Calibración de equipos de citometría para empleo de paneles EuroFlow

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1 Calibración de equipos de citometría para empleo de paneles EuroFlow Departamento de Medicina, Centro de Investigación del Cáncer y Servicio de Citometría. Universidad de Salamanca, Salamanca, España. EuroFlow TM Consortium Curso avanzado de Actualización en Onco Hematología. Buenos Aires, Mayo, 30 a Junio, 1, 2011

2 Standardization in diagnostic flow cytometry Standardization according to literature generally refers to: lists of CD codes and markers per disease category rarely a specific antibody is recommended and (almost) never a fluorochrome is proposed HOWEVER: Standardization according to GLP guidelines demands for much higher levels of standardization EuroFlow standardization aims at: usage of comparable flow cytometers (3 lasers and 8 colors) full standardization of instrument settings (e.g. based on standard beads) standardized laboratory protocols and immunostaining procedures (SOP s) careful selection of optimal antibody clones per marker/cd code selection of optimal 8-color antibody combinations and fluorochromes design of combinations of multiple 8-color tubes: estimation and APS view new software for fast and easy data analysis with automated pattern recognition recognition of normal and abnormal leukocyte subsets (complete differentiation pathways) with the same immunostaining protocols mapping of new patient samples against large data base of earlier collected patient samples, analyzed with the same immunostaining protocol

3 Standard operating procedures: What for? Get optimal, reproducible & comparable results

4 Standard operating procedures: What for? Get optimal, reproducible & comparable results

5 Standard operating procedures: What for? Get optimal, reproducible & comparable results

6 Standard operating procedures: How? Get optimal, reproducible & comparable results Instruments and set-up Fluorochromes selection & compensation issues Standardization Antibody reagents Sample preparation protocols

7 Standard operating procedures: How? Get optimal, reproducible & comparable results Instruments and set-up Fluorochromes selection & compensation issues Standardization Antibody reagents Sample preparation protocols

8 Instruments Optical configuration: Channel Laser Dichroic filter FACSCantoII (BD Bioscience) FACSAria (BD Bioscience) Emission filter LSRII (BD Bioscience) Cyan ADP (Dako/Beckman Coulter) 1 Violet 450/50 450/50 450/50 450/50 2 Violet /50 530/30 525/50 530/40 3 Blue /30 530/30 530/30 530/40 4 Blue /42 585/42 575/26 585/42 5 Blue 613/20 6 Blue LP 695/40 695/40 680/30 7 Blue /60 780/60 780/60 E750LP 8 Red 660/20 660/20 660/20 665/20 9 Red /60 780/60 780/60 E750LP

9 Instrument set up: Window of analysis SSC-A Exp-SSC Low CD8-APCH7 FSC-A LINEAR CD16-PECy7 Stable, reliable system reproduce, share and monitor Normal peripheral (PB) sample processed according to EuroFlow sample preparation protocol

10 Instrument set-up: FSC & SSC SSC-A Exp-SSC Low FSC-A LINEAR Mean FSC channel: 55,000 (± 5,000) Mean SSC channel: 13,000 (± 2,000) Fixed FSC threshold at 10,000 Normal peripheral PB sample processed according to EuroFlow sample preparation protocol

11 Instrument set up & monitoring: fluorescence APC H7-A: CD8 LOGICAL Reference value PE Cy7-A: CD16 LOGICAL - No bright positive population felt out of the window of analysis - Reference values in the plateau part of the curve in all instruments

12 Instrument set up & monitoring: fluorescence Target fluorescence channels for the 8 th Rainbow bead peak (lot: X02) Target values (lot: X02 of 8 th Rainbow bead peak) Target MFI PacB PacO FITC PE PerCPCy PECy APC APCH

13 Instrument set up & monitoring: fluorescence Reference values for the 8 th peak of Rainbow beads (lot: X02) Target MFI PacB PacO FITC PE PerCP Cy PECy APC APCH Range (MFI±1 5%) Population 1 MFI FITC PE PerCPCy PECy APC APCH PacB PacO Infinicyt TM automated bead gating and analysis tool

14 Fluorochrome selection Excitation & emission profile Instrument optical configuration APCCy7 Panel aim Relative brightness Alexa Fluor 647 APC PE Pacific Orange Availability AmCyan Type of sample/cells Spillover into other FL channels

15 Fluorochrome selection Optical configuration of the flow cytometer FL Channel Laser Dichroic mirror FACSCantoII (BD Biosciences) FACSAria (BD Biosciences) Emission filter LSRII (BD Biosciences) Cyan ADP (Dako/Beckman Coulter) 1 Violet 450/50 450/50 450/50 450/50 2 Violet /50 530/30 525/50 530/40 3 Blue /30 530/30 530/30 530/40 Commonly available fluorochromes Pacific Blue HORIZON V450 AmCyan Pacific Orange HORIZON V500 FITC Alexa Fluor Blue /42 585/42 575/26 585/42 PE 5 Blue 613/20 PETxRed 6 Blue LP 695/40 695/40 680/30 PerCPCy5.5 PerCP 7 Blue /60 780/60 780/60 E750LP PECy7 8 Red 660/20 660/20 660/20 665/20 9 Red /60 780/60 780/60 E750LP ** Alexa Fluor 700 requires a 710/50 emission filter APC Alexa Fluor 647 APCCy7 APCH7 Alexa Fluor 700**

16 Fluorochrome selection Initial selection Further comparisons FL Channel Laser Commonly available fluorochromes FL Channel Laser Commonly available fluorochromes 1 Violet 2 Violet 3 Blue Pacific Blue HORIZON V450 AmCyan Pacific Orange HORIZON V500 FITC Alexa Fluor Blue PE 5 Blue PETxRed 6 Blue PerCPCy5.5 PerCP 7 Blue PECy7 8 Red 9 Red APC Alexa Fluor 647 APCCy7 APCH7 Alexa Fluor Violet 2 Violet 3 Blue Pacific Blue HORIZON V450 AmCyan Pacific Orange HORIZON V500 FITC Alexa Fluor Blue PE 5 Blue PETxRed 6 Blue PerCPCy5.5 PerCP 7 Blue PECy7 8 Red 9 Red APC Alexa Fluor 647 APCCy7 APCH7 Alexa Fluor 700

17 Fluorochrome selection: further comparisons FL Channel Fluorochrome conjugate MFI* SI* Main Overlap Availability Violet 1 CD4 Pacific Blue (BD Biosciences) CD4 HORIZON V450 (BD Biosciences) 9, , Violet 2 (AmCyan/Pacific Orange/ Horizon V500) Violet 2 (AmCyan/Pacific Orange/ Horizon V500) * Intensity of staining and staining index (SI) obtained for reagents evaluated in normal PB samples (n=5). Overlay histograms of normal PBL stained with both reagents Blue line: Pacific Blue. Green line: HORIZON V450 % FL compensation requirements in other fluorescence channels Channel PB HV ± ± Violet 1-A Logical

18 Fluorochrome selection: further comparisons FL Channel Fluorochrome conjugate MFI* SI* Main Overlaps Availability Violet 2 CD45 Pacific Orange (Invitrogen) CD45 HORIZON V500 (BD Biosciences) CD45 AmCyan (BD Biosciences) 5, , , Violet 1 (Pacific Blue/Horizon V450) Blue 1 (FITC) Violet 1 (Pacific Blue/Horizon V450) Blue 1 (FITC) Violet 1 (Pacific Blue/Horizon V450) Blue 1 (FITC) * Intensity of staining and staining index (SI) obtained for reagents evaluated in normal PB samples (n=5). Overlay histograms of normal PBL stained with both reagents Blue line: Pacific Orange Green line: HORIZON V500 Yellow line: AmCyan Violet 2-A Logical % FL of compensation requirements in other fluorescence channels Channel Pacific HORIZON Orange V500 AmCyan 1 2.2± ± ±1.5 2 NA NA NA 3 0.8± ± ± ± ± ± ± ± ± ±0.1 NR NR 7 0.3±0.4 NR NR 8 0.1±0.2 NR NR

19 Fluorochrome selection: further comparisons FL Channel Fluorochrome conjugate MFI* SI* Main Overlaps Availability CD4 APCCy7 (BD Bioscience) 13, Blue 4 (PECy7) Red 1 (APC) Red 2 CD4 APCH7 (BD Bioscience) 9, Blue 4 (PECy7) Red 1 (APC) CD4 Alexa Fluor 700º (BD Bioscience) 4, Blue 3 (PerCPCy5.5) Blue 4 (PECy7) *Intensity of staining and staining index (SI) obtained for reagents evaluated in normal PB samples (n=5). ºInvolves modification in the optical configuration of the instrument Overlay histograms of normal PBL stained with both reagents Blue line: APC Cy7 Green line: APC H7 Yellow line: Alexa Fluor 700 Red 2-A Logical % FL of compensation requirements in other fluorescence channels Channel APC Cy7 APC H7 Alexa Fluor ± ± ± ± 0.4 NR 0.1 ± ± ± ± ± ± 0.2 NR ± ± ± ± ± ± ± ± ± NA NA NA

20 Final fluorochrome selection FL Channel Laser Fluorochromes 1 Violet Pacific Blue 2 Violet Pacific Orange 3 Blue FITC 4 Blue PE 6 Blue PerCPCy5.5 7 Blue PECy7 8 Red APC 9 Red APCH7

21 Fluorescence overlaps: Violet laser line Blue laser line Red laser line Spectrum Viewer at

22 Fluorescence compensation Single fluorochrome dyes Tandem dyes Pacific Blue Pacific Orange FITC PE APC PerCPCy5.5 PECy7 APCH7 Generic (reagent independent) FL compensation Reagent conjugate & lot specific needs

23 Fluorescence compensation Generic and reagent/lot-especific compensation tubes Generic PECy7 conjugates APCH7 conjugates CD20 PacB CD2 CD3 CD45 PacO CD8 CD4 CD8 FITC CD1 0 CD8 CD8 PE CD16 CD9 CD5 PerCPCy5-5* CD19 CD1 0 CD8 APC CD45RA CD14 CD45RO CD19 CD56 CD24 *Tandem dye CD117 HLADR CD38 CD43 CD49d CD71 CD81 Anti- Igλ. Matched negative and positive reference populations. Use of beads when reference cell population is not possible

24 Fluorescence compensation Primary Fluorescence Channel Summary of % FL compensation requirements obtained from data files (n=14) generated in 7 different flow cytometry instruments Secondary Fluorescence Channel PB PO FITC PE PerCP- Cy5.5 PE-Cy7 APC APC-H7 MIN 24.3% 0.0% 0.0% 0.0% PB MED 27.7% 0.0% NR 0.0% 0.0% NR NR MAX 31.0% 0.2% 0.6% 0.1% MIN 1.9% 0.2% 0.0% 0.0% 0.0% 0.0% 0.0% PO MED 2.4% 0.4% 0.2% 0.3% 0.0% 0.0% 0.0% MAX 2.9% 0.5% 0.3% 0.5% 0.1% 0.3% 0.5% MIN 0.0% 4.8% 10.0% 3.0% 0.2% 0.0% 0.0% FITC MED 0.0% 5.6% 12.0% 3.5% 0.3% 0.0% 0.0% MAX 0.1% 6.4% 16.0% 4.0% 0.5% 0.2% 0.2% MIN 0.0% 0.0% 0.2% 30.1% 2.2% 0.0% PE MED 0.0% 0.1% 1.3% 32.9% 2.5% 0.1% NR PerCP- Cy5.5 MAX 0.1% 0.3% 1.7% 38.9% 2.8% 0.1% MIN 0.0% 0.0% 0.0% 0.0% 12.5% 1.6% 1.0% MED 0.0% 0.0% 0.0% 0.0% 16.5% 2.4% 5.5% MAX 0.9% 0.9% 0.2% 0.1% 18.8% 3.7% 8.0% MIN 0.0% 0.0% 0.0% 0.2% 0.6% 0.0% 3.2% PE-Cy7 MED 0.0% 0.0% 0.1% 0.7% 2.9% 0.0% 6.8% MAX 0.4% 0.6% 0.5% 13.1% 5.7% 0.9% 9.1% MIN 1.0% 0.1% 8.5% APC MED NR NR NR NR 1.2% 0.1% 9.6% MAX 1.4% 0.2% 11.6% MIN 0.0% 0.0% 0.0% 0.0% 0.0% 1.3% 1.3% APC-H7 MED 0.0% 0.0% 0.0% 0.0% 0.0% 1.5% 1.8% MAX 0.2% 0.1% 0.2% 0.1% 0.2% 2.0% 3.9% NR: never required

25 Reagent s evaluations CD19 PECy7 example Population CD19 PC7 MFI (Beckman/Coulter) CD19 PECy7 MFI (BD Bioscience) Monocytes B-cells Non B-cells Stain Index Intensity of staining and staining index (SI) obtained for reagents evaluated in normal PB samples (n=7). Ilustrative bivariate dot plots CD19 PC7 (Beckman/Coulter) CD19 PECy7 (BD Bioscience)

26 Sample preparation protocol

27 Sample preparation protocol Red cell lysis Amonium Chloride Versalyse (Beckman-Coulter) Quicklysis (Cytognos) FACS Lyse (BD Biosciences) Protocol sequence: Stain-lyse-non-wash Stain-lyse-wash Stain-lyse-wash-fix Time for acquisition Immediate 1 hour 3 hours 24 hours (SLW) (SLNW) (SLWF)

28 Sample preparation protocol GOALS: * Adequate scatter resolution of major cell populations * Minimal cell loss * Preservation of fluorochrome brightness * Low impact on stability of fluorochrome tandems * Easy and fast to use * Reduced data acquisition time

29 Sample preparation protocol Scatter (FSC vs SSC) characteristics of PBL

30 Impact of the sample preparation protocol Cell loss Fluorescence intensity (MFI)

31 Sample preparation protocol & lysing reagent Fluorescence intensity & stability

32 Sample preparation protocol & lysing reagent NH 4 CL FACS Lyse MFI of CD19 + PECy7 cells in PE SLNW SLW SLWF 0h 1h 3h 24h 0h 1h 3h 24h Fluorescence intensity & stability

33 Sample preparation protocol Red cell lysis Amonium Chloride Versalyse (Beckman-Coulter) Quicklysis (Cytognos) FACS Lyse (BD Biosciences) Protocol sequence: Stain-lyse-non-wash Stain-lyse-wash Stain-lyse-wash-fix Time for acquisition Immediate 1 hour 3 hours 24 hours (SLW) (SLNW) (SLWF)

34 Sample preparation protocol 10 µl 7 µl Titration of reagents: - Final volume of 100 µl 5 µl 2,5 µl 1,25 µl - Saturation conditions for variable volumes (e.g. 2x) 0,625 µl 0 µl

35 Sample preparation protocol

36 Sample preparation protocol Initial steps (B-CLPD): - Wash twice (SmIgs) - Premix & stain BB markers first

37 Thanks!! J. Flores-Montero 1,T. Kalina 2, Q. Lecrevisse 1, J.J Pérez 3,M. Cullen 4,L. Lhermitte 5, L. Sedek 6, A. Mendonça 7, S. Bötcher 8, J. te Marvelde 9, E. Mejstříková 2, O. Hrušák 2, J.J.M. van Dongen 9 and A.Orfao 1 1, Department of Medicine, Cancer Research Centre and Cytometry Service, University of Salamanca, Salamanca, ES; 2, Department of Pediatric Hematology and Oncology, Charles University, Prague, Czech Republic; 3, Department of Hematology, University Hospital of Salamanca, Salamanca, ES; 4,St. James University Hospital, Leeds, UK; 5, Department of Hematology, Hôpital Necker, Paris, FR; 6, Department of Pediatric Hematology and Oncology, Medical University of Silesia, Zabrze, PL; 7, Department of Hematology, Instituto Portugues de Oncologia, Lisbon, PT; 8, 2nd Department of Medicine, University Klinik Schleswig- Holstein, Kiel, DE; 9, Department of Immunology, Erasmus MC, Rotterdam, NL

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