From Innovative Technologies To Global Solutions

Transcription

1 From Innovative Technologies To Global Solutions Antoine PACHECO Marketing Group Manager Beckman Coulter France Mol, May BVAC/ABCA - Molecular Biology and Cytometry Course

2 Overview of Beckman Coulter Solutions 2

3 Program 1. HematoFlow/Cytodiff Solution 2. Gallios/Navios Cytometer 3. Multicolor Strategies (some combinations to win) 4. High Throughput Solutions (Cyan + HyperCyt + Biomek) 5. Cocktail Automation (Biomek ACP) 6. Cell Sorting Revolution (Moflo Astrios) 7. Single Cell Analysis (Advalytics AmpliSpeed and Ampligrid) 8. Quantum Cell Expansion System 9. Kaluza Software 3

4 1. Hematoflow/Cytodiff Solution Hema to Flow 4

5 HematoFlow CytoDiff - A Definition An unique Cellular Analysis Solution that brings routine Flow Cytometry testing into the Hematology laboratory 5 color, 6 monoclonal antibody reagent cocktail which yields a 9-part CE marked flow differential, when used in the HematoFlow solution 5

6 HematoFlow vs CytoDiff CytoDiff Reagent 6

7 HematoFlow vs CytoDiff HematoFlow BC Hematology Analyser LIS Remisol Middleware RBC Morphology FC 500 Flow Cytometer Sample Preparation System 7

8 We use the best of each technology HematoFlow Microscopy Hematology Flow Cytometry 8

9 CytoDiff Panel (5-colors, 6-Markers) Marker CD36-FITC CD2-PE CD294 PE CD19 ECD CD16 PC5 CD45 PC7 Cellular Expression Monocytes Activated T Lymphocytes, NK cells Eosinophils, Basophils, Activated T cells B lymphocytes and B Precursors Mature and Immature Granulocytes, T Cytotoxic Lymphocytes All Leukocytes Currently a CE Cleared Product Only 9

10 Motivation to Create HematoFlow HematoFlow is an unique Cellular Analysis solution, which: - Reduces Microscopic Review - Improve Quality of Results - Improve TAT 10

11 History of Innovation in Hematology Coulter Model A Coulter S-Plus Series Coulter VCS Technology Coulter MAXM Coulter GenS Coulter AcT Diff II Coulter AcT 5 Diff Coulter LH 1500 Series Coulter DxH 800 CytoDiff CXP Coulter Model S Coulter STKR, STKS Coulter MD Series Coulter AcT Diff Coulter HmX Coulter LH 700 Series Coulter LH 500 HematoFlow CytoDiff 11

12 What s new in 2011? CytoDiff CXP Software + Automated CytoDiff gating under CXP Uses sophisticated proprietary algorithm The most sophisticated flow software according to Francis Lacombe (Bordeaux France) 12

13 Autogating Algorithm Advantages Automatic Gating Algorithm Ease of Use in Routine Hematology Lab Workflow Improvement Advanced Population Separation 13

14 General Gating Logic Flow 14

15 CytoDiff Single View classifier on 2D Histograms 15

16 Algorithm Gating Strategy All Cells Previous less B Cell Previous less Non-WBC Previous less Mature Ne Previous less Mono Previous less IG & Eo Previous less Ly T & NK Previous less BlastnBnT 16

17 Algorithm Gating Strategy Continued From CD19 gate From LyT&NK gate From IG & Eo gate From CD36 gate From Mo gate 17

18 Complete Algorithm Overview B CD19+ Leucocytes CD45+ Neutrophil CD16+ Mono CD36+ EO + IG CD 16-/SS High T + NK CD45+ Baso + Xt CD2/CRTH2 - Baso & Xt FS High EO & IG CD2/CRTH2+ LyB & Xb CD45+ Mono & Xm CD 45+ Mono & ProI CD 16+/- LyTC+NK & LyTnC CD16+Tc+NK/-TNc 18

19 CytoDiff Algorithm In an average CytoDiff run, between and events with 7 dimensions (i.e., FS, SS, CD45, CD36, CD2+CRTH2, CD 19, CD16) are acquired The CytoDiff algorithm classifies data points acquired by the FC500 running the CytoDiff Reagent Kit in a matter of second. Each classified histogram produces information that is reported or used as input for the remaining stages. 19

20 Benefits for the Laboratory Reduction of Microscopic Evaluations Reduction of Manual Tasks Objectivity and Reliability of Results Extensive Decision Support Increased Standardisation Saves Time and Money 20

21 Beckman Coulter Seeing Beyond the Horizon XL 4C 1L Quanta 3C 2L Cellios 5C 1L End of 2011 MoFlo XDP 18C 3L Cyan ADP 9C 3L Astrios 49C 7L FC500 5C 2L Gallios 10C 3L Navios 10C 3L 21

22 Gallios Navios RUO CE - IVD A new frontier in Flow Cytometry 22

23 2. Gallios Navios Cytometer 3 configurations 6 color 2 Lasers (5+1) 8 color 2 Lasers (5+3) 10 color 3 Lasers (5+3+2) 638nm 488nm 405nm Modular to Fit your Needs, Today and Tomorrow. 23

24 Submicron Resolution A = Liquid => Max 19.0 B = Quartz => Max 23.0 C = Air => Max 24.8 Alimentation +15 Volts Forward Scatter optimisation for analysis of microparticules. 24

25 Submicron Resolution A = Liquide => Max 19.0 B = Quartz => Max 23.0 C = Air => Max 24.8 Alimentation +15 Volts Alimentation +30 Volts (W 2 ) Forward Scatter optimisation for analysis of microparticules. 25

26 Side Scatter Submicron Resolution Gallios-Navios: extreme resolution on small particules Innovative W 2 detector for Forward Scatter Low optical noise on blue laser Low Scatter noise Forward Scatter Forward Scatter Forward Scatter optimisation for analysis of microparticules. 26

27 Microparticles : Megamix Beads ISTH : standardisation of analysis protocol (Plasma) Microparticle size between 0.1µm et 1µm But recommended size range between0.5µm et 1µm 27

28 Microparticles : Platelets and Endothelials ISTH : standardisation of analysis protocol (Plasma) Microparticle size between 0.1µm and 1µm But recommended size range between 0.5µm and 1µm Normal Patient 1 Patient 2 28

29 Navios / Gallios : Multicolor Exemple 10C : 8-203c / DR-45 29

30 3. Multicolor Strategies Some combinations to win : T/B/NK 9-Color Combination 9C Treg Combination 10C NK-Cell Combination for KIR Profiling 10C B-Cell Tube with CD19+CD5+Kappa+ Example 30

31 T/B/NK 9-Color Combination Target: Identify the following cell populations: B cells, NK cells, NKT cells, CD4 + and CD8 + T cells Measure the expression of activation marker such as CD38, PD-1 and HLA-DR Staining strategy: PB405 Excitation KrO FITC PE ECD 488 Excitation PC5.5 PC7 APC APC-A Excitation APC-A750 PacBlue KrO FITC PE ECD PC5.5 PC7 APC APC-AF700 APC-AF750 CD8 CD45 CD3 CD38 CD56 CD19 A07758 A96416 IM1281U IM2371U A82943 A66328 A78885 IM2468U A89312 PD1 CD4 CD16 CD8 CD45 CD3 CD38 CD56 CD19 HLA-DR CD4 CD16 Procedure: TQ-Prep no wash A

32 Results MNC SS lo CD45++ CD19+ CD56+ CD3+ CD16+ NKT CD4+ CD8+ CD38, CD279 (PD-1), HLA-DR 32

33 CD4+ CD8+ CD38 CD38-PE CD279 (PD-1) CD279 (PD-1)-PC7 HLA-DR HLA-DR-PC7 33

34 9C Treg Combination Target: Identify the following cell populations: T cells: CD8 + T cells; T Regs; Naïve and central memory CD4+T cells; Effector CD4 + T cells; NK cells; NKT cells and B cells Measure the expression of an activation marker: HLA-DR Staining strategy: PB KrO FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750 HLA-DR CD25 CD127 CD45 CD19 CD56 CD4 CD8 CD3 A74781 IM0478U IM1980U IM2710U A66328 A51078 IM2468U A66332 A66329 Procedure: VersaLyse + IOTest 3 Fixative with wash 34

35 B cells CD4+ T cells NK/NKT cells 35

36 10C NK-Cell Combination for KIR Profiling PB KrO FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750 CD57 CD45 CD16 CD158e1/2 CD56 CD158b1/2 CD158a/h CD158i CD25 CD33/CD3 CD45 Pan-WBC marker; on all differentiated hematopoietic cells, except RBC, plasma cells CD16 Low affinity Fc receptor; Fc RIIIa and b; on NK, neutrophils, mono and macrophages CD56 NCAM neural cell adhesion molecule; expressed on NK cells, neurons, muscle cells CD57 Human natural killer protein-1; on subsets of NK and T cells CD3 Pan-T cell marker; composed of CD3, CD3, CD3 CD33 Myeloid lineage marker; sometimes also found on lymphatic cells CD25 Alpha chain of IL-2 receptor; on activated B and T cells; high on T regs CD158a/h Killer-cell immunoglobulin-like receptor (KIR); found on NK cells CD158b1/2 Killer-cell immunoglobulin-like receptor (KIR); found on NK cells CD158e1/2 Killer-cell immunoglobulin-like receptor (KIR); found on NK cells CD158i Killer-cell immunoglobulin-like receptor (KIR); found on NK cells 36

37 10C NK-Cell Combination for KIR Profiling Gating Strategy LY w/o CD3+/ or CD56+ CD16+ Boolean Gate for NK Cells CD158b1/2;e1/2;a/h;i 37

38 10C NK-Cell Combination for KIR Profiling 38

39 10C B-Cell Tube PB KrO FITC PE ECD PC5.5 PC7 APC APC-A700 APC-A750 CD20 CD45 Kappa Lambda CD19 CD5 CD56 CD4 CD8 CD3 CD45 CD3 CD4 CD8 CD56 CD19 CD20 CD5 Kappa Lambda Pan-WBC marker; on all differentiated hematopoietic cells, except RBC, plasma cells Pan-T cell marker; composed of CD, CD3, CD3 Co-receptor for the T cell receptor; found on T cells, NK cells, dendritic cells; also Mono Co-receptor for the T cell receptor; binds MHC class I; found on T cells, NK, DC NCAM neural cell adhesion molecule; expressed on NK cells, neurons, muscle cells From earliest recognizable B-lineage cells through mature B-cells; not on plasma cells B-cell marker; from pre-pre B-cells through memory B-cells; not on plasma cells T-cell marker; upregulated upon activation; also found on IgM-secreting B-cells One of the two immunoglobulin light chains expressed on the surface of B-cells One of the two immunoglobulin light chains expressed on the surface of B-cells 39

40 10C B-Cell Tube with CD19+CD5+Kappa+ Example Gating Strategy LY CD3+ CD4, CD8, CD5 CD56+ CD19+ CD5, CD20, Kappa, Lambda 40

41 10C B-Cell Tube with CD19+CD5+Kappa+ Example 41

42 4. HyperCyt Module for CYAN and GALLIOS Cyan + HyperCyt A new reference in speed 42

43 4. High Throughput Solutions HyperCyt Rapid Sample Transfer System 96 wells in 2.5 minutes 384 wells in 10 minutes As little as 1 μl sampling No dead volume State of the Art Flow Cytometer Multiple cytometers Easy connection Full cytometer capabilites 1000s cells/second Multiplexing Sensitivity HyperView Data Analysis Software Select populations Plate level statistics Quickly visualize hits/trends Export well FCS data 43

44 Cyan + HyperCyt Sample Probe Sample Tubing Buffers & Cleaning Stations To Gallios/CyAn Peristaltic pump Plateholder Orbital Shaker 44

45 Cyan + HyperCyt Number of Events Sample Plugs and Air Gaps HyperCyt Tubing To Flow Cytometer HyperCyt Probe HyperView Well Identification Well A1 Well A2 Well A3 Well A4 Time 45

46 FCS File 46

47 CD4 Negative CD4 Positive 47

48 48

49 49

50 High Throughput Flow Cytometry Analysis time for 96 well plate : 2.5 min Analysis time for 384 well plate : 10 min Carry Over < 1.0% without rinse and <0.1% with rinse Dedicated software for data analysis (HyperView) 50

51 Cyan + HyperCyt 51

52 HTFC : Cyan + HyperCyt + Biomek Price / CyAn 9 cl + HyperCyt + BIOMEK NX CyAn 9 cl + HyperCyt + BIOMEK 3000 CyAn 9 cl + HyperCyt Speed (Wells / 8 h) 52

53 High Throughput Flow Cytometry Applications sirna knockdowns Antibody Screening Transfection and Expression studies Stem Cell screening Small molecule screening Cell viability/cytotoxicity/apoptosis Cell cycle analysis Cell signaling Multiplexing Receptor - ligand binding Yeast library screening T-cell activation Mediating sirna interference in cultured mammalian cells 53

54 5. Cocktail Automation BIOMEK ACP SOLUTION For Coktails or Large PANELS of AntiBodies 54

55 The Biomek ACP Workstation - Based on World Leading Liquid Handling Technology Tube base Caroussel Base Microplate Base 8 probes for B.NX 1 probe for B.3000 mab onboard 24h/day T control In the dark 55

56 The Biomek ACP Workstation - Safe Reagent Storage During Operation and Standby Dark and cooled reagent racks allow for continuous workflow without repeated physical instrument reagent setup 56

57 The Biomek ACP Workstation - Flexible Operation Meets Ease of Use 57

58 The Biomek ACP Workstation - Flexible Operation Meets Ease of Use User ID mab Lot nb Peremption date Level detection Estimate mab volume Linked to the Flow Cytometer panels (FC500, Navios, Gallios) 58

59 The Biomek ACP Workstation - Flexible Operation Meets Ease of Use 59

60 The Biomek ACP Workstation - Flexible Operation Meets Ease of Use Reagent Track 60

61 The Biomek ACP Workstation - Flexible Operation Meets Ease of Use 61

62 The Biomek ACP Workstation - Flexible Operation Meets Ease of Use Reports for tracability 62

63 6. MoFlo ASTRIOS Disponibilité 2 ème semestre 2009 A New Dawn 63

64 6. Moflo Astrios : Cell Sorting Revolution Features Up to 7 separated lasers Up to 49 PMT 7 PMTs / Laser 6 way sorting Sorting Plate Module Sorting : cells/sec With purity > 99% and recovery > 90% ZERO Dead Time 32 bit resolution Automated delay calculation Customer Drive the Design 64

65 Optics Adjustment Fiber Coupled Lasers Pass through BSO Flat-Top Beam focuses laser power Greater laser power at the stream Less sensitivity to laser or core stream shifts Ease of alignment Elliptical Beam Flat-Top Beam Increasing Sample pressure does not affect the CV => High speed with lower concentrated samples Simple sustained alignment for minimal hands on time and consistent results 65

66 Biosafety Cabinet Biosafety Level II Specifically designed External touch screen Reducing need to place hands inside cabinet Protecting you - Protecting your sample 66

67 Start-Up Scheduling Warm up fluidics and lasers before you arrive to the laboratory Why waste your time waiting for lasers to warm up? 67

68 7. Single Stem Cells and Tumour Cells Analysis AmpliGrid Single Cells AmpliSpeed Thermal Cycling 68

69 Stem Cells and Tumour Cells Astrios / XDP Cell Sorting Evaluation AmpliGrid Single Cells Tumour Markers PCR Amplification icys Research Imaging Cytometer Cell Cycle 69

70 8. QUANTUM CELL EXPANSION SYSTEM Closed, Automated, Self Contained Cell Expansion System 70 70

71 DISPOSABLE QUANTUM CELL EXPANSION SET Oxygenator bioreactor Disposable cassette enables quick loading 71 71

72 QUANTUM CELL EXPANSION SYSTEM : COMPONENTS HOLLOW FIBER BIOREACTOR: Cells can be grown: Inside the hollow fibers Outside the hollow fibers Simultaneously on both sides 72 72

73 QUANTUM CELL EXPANSION SYSTEM : COMPONENTS HOLLOW FIBER BIOREACTOR: good biocompatibility with no cell growth inhibition Surface area around 2 m² Contains around 10,000 fibers Nutrients, metabolites and gases easily diffuse across fibers 73 73

74 QUANTUM SAMPLING METHODS TAKING A MEDIA SAMPLE Sampling from the sample port Use these samples to measure glucose and lactate levels 74 74

75 QUANTUM TOUCH SCREEN USER INTERFACE 75 75

76 QUANTUM CELL EXPANSION SYSTEM : COMPONENTS 76 76

77 9. Kaluza Analysis Software Version 1.2 coming soon Explore new dimensions in flow analysis 77

78 Version 1.2 Design Goals Promise of the Platform Facilitate comp autorecognize controls Continuous Innovation Correlation Plot Radar gates Capture Existing Base Advanced Color Precedence Expand Base Batch capability Gate hierarchy/population statistics 78

79 Correlation Plot 79

80 Radar gates 80

81 Black backgrounds 81

82 New Comp methods Ability to autodetect single color controls 82

83 New Website Visit:

84 Beckman Coulter Seeing Beyond the Horizon XL 4C 1L Quanta 3C 2L Cellios 5C 1L End of 2011 MoFlo XDP 18C 3L Cyan ADP 9C 3L Astrios 49C 7L FC500 5C 2L Gallios 10C 3L Navios 10C 3L 84