes and myeloid cells. Such cocktails can frequently be unstable due to the tendency of one or more components to lose structural integrity, photobleach, or develop unwanted affinities for another component. However, the cocktails employed in this study enabled several different applications to be run and established that multicolor reagent mixtures containing V450–antibody conjugates are functional and stable.ViewShow abstractSelecting Fluorochrome Conjugates for Maximum SensitivityArticleDec 2004CYTOM PART AHolden T MaeckerTom FreyLaurel NomuraJoe TrotterViewImmunophenotyping of acute leukemia and lymphoproliferative disorders: A consensus proposal of the European LeukemiaNet Work Package 10ArticleJan 2011 Marie Bene Thomas Nebe Peter Bettelheim Anna PorwitThe European LeukemiaNet (ELN), workpackage 10 (WP10) was designed to deal with diagnosis matters using morphology and immunophenotyping. This group aimed at establishing a consensus on the required reagents for proper immunophenotyping of acute leukemia and lymphoproliferative disorders. Animated discussions within WP10, together with the application of the Delphi method of proposals circulation, quickly led to post-consensual immunophenotyping panels for disorders on the ELN website. In this report, we established a comprehensive description of these panels, both mandatory and complementary, for both types of clinical conditions. The reason for using each marker, sustained by relevant literature information, is provided in detail. With the constant development of immunophenotyping techniques in flow cytometry and related software, this work aims at providing useful guidelines to perform the most pertinent exploration at diagnosis and for follow-up, with the best cost benefit in diseases, the treatment of which has a strong impact on health systems.ViewShow abstractData Analysis in Flow Cytometry: The Future Just StartedArticleJul 2010Cytometry Enrico LugliMario Roederer Andrea CossarizzaIn the last 10 years, a tremendous progress characterized flow cytometry in its different aspects. In particular, major advances have been conducted regarding the hardware/instrumentation and reagent development, thus allowing fine cell analysis up to 20 parameters. As a result, this technology generates very complex datasets that demand for the development of optimal tools of analysis. Recently, many independent research groups approached the problem by using both supervised and unsupervised methods. In this article, we will review the new developments concerning the use of bioinformatics for polychromatic flow cytometry and propose what should be done to unravel the enormous heterogeneity of the cells we interrogate each day.ViewShow abstractFlow Cytometry APC-Tandem Dyes Are Degraded Through a Cell-Dependent MechanismArticleOct 2009Cytometry Christine Le Roy Nadine Varin-BlankFlorence Ajchenbaum-Cymbalista Remi LetestuTechnological developments of multiparametric flow cytometry come along with the generation of new dyes. The APC-tandem dyes, which combine the fluorophores APC and Cy7/H7, allow the detection of a specific signal in the APC-Cy7/H7 channel along with an unexpected nonspecific signal in the APC channel. Depending on the magnitude of the latter, it may be a handicap for interpreting the data of multicolor labeling experiments. We investigated the alteration of the APC-tandem dyes by labeling peripheral blood cells with antibodies directed toward leukocyte surface proteins and by analyzing cells by flow cytometry. Our results show that the APC-Cy7/H7 tandem fluorochromes degraded over time. Nonspecific APC signal was observed with the various antibodies tested only upon cell attachment but not under bead linkage. Moreover, the percentage of degradation of the APC-Cy7/H7 dyes was dependent on the cell type analyzed. Interestingly, nonspecific APC signal strongly decreased when the metabolic activity of immunolabeled cells was inhibited or when cells were incubated with vitamin C. This study demonstrates that the APC-tandem dyes are the target of cell-dependent degradation, which may be antagonized. These findings will allow cytometer users to optimize their multicolor panels.ViewShow abstractHarmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface-only versus intracellular antigens with the Fix Perm™ reagentArticleJan 2009Cytometry B Clin Cytometry Elaine S CostaRodrigo T. PeresJulia Almeida Alberto OrfaoStaining for intracellular markers with the Fix Perm reagent is associated with variations in the scatter properties of leucocytes, limiting automated analysis of flow cytometry (FCM) data. Here, we investigated those variables significantly contributing to changes in the light scatter, autofluorescence, and bcl2 staining characteristics of peripheral blood (PB) leucocytes, after fixation with Fix Perm. Our major aim was to evaluate a new mathematical approach for automated harmonization of FCM data from datafiles corresponding to aliquots of a sample treated with cell-surface-only versus Fix Perm intracellular staining techniques. Overall, neither the anticoagulant used nor sample storage for 24 h showed significant impact on the light scatter and fluorescence properties of PB leucocytes; similarly, the duration of the fixation period (once 15 min were used) had a minimum impact on the FCM properties of PB leucocytes. Conversely, changes in cell/protein concentrations and the fixative/sample (vol/vol) ratio had a clear impact on the light scatter features of some populations of leucocytes. Accordingly, lower cell/protein concentrations were associated with lower scatter values, particularly for the neutrophils. Such changes could be partially corrected through the use of higher fixative to sample volume ratios. Despite the variable changes detected between aliquots of the same sample treated with cell surface-only versus intracellular staining procedures, the new mathematical approach here proposed and evaluated for automated harmonization of common parameters in both datafiles, could correct the FCM profiles of leucocytes derived from cells undergoing conventional fixation/permeabilization procedures, and made them indistinguishable from those corresponding to aliquots of the same sample treated with cell-surface-only staining techniques.ViewShow abstractShow moreAdvertisementRecommendationsDiscover moreProjectFAB CD81 cell isolotation Ondřej Pelák Tomas Kalina Jan StuchlýView projectProjectMicrosphereAffinityProteomics Fridtjof Lund-Johansen Tomas Kalina Jan Stuchlý[...] Kjetil RøyslandDevelop a unified platform for high-resolution antibody array analysis and MS-based proteomics View projectProjectEuroFlow project Ludovic Lhermitte Vincent H J van der Velden Alberto Orfao[...] Anne E BrasView projectProjectCVID project Veronika Kanderová Tomas Kalina Anna Sediva[...] Tomas FreibergerView projectArticleFull-text availableEuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocolsSeptember 2012 · Leukemia Tomas KalinaJuan Flores-Montero Vincent H J van der Velden[...] Alberto OrfaoThe EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of ... [Show full abstract] compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.View full-textArticleFluorochrome choices for multi-color flow cytometryJune 2019 · Journal of Immunological MethodsJuan Flores-Montero Tomas KalinaAlba Corral-Mateos[...]Alberto OrfaoFluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the fluorescence profiles (excitation and emission), ii) ... [Show full abstract] relative brightness, iii) fluorescence overlap, iv) fluorochrome stability, and v) reproducible conjugation to antibodies. Here we used 75 samples (45 bone marrow and 30 blood) to illustrate EuroFlow strategies for evaluation of compatible fluorochromes, and how the results obtained guide fluorochrome selection as a critical step in the antibody-panel building process. Our results allowed identification of optimal fluorescence profiles (e.g. higher fluorescence intensity and/or resolution with limited fluorescence overlap into neighbor channels) for brilliant violet (BV)421 and BV510 in the violet laser and allophycocyanin (APC) hilite 7 (H7) or APC C750 in the red laser vs. other candidate fluorochromes generally applied for the same detectors and here evaluated. Moreover, evaluation of the same characteristics for another group of fluorochromes (e.g. BV605, BV650, PE CF594, AF700 or APC AF700) guided selection of the most appropriate fluorochrome conjugates to be combined in a multi-color antibody panel. Albeit this is a demanding approach, it could be successfully applied for selection of fluorochrome combinations for the EuroFlow antibody panels for diagnosis, classification and monitoring of hematological malignancies and primary immunodeficiencies. Consequently, sets of 8-, 10- and 12-color fluorochrome combinations are proposed as frame of reference for initial antibody panel design.Read moreArticleFull-text availableEuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal,...May 2012 · Leukemia J.J.M. van Dongen Ludovic LhermitteSebastian Böttcher[...] Alberto OrfaoMost consensus leukemia lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by ... [Show full abstract] clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.View full-textArticlePitfalls in the use of multicolour flow cytometry in haematologyMarch 2011 · Journal of Clinical Pathology Ulrika JohanssonMarion MaceyMulticolour flow cytometry in haematology has developed considerably in recent years. The ability to analyse eight or more colours of fluorescence on millions of cells in a matter of minutes has enabled the provision of rapid and reliable measures of minimal residual disease for clinicians. The use of multicolour analysis has also enabled more specific characterisation of presenting leukaemias ... [Show full abstract] and lymphomas. However, there has not been a concomitant increase in the knowledge and experience of the flow cytometrists to deal with certain problems associated with this more complex analysis.Read moreDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! Please log in.Email · HintTip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleNo account? Sign upCompanyAbout usNewsCareersSupportHelp CenterBusiness solutionsAdvertisingRecruiting© 2008-2021 ResearchGate GmbH. All rights reserved.TermsPrivacyCopyrightImprint