Human Cell Analysis The Technology Behind The World s Most Common Diagnos8c Test
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1 Human Cell Analysis The Technology Behind The World s Most Common Diagnos8c Test IEEE- CNSV November 4, 2014 Giacomo Vacca, Ph.D. Founder & President Kine?c River Corp. Proprietary 1
2 OVERVIEW Blood Components of blood Blood diagnos?cs Coun?ng blood cells Principles of flow cytometry Flow cytometry systems Flow cytometry system architecture Developing flow cytometers If there s 8me Flow cytometry data analysis Disrup?ve innova?ons Proprietary 2
3 Giacomo Vacca, Ph.D. Kine8c River (President) BeamWise (CSO) AbboT Labs (Res. Fellow) Picarro Lightwave Micro Exxon Research & Engr. 28 patents issued/pending Picarro GEN Abbott Laboratories Proprietary 3
4 BLOOD Proprietary 4
5 What Is It About Blood? Newsurg buzzfeed.com Proprietary 5
6 Blood thinkhealthypz.blogspot.com Proprietary 6
7 6 Quarts thinkhealthypz.blogspot.com cliparts101.com Proprietary 7
8 It s In Your Blood Newsurg CC BY 3.0 Proprietary 8
9 COMPONENTS OF BLOOD Proprietary 9
10 White Blood Cells (WBCs) Proprietary 10
11 WBCs (aka Leukocytes) Proprietary 11
12 WBC Facts typical adult: ~ 30,000,000,000 (30 billion) WBCs ~ 5 x 10 3 / ul ~ 5 15 um in size 3 major subtypes lymphocytes monocytes granulocytes all have nucleus part of body s immune system: detect, recognize, fight invaders Proprietary 12
13 Platelets (aka Thrombocytes) Proprietary 13
14 Platelet Facts typical adult: ~ 150,000,000,000 (150 billion) platelets ~ 250 x 10 3 / ul ~ 2 3 um in size not actually cells splintered fragments of precursors: megakaryocytes aggregate to stop bruising and bleeding Proprietary 14
15 Red Blood Cells (RBCs) Proprietary 15 Annie Cavanagh, Wellcome Images
16 RBCs (aka Erythrocytes) Proprietary 16
17 RBCs Proprietary 17
18 RBC Facts typical adult: ~ 30,000,000,000,000 (30 trillion) RBCs 3 million RBCs produced per second 120 days life?me ~ 5 x 10 6 / ul ~ 45% of blood by volume ~ 5 um in size characteris?c biconcave shape no nucleus! hemoglobin bags carry O 2 from lungs to?ssues, carry back CO 2 Proprietary 18
19 The Three Amigos NCI Frederick Proprietary 19
20 BLOOD DIAGNOSTICS Proprietary 20
21 Blood- Based Diagnos?cs James Anness/The Record, via The Associated Press Proprietary 21
22 Blood- Based Laboratory Diagnos?cs Cell assays Complete Blood Count (CBC) hematocrit hemoglobin WBC differen?al platelet count immature RBCs Plasma assays glucose electrolytes lipids Other diagnos?c assays molecular diagnos?cs blood typing DNA sequencing blood gases proteins enzymes Proprietary 22
23 Blood- Based Laboratory Diagnos?cs Cell assays Complete Blood Count (CBC) hematocrit hemoglobin WBC differen?al platelet count immature RBCs Plasma assays glucose electrolytes lipids blood gases proteins enzymes Other diagnos?c assays molecular diagnos?cs blood typing DNA sequencing Proprietary 23
24 Blood- Based Diagnos?cs Proprietary 24 Sysmex
25 Complete Blood Count (CBC) ~ 200 million CBCs / yr worldwide CBCs used for diagnosis of: general health (screening) anemia (low RBCs, small RBCs, low hemoglobin concentra?on) bacterial infec?on (high WBCs) drug toxicity (low platelets) leukemia (increased subtypes of WBCs) allergy (increased subtype of WBCs) Proprietary 25 Sysmex
26 COUNTING BLOOD CELLS Proprietary 26
27 Blood Smears GreatScopes Frederick Wasti Proprietary 27
28 Blood Smears Frederick Wasti Proprietary 28
29 Blood Smears Proprietary 29 pdbio325.blogspot.com
30 Hemocytometer: Manual Coun?ng wetnf.org Hausser Scientific Proprietary 30
31 Par?cle Sizing: The Coulter Principle Pulver Technology Beckman Coulter Proprietary 31
32 Cell Iden?fica?on: Flow Cytometry Physikalisch-Technische Bundesanstalt Proprietary 32
33 PRINCIPLES OF FLOW CYTOMETRY Proprietary 33
34 The Op?cal Bench: Cri?cal Elements spectral filters light source(s) flow cell side- scater detector(s) fluorescence detector(s) forward- scater detector(s) Life Tech Proprietary 34
35 Hydrodynamic Focusing core stream sheath core stream flow rate: ~ 1 µl/s core stream speed at nozzle: ~ 1 mm/s core stream speed in flowcell: ~ 10,000 mm/s 2011 Life Tech Proprietary 35
36 Flowcell: Where It All Comes Together Nihon Kohden Proprietary 36
37 Cell Interroga?on: Rela?ve Sizes 1/e 2 laser beam waist ~70 um ~20 um 10 um Life Tech Proprietary 37
38 Par?cle Detec?on: ScaTering Signal Proprietary 38
39 Forward ScaTering Histogram Life Tech Life Tech Proprietary 39
40 Side ScaTering Histogram Life Tech Life Tech Proprietary 40
41 Building the 2- D ScaTergram side scater Life Tech forward scater Proprietary 41
42 Two Methods: ScaTering & Fluorescence ScaTering: mainly hematology analysis FC cell size morphology (shape of cell and nucleus) complexity (presence of crystals, granules) Fluorescence: mainly non- hematology FC DNA, RNA staining surface an?gens (immune system) e.g.: CD4+ lymphocytes for HIV monitoring intracellular biomarkers Proprietary 42
43 Two Methods: ScaTering & Fluorescence ScaTering: mainly hematology analysis FC cell size morphology (shape of cell and nucleus) complexity (presence of crystals, granules) Fluorescence: mainly non- hematology FC DNA, RNA staining surface an?gens (immune system) e.g.: CD4+ lymphocytes for HIV monitoring intracellular biomarkers Proprietary 43
44 This Seminar: ScaTering, Hematology ScaTering: mainly hematology analysis FC cell size morphology (shape of cell and nucleus) complexity (presence of crystals, granules) Fluorescence: mainly non- hematology FC DNA, RNA staining surface an?gens (immune system) e.g.: CD4+ lymphocytes for HIV monitoring intracellular biomarkers $3B/yr Proprietary 44
45 FLOW CYTOMETRY SYSTEMS Proprietary 45
46 From Very Small Courtesy of Peter Kiesel, PARC Proprietary 46
47 To Very Large Academia Sinica Proprietary 47
48 Hematology Analyzer Examples ABX Orphee Beckman Coulter Abbott Laboratories Proprietary 48
49 Flow Cytometer Examples BD Beckman Coulter Partec / Sysmex EMD Millipore Proprietary 49
50 FLOW CYTOMETRY SYSTEM ARCHITECTURE Proprietary 50
51 Flow Cytometer HW Modules 2014 Kinetic River Corp. Proprietary 51
52 Flow Cytometer HW Modules sample reagents waste sample, reagents 2014 Kinetic River Corp. Proprietary 52
53 Flow Cytometer HW Modules aspira?on flowcell autoloader (op?onal) fluidics/ pneuma?cs incuba?on (op?onal) 2014 Kinetic River Corp. fluidics/pneuma?cs Proprietary 53
54 Flow Cytometer HW Modules excita?on collec?on flowcell 2014 Kinetic River Corp. op?cs Proprietary 54
55 Flow Cytometer HW Modules embedded control signal processing algorithms, database, UI 2014 Kinetic River Corp. electronics Proprietary 55
56 Flow Cytometer HW Modules Laboratory Automa8on System (op8onal) Laboratory Informa8on System (op8onal) 2014 Kinetic River Corp. Proprietary 56
57 Flow Cytometer HW Modules liquids photons data controls sample, reagents fluidics/pneuma?cs 2014 Kinetic River Corp. op?cs electronics Proprietary 57
58 DEVELOPING FLOW CYTOMETERS Proprietary 58
59 Flow Cytometers Are Complex Systems e.g.: Kine?c River s Danube FC op?cs, microfluidics, control SW: KRC fluidic management electronics signal processing PARTNERS Proprietary 59
60 Kine?c River Key Exper?se Op?cs System Design Life Sciences & Diagnos?cs LiveIdeas Microfluidics 2D / 3D CAD Abbott Laboratories BeamWise Proprietary 60
61 Flow Cytometer HW Modules liquids photons data controls sample, reagents fluidics/pneuma?cs 2014 Kinetic River Corp. op?cs electronics Proprietary 61
62 Kine?c River Core Competencies liquids photons data controls sample, reagents microfluidics 2014 Kinetic River Corp. op?cs signal processing Proprietary 62
63 Partner Core Competencies liquids photons data controls sample, reagents fluidics/pneuma?cs 2014 Kinetic River Corp. op?cs electronics Proprietary 63
64 RESOURCES, Q&A Flow cytometry tutorials at Purdue University PPTs Prac8cal Flow Cytometry, 4 th ed., H.M. Shapiro also online at Flow Cytometry: An Introduc8on, M.G. Ormerod also online at GVacca@Kine?cRiver.com Proprietary 64
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