Practical flow cytometry / Howard M. Shapiro.

Format:

Book

Author/Creator:

Shapiro, Howard M. (Howard Maurice), 1941-

Language:

English

Subjects (All):

Flow cytometry.

Flow Cytometry.

Medical Subjects:

Flow Cytometry.

Physical Description:

l, 681 pages : illustrations ; 29 cm

Edition:

Fourth edition.

Place of Publication:

Hoboken, N.J. : Wiley-Liss, [2003]

Summary:

This complete reference provides the background science and discussion of the vast biomedical applications quantitative analytical cytology using laser-activated detection and cell sorting. Now in its fourth edition, thishas been expanded to provide full coverage of the broad spectrum of applications in molecular biology biotechnology today. New to this edition are chapters automated analysis of array technologies, compensation, high-speed sorting, reporter molecules, and multiplex apoptosis assays, along with fully updated and revised references and a list of suppliers. The Fourth Edition of the discipline's benchmark text offers a variety of new features, including ... ......... Expanded coverage of new commercial instrumentation with detailed descriptions of new analog and digital techniques for signal processing and fluorescence compensation; A completely revised introduction, emphasizing the interrelationships of flow and static cytometry and microscopy; Links to supplementary online resources

Contents:

1.1 What (And What Good) Is Flow Cytometry? 1

Tasks and Techniques of Cytometry 1

Some Notable Applications 1

What is Measured: Parameters and Probes 2

1.2 Beginnings: Microscopy And Cytometry 2

A Little Light Music 4

Making Mountains out of Molehills: Microscopy 6

Why Cytometry? Motivation and Machinery 9

Flow Cytometry and Sorting: Why and How 10

1.3 Problem Number One: Finding The Cell(s) 14

Flow Cytometry: Quick on the Trigger 16

The Main Event 17

The Pulse Quickens, the Plot Thickens 17

1.4 Flow Cytometry: Problems, Parameters, Probes, and Principles 18

Counting Cells: Precision I (Mean, S.D., CV) 18

And Now to See with Eye Serene the Very Pulse of the Machine: Display, Digitization, and Distributions 21

DNA Content Analysis: Precision II (Variance) 21

Two-Parameter Displays: Dot Plots and Histograms 26

Identifying Cells in Heterogeneous Populations: Lift Up Your Heads, Oh Ye Gates! 33

Sorting Sorting Out 40

Parameters and Probes II: What is Measured and Why 42

1.5 What's In the Box: Flow Cytometer Anatomy, Physiology, and Pathology 49

Light Sources for Microscopy and Flow Cytometry 49

Instrument Configurations: The Orthogonal Geometry 50

Laser Beam Geometry and Illumination Optics 50

Flow Chamber and Forward Scatter Collection Optics 51

Fluorescence and Orthogonal Scatter Optics 52

Optical Filters for Spectral Separation 52

Multistation Flow Cytometers 54

Photomultipliers and Detector Electronics 54

Putting the Flow in Flow Cytometry 55

Signal Processing Electronics 57

Is It Bigger than a Breadbox? 57

Flow Cytometer Pathology and Diagnostics 58

1.6 Alternatives to Flow Cytometry; Cytometer Ecology 59

Lis(z)t Mode 60

2. Learning Flow Cytometry 61

Learning from History: Take One 61

Books on Flow Cytometry in General 62

Books on Flow Cytometric Methodology and Protocols 62

Clinical Flow Cytometry Books 63

Flow's Golden Oldies 63

2.2 The Reader's Guide To Periodical Literature 64

Flow Cytometer Manufacturers 66

The International Society for Analytical Cytology 66

The Clinical Cytometry Society 66

The National Flow Cytometry Resource 66

"The Annual Courses" and Others 67

Other Societies and Programs 67

The Purdue Mailing List, Web Site, and CD-ROMs 68

2.4 Exploring The Foundations 68

Optics and Microscopy 68

Electronics 69

Computers: Hardware and Software 69

Digital Signal Processing 70

Data Presentation and Display 70

Spectroscopy, Fluorescence and Dye Chemistry 71

Cell and Molecular Biology and Immunology 71

2.5 Alternatives To Flow Cytometry 71

3. History 73

3.1 Ancient History 73

Flow Cytometry: Conception and Birth 73

Staining Before and After Paul Ehrlich 74

Origins of Modern Microscopy 75

Making Cytology Quantitative: Caspersson et al 75

Origins of Cancer Cytology: The Pap Smear 76

The Fluorescent Antibody Method 77

Blood Cell Counting: Theory and Practice 77

Video and Electron Microscopy 78

Optical Cell Counters and the Coulter Orifice 78

3.2 Classical History 79

Analytical Cytology in the 1950's 79

The Cytoanalyzer 79

Acridine Orange as an RNA Stain: Round One 79

How I Got Into this Mess 79

The Rise of Computers 80

Computers in Diagnosis: A Central Problem 80

Diagnosis and Classification: Statistical Methods 80

Cytology Automation in the 1960's 81

First Steps toward Automated Differentials 81

Pattern Recognition Tasks in Cell Identification 82

Differential Leukocyte Counting: An Early Flow Systems Approach 83

Kamentsky's Rapid Cell Spectrophotometer 84

Fulwyler's Cell Sorter 85

3.3 Modern History 85

Cell Cycle Analysis: Scanning versus Flow Systems 85

Cancer Cytology: Scanning versus Flow Cytometry 86

Early Commercial Flow Cytometers 87

Not Quite Commercial: The Block Projects 89

The Evolution of Flow Cytometers in the 1970's 90

Dog Days: The Genesis of Cytomutts 93

The 1980's: Little Things Mean a Lot 94

Measurements in the Main Stream 95

Clinical Uses of Fluorescence Flow Cytometry 98

The End of History? 99

4. How Flow Cytometers Work 101

4.1 Light and Matter 101

Photometry versus Radiometry: What's in a Name? 101

Physical Measurement Units 101

Light in Different Lights 102

It's All Done With Photons 102

A Few Warm Bodies 103

Polarization and Phase; Interference 104

Light Meets Matter: Rayleigh and Mie Scattering 105

A Time for Reflection-and Refraction: Snell's Law 107

Polarization by Reflection; Brewster's Angle 107

Dispersion: Glass Walls May Well a Prism Make 108

Interference in Thin Films 108

Interference and Diffraction; Gratings 108

Optical Activity and Birefringence 109

Matter Eats Light: Absorption 109

Absorption: Counting the Calories 110

A Selective Diet 110

The Chance of a Lifetime 110

Spinning a Tale of Degeneracy 111

Facing Extinction: Cross Section and Optical Density 111

Unexciting Times: Emigrating from the Excited States 112

Fluorescence: Working the Stokes Shift 112

Phosphorescence 113

Fluorescence Polarization 114

Stimulated Emission 114

Resonance Energy Transfer 115

Quenching, Bleaching, and Photon Saturation 115

Quantum Flotsam and Jetsam 118

4.2 Optical Systems 119

Light Propagation and Vergence 119

Image Formation by Optical Systems: Magnification 119

Lens Types and Lens Aberrations 120

Numerical Aperture and Lens Performance 121

Gradient Index, Fresnel, and Cylindrical Lenses 122

The Helmholtz Invariant and Throughput 123

Photons in Lenses: See How They Run 123

Aperture and Field Stops: The f Number 124

Depth of Field and Focus and Resolution of Lenses 124

4.3 Light Sources 124

The Best and the Brightest 124

Harc, Harc, the Arc! 126

Quartz Halogen Lamps 127

Light Emitting Diodes (LEDs) 127

Illumination Optics for Lamps and LEDs 127

Arc Source Epiillumination for Flow Cytometry 128

Lasers as Light Sources for Flow Cytometers 129

Laser Illumination: Going to Spot 130

Shedding Light on Cells: Lasers, Lamps, and LEDs 131

Lasers: The Basic Physics 133

Lasers Used and Usable in Cytometry 138

Laser and Light Source Noise and Noise Compensation 147

Danger!!! Laser!!! Hazards and Haze 148

4.4 Light Collection 149

Microscope Objectives 149

Looking at the Observation Point 150

Stops versus Blockers 150

Signal versus Noise: To See or Not to See 150

Spectral Selection: Monochromators versus Filters 152

Neutral Density Filters 156

Beamsplitters; Ghosts and Ghostbusters 156

Optics for Polarization Measurements 156

Tunable Filters 157

Fiber Optics and Optical Waveguides 157

Through a Glass Darkly: Light Lost (and Found) in Optical Components 158

Collection Optics for Forward Scatter Signals 159

4.5 Detectors 160

Silicon Photodiodes 160

Photomultiplier Tubes (PMTs) 161

Sensitivity Training: Photodiode versus PMT 163

Single Photon Counting 164

Avalanche Photodiodes (APDs) 164

PMTs: Picking a Winner 165

Photomultipliers: Inexact Science 166

Charge Transfer Devices: CCDs, CIDs, Etc. 166

4.6 Flow Systems 166

Flow System Basics 167

When You've a Jet 174

Core and Sheath: Practical Details 175

Grace Under Pressure: Driving the Sheath and Core 175

Perfect Timing: Fluidics for Kinetic Experiments 177

Oriented and Disoriented Cells 178

Matchmaker, Matchmaker, Make Me a(n) Index Match! 178

Flow Unsheathed 178

Flow Systems: Garbage In, Garbage Out 178

4.7 Electronic Measurements 180

Electricity and Electronics 101 180

The Coulter Principle: Electronic Cell Sizing 182

4.8 Analog Signal Processing 183

Beam Geometry and Pulse Characteristics 183

Electronics 102: Real Live Circuits 184

Detector Preamplifiers and Baseline Restoration 190

Analog Pulse Processing: Front Ends and Triggering 191

Dead Times, Doublets, and Problem Pulses 196

Trigger Happy? 196

Analog Linear,

Log and Ratio Circuits 197

4.9 Digital Signal Processing 204

Analog-to-Digital Conversion 204

Digital Pulse Processing and DSP Chips 209

Digitization: Tying it All Together 214

4.10 Performance: Precision, Sensitivity, and Accuracy 214

Precision; Coefficient of Variation (CV) 214

Sensitivity I Minimum Detectable Signal 215

Sensitivity II MESF Units 216

Accuracy I Linearity and Nonlinearity 217

Sensitivity III What's All the Noise About? 217

Sensitivity IV More Photons Give Better Precision 218

Sensitivity V Background Effects 218

Sensitivity VI Electrons Have Statistics, Too 218

Source Noise Fluctuations and Performance 219

I Blurred It Through the Baseline 219

Restoration Comedy: The Case of the Disappearing Leukocytes 220

Top 40 Noise Sources 221

Sensitivity 007: Q and B (Dye Another Day?) 221

5. Data Analysis 225

5.1 Goals and Methods in Data Analysis 225

Cell Counting 225

Characterization of Pure Cell Populations 226

Identification of Cells in Mixed Populations 226

Characterization of Cell Subpopulations 226

Data Analysis Hardware and Software Evolve 226

5.2 Computer Systems for Flow Cytometry 227

The Beginning 227

The End of the Beginning 227

Data Rates and Data Acquisition Systems 228

5.3 Primary Data: Frequency Distributions 231

You Say You Want a Distribution 231

Distributions Have Their Moments 233

Some Features of the Normal Distribution 234

Measures of Central Tendency: Arithmetic and Geometric Means, Median, and Mode 235

Measures of Dispersion: Variance, Standard Deviation, CV, and Interquartile Range 235

Robustness in Statistics; the Robust CV 235

Calculating and Displaying Histograms 236

Bivariate and Multivariate Distributions and Displays 237

5.4 Compensating Without Decompensating 242

5.5 Dealing With the Data 244

Comparing and Analyzing Univariate Histograms 244

Deconvoluting Single-Parameter Histograms 246

Analysis of Two-Parameter Data 246

Analysis of Two-Parameter Distributions 247

5.6 Multiparameter Data Analysis 248

Multiparameter versus Multivariate Analysis 248

Multiparameter Analysis of Leukocyte Types: 1974 248

Multiparameter Analysis of Leukocyte Types: 2002 250

Procedures for Automated Classification 250

5.7 Analysis of Collected Data: How Much Is Enough/Too Much? 253

Data Storage 254

Linear and Log Scales and Ratios: Proceed with Care! 255

6. Flow Sorting 257

6.1 Sort Control (Decision) Logic 257

6.2 Preselected Count Circuits and Single Cell Sorting 258

6.3 Droplet Sorting, High-Speed and Low 258

Droplet Generation 259

Drop Charging and Deflection 260

Determining Droplet Delay Settings 262

Transducers and Transducer Drive Signals 263

Improving Droplet Sorting 263

Sorting Large Objects with Droplet Sorters 263

6.4 Fluidic Switching Cell Sorters 264

Sorting Large Objects Using Fluidic Switching 265

Sorting Very Small Objects: Microfluidic Switching 266

6.5 Cell Manipulation By Optical Trapping 266

6.6 Cell Damage Cell Selection ("Cell Zapping") 266

Photodamage Cell Selection 266

Sorting (Zapping) Without Flow (Gasp!) 267

Electrodamage Cell Selection in Flow 267

6.7 Measures of Cell Sorter Performance: Purity, Recovery (Yield), and Efficiency 267

Coincidence Effects on Performance 267

Doing the Math 268

Speed Limits: The Reynolds Rap 269

Instrument Utilization 269

Monitoring versus Sorting for Cell Preparation 269

Collection Techniques: Life and Death Decisions 269

Dilutions of Grandeur 270

Can Getting Sorted Be Hazardous to Cells' Health? 270

6.9 Biohazard Control and Biosafety in Flow Cytometers and Sorters 271

7. Parameters and Probes 273

7.1 Physical Parameters and Their Uses 273

Electrical Parameters 273

Acoustic Measurements of Cells in Flow 274

Optical Parameters: Light Scattering 274

Optical Parameters: Absorption 281

Optical Parameters: Extinction 282

Other Transmitted Light Measurements 282

Optical Parameters: Fluorescence 283

Optical Parameters: Fluorescence

7.2 Intrinsic Cellular Parameters 285

Cell Size 285

Cell Shape and Doublet Discrimination 289

Measurement of Intrinsic Parameters Using Absorption and Extinction Signals 290

Fluorescence Measurement of Intrinsic Parameters 290

7.3 Probes, Labels, and [Not] Protocols for Extrinsic Parameter Measurements 293

Probes, Labels, and Dyes 293

Dyes and Quality Control: Gorillas in the NIST 294

Mechanisms of Staining by Fluorescent Dyes 298

"Vital" Staining 299

Fixation

Why and How 302

Red Blood Cell Lysis: The Distilled Essence 306

7.4 Nucleic Acid Dyes and Their Uses 306

DNA Content Measurement 306

DNA Base Composition 317

Chromatin Structure; Identifying Cells in Mitosis 319

RNA Content 320

7.5 Fluorescent Labels and Protein Dyes 326

Estimating Total and Basic Protein Content of Cells 327

Covalent Labels for Antibodies and Other Molecules 328

Future Tandems: Heterocycles Built for Two? 335

Cyanine Dye Labels: From Cy-Fi to Hi5 for Cy5 336

Blue Notes: AMCA and Cascade Blue 337

Hey, BODIPY! 337

Alexa Dyes: Some Thoughts on Dyemographics 338

Other Organic Fluorescent Labels: A Dye Named Joe, etc. 338

Quantum Dots 339

Getting Labels Onto Molecules of Interest 340

7.6 Improving Signals from Labels: Amplification and Other Techniques 340

Limits to Sensitivity: Autofluorescence 341

Raman Scattering Effects on Sensitivity 342

Increasing Sensitivity: Amplification Techniques 343

Improving Sensitivity: Time-Resolved Fluorescence 345

7.7 Measuring Cell Surface and Intracellular Antigens 345

History and Background 345

Antibody Reagents and Staining Procedures 348

Fluorescence Measurements: Lurching Toward Quantitation 353

7.8 Nucleic Acid Sequence Detection 361

Peptide Nucleic Acid (PNA) Probes 362

7.9 Probes for Various Cell Constituents 362

Surface Sugars (Lectin Binding Sites 362

Analysis of Total Carbohydrate Content 363

Specific Detection of Cellulose 363

A Probe for Cell Surface Aldehydes 363

Probes for Lipids and Cholesterol 364

Probes for Cytoskeletal Organization/Actins 364

7.10 Time as a Parameter: Kinetic Measurements 364

Sample Handling for Kinetic Measurements 365

Time as a Quality Control Parameter 366

Slooowww Flooowww 366

7.11 Labeled Ligand Binding 366

Labeling Strategies 367

Formal Analysis of Ligand binding 367

Labeled Ligands versus Anti-Receptor Antibodies 368

Ligand Binding Detected by Functional Changes 368

Fluorescent Ligand Binding: Some Examples 368

7.12 Functional Parameters I 369

Cell Surface Charge 369

Cell Membrane Characteristics 369

Enzyme Activity 378

Sulfhydryl (Thiol) Groups; Glutathione 381

7.13 Functional Probes II: Indicators of Cell Activation 381

Changes in the Cellular Ionic Environment Following Activation by Ligand Interaction with Cell Surface Receptors 382

"Structuredness of Cytoplasmic Matrix" (SCM) and the Cercek Test for Cancer 383

Optical Probes of Cell Membrane Potential 385

Optical Probes of Intracellular Calcium 402

Flow Cytometric Probes of Intracellular pH 405

The Hat Trick: Multiparameter Approaches to Ion Flux Measurements in Cell Activation 407

NOsing Around for Nitric Oxide 408

Other Ions in the Fire 408

7.14 Reporter Genes 408

Somebody Cloned My Gal: Enzymes as Reporter Genes 408

Green Fluorescent Protein (GFP) et al 409

Minority Report(er)? 410

8. Buying Flow Cytometers 411

8.2 History 411

8.3 BD Biosciences 412

The BD FACS Vantage SE Cell Sorter 413

The BD FACSCalibur Analyzer 414

The B-D LSR II Analyzer 416

The B-D FACSAria Cell Sorter 417

The B-D FACSCount 418

8.4 Beckman Coulter, Inc. 418

Background

and Signal-to-Background 418

The Beckman Coulter EPICS ALTRA Cell Sorter 419

The Beckman Coulter Cytomics FC 500 Analyzer 420

The EPICS XL and XL-MCL Analyzers 422

8.5 DakoCytomation 423

The MoFlo Cell Sorter 423

The CyAn Flow Cytometer 424

8.6 Cytopeia 425

The InFlux Cell Sorter 425

8.7 Optoflow AS 426

The MICROCYTE Flow Cytometer 426

8.8 Partec GmbH 427

The CyFlow and CyFlow ML Flow Cytometers 427

The PAS, PAS II, and PAS III Flow Cytometers 428

PA Ploidy Analyzer and CCA Cell Counter Analyzer 429

8.9 Some Other Flow Cytometer Companies 429

Advanced Analytical Technologies, Inc.

(AATI) 429

Agilent Technologies, Inc. 429

Apogee Flow Systems Ltd. 430

Bentley Instruments 430

Chemunex SA 430

CytoBuoy b.v. 430

Delta Instruments bv 431

Fluid Imaging Technologies, Inc. 431

FOSS Electric A/S 431

Guava Technologies, Inc. 431

Howard M. Shapiro, M.D., P.C. 431

iCyt

Visionary Bioscience 431

International Remote Imaging Systems 431

Luminex Corporation 431

NPE Systems, Inc. 432

Union Biometrica, Inc. 432

8.10 Hematology Instruments, Etc. 433

8.11 Little Orphan Analyzers (And Big Orphan Sorters) 434

Bio/Physics and Ortho: Cytofluorograf to Cytoron 434

HEKA Elektronik GMBH: The FLUVO II Analyzer 435

The Kratel Partograph 435

The ODAM ATC 3000 435

Also Among the Missing 436

Flow Cytometer Rehabilitation; Used Instruments 436

Following Suit 436

8.12 Third Party Software 437

8.13 The Selling of Flow Cytometers: Hype and Reality 437

8.14 Applying for a Grant for a Cytometer 438

9. Building Flow Cytometers 441

9.1 Why Buy a Flow Cytometer? 441

9.2 Why Build a Flow Cytometer? 441

9.3 Learning to Build Your Own 442

10. Using Flow Cytometers: Applications, Extensions, and Alternatives 443

10.1 The Daily Grind 443

Keeping the Instrument Running: Diet and Exercise 443

Particulars: Drawing a Bead on Flow Cytometer Alignment, Calibration, and Standardization 444

Rose Colored Glasses: Optical Filter Selection 446

Experimental Controls 447

Shake Well Before Using: When Controls Won't Help 447

10.2 Significant Events in the Lives of Cells 448

Taking the Census: Cell Counting 448

The Doubled Helix: Reproduction 448

Memento Mori: Detecting Cell Death 462

Die Another Day: Cytometry of Telomeres 464

10.3 Identification of Cells in Mixed Populations 464

Mixed Genotypes versus Mixed Phenotypes 464

No Parameter Identifies Cancer Cells 464

Many Parameters Identify Blood Cells 464

Flow Cytometric Parameters Useful for Blood Cells 464

Specific Gene Products Identify Cell Types 465

Maturation Processes and "Missing Links": The "Ginger Root" Model 465

Practical Multiparameter Gating: Color Wars 467

Finding Rare Cells 469

10.4 Tricks and Twists: Odd Jobs for Flow Cytometry 471

Single Molecule Detection 471

DNA Sizing, if not Sequencing, in Flow 471

Solid Phase (Bead) Assays Using Flow Cytometry 473

Cells in Gel Microdroplets and on Microspheres 474

10.5 Single Cell Analysis: When Flow Won't Do 475

10.6 Applications of Flow Cytometry 476

Cell Differentiation, Ab Ovo and De Novo 476

Somatic Cell Genetics and Cell Hybridization 477

Chromosome Analysis and Sorting and Flow Karyotyping 477

Probing Details of Cellular Structures and Inter- and Intramolecular Interactions 479

Clinical Flow Cytometry: Turf and Surf 480

Hematology 480

Immunology 489

Cancer Biology and Clinical Oncology 502

Analysis of Sperm 508

Isolating Fetal Cells from the Maternal Circulation for Prenatal Diagnosis 509

The March of Time: Circadian Rhythms, Aging, and Atherosclerosis 510

Clinical Application: Urine Analysis 510

The Animal Kingdom 510

Big Stuff, Vegetable, Animal or Mineral 512

Flow Cytometry of Plant Cells and Chromosomes 512

Microbiology, Parasitology and Marine Biology 514

Pharmacology and Toxicology 537

Food Science 538

Biotechniques and Biotechnology 539

Alternatives: Microfluidic Cytometers, Flow and Static 541

Cytometry Afield 541

11. Sources of Supply 543

11.1 Resources, Societies, Journals 543

11.2 Optical Supply Houses 543

11.3 Probes and Reagents 544

11.4 Calibration Particles/Cytometry Controls 548

11.5 Flow Cytometers 549

Hematology Instruments 551

11.6 Data Analysis Software/Systems 551

Hardware and Software 551

Commercial Software Sources 552

Noncommercial Software Sources 552

11.7 Cytometer Rehabilitation/Add-ons 553

11.8 Flow Cytometer Parts 553

Flow System Plumbing 553

Photodetectors 554

DC-DC Converter Modules for HV Power Supplies 555

Power Supplies (Low Voltage) 555

Other Electronics 555

11.9 Lasers 555

Laser Trade Publications 555

Laser Manufacturers 555

11.10 Optical Filters 556

Color Glass Filters 556

Interference Filters 557

Neutral Density Filters 557

Polarizing Filters and Optics 557

Tunable Filters 557

11.11 Aids to Troubleshooting Flow Cytometers When All Else Fails 557

11.12 Proficiency Testing 557

11.13 Sex Selection 557

11.14 Alternative Technology 558

12.1 Dotting i's and Crossing t's 561

12.2 Late Breaking News 561

New Book 561

New Protein Stain 561

Caveat on Fluorescent Caspase Inhibitors 561

Polyamide Probes 561

Tearing Down the (Picket) Fences 562

New Instrument: The BD FACSArray 563

Science Special Section: Biological Imaging 563

Cytomics in Predictive Medicine: a Clinical Cytometry Special Issue and Other Recent Citings and Sightings 563

12.3 Analytical Biology, Such as it Isn't: Is This Any Way to Run a Science? 563

12.5 Unfinished Business 565

AIDS and Infectious Disease in the Third World 565

A Center for Microbial Cytometry 565

A Nobel Prize for Herzenberg and Kamentsky? 566

12.6 Flow and the Human Condition 566

There's No Business Like Flow Business 566.

Notes:

Includes bibliographical references (pages 567-654) and index.

ISBN:

0471411256

OCLC:

49221931