Flow Cytometry Today: Everything You Need to Know about Flow Cytometry

Foreword
Preface
Contents
Abbreviations
Chapter 1: General Principles
1.1 Flow Cytometers General Layout
1.2 Flow Cytometers Features
1.3 Parameters and Signals
1.4 Time
References
Chapter 2: Signals: Scattering
2.1 Forward Scatter (FSC)
2.2 Side Scatter (SSC)
2.3 Raman Scattering
2.4 Depolarized Scatter
References
Chapter 3: Signals: Fluorescence, Phosphorescence, Impedance, Extinction
3.1 Fluorescence
3.1.1 Depolarized Fluorescence
3.1.2 Autofluorescence
3.2 Phosphorescence
3.3 Impedance
3.4 Axial Extinction
References
Chapter 4: Fluidics
4.1 Overview on Fluids
4.1.1 Laminar Flow and Turbulent Flow
4.1.2 Hydrodynamic Focusing
4.2 Cytometer Fluidics
4.2.1 Sheath and Core
4.2.2 Flow System Components
4.2.3 Flow Rate Control
4.2.4 Sample Injection
4.2.4.1 Sample Differential Control
4.2.4.2 Absolute Counts
4.2.5 Event-Light Interaction: The Interrogation Point
4.2.5.1 In-Cuvette Interaction
4.2.5.2 Stream-in-Air Interaction
4.2.5.3 Interrogation on an Open Surface
4.2.5.4 Systems Based on Acoustic Focusing
4.2.5.5 Sheathless Systems
References
Chapter 5: Light Sources
5.1 Arc Lamps
5.2 Lasers
5.2.1 Gas Lasers
5.2.1.1 Argon Ion Lasers
5.2.1.2 Krypton Ion Lasers
5.2.1.3 Mixed-Gas Ion Lasers (Argon/Krypton)
5.2.1.4 Helium-Neon Atom Lasers
5.2.1.5 Helium-Cadmium Ion Lasers
5.2.1.6 Helium-Silver and Neon-Copper Metal Vapor Lasers
5.2.2 Solid-State Lasers (SSLs)
5.2.2.1 Ultraviolet (UV) Emitting SSLs
Deep UV
320 nm
355 nm
5.2.2.2 Near-Ultraviolet Emitting SSLs
5.2.2.3 Violet Emitting SSLs
5.2.2.4 Blue Emitting SSLs
Deep Blue
Blue-Green
5.2.2.5 Green and Yellow-Green Emitting SSLs
532 nm
552 nm
561 nm
5.2.2.6 Orange Emitting SSLs
5.2.2.7 Red Emitting SSLs
Short Red
Full Red
5.2.2.8 Infrared (IR) Emitting SSLs
5.2.2.9 Supercontinuum White Light Emitting SSLs
5.2.3 Liquid State Lasers (Dye Lasers)
5.3 Light Emitting Diodes (LEDs)
References
Chapter 6: Optical Benches
6.1 From the Light Source(s) to the Interrogation Point(s)
6.2 From the Interrogation Point(s) to the Detector(s)
6.3 Optical Bench Components
6.3.1 Absorption Filters
6.3.2 Interference Filters
6.3.3 Neutral Density Filters
6.3.4 Polarizing Filters
6.3.5 Beam Splitters
6.3.6 Wavelength Division Multiplexing (WDM)
6.3.7 Prisms, Gratings, Coarse WDM (CWDM)
6.4 Optical Bench Layouts
6.4.1 Transmission Benches
6.4.2 Reflection Benches
6.4.3 Multilaser Benches
6.4.3.1 Spaced Lasers and Separate Pathways/Detectors
6.4.3.2 Spaced Lasers and Shared Pathways/Detectors
6.4.3.3 Time Delay
6.4.3.4 Continuous Collinear Lasers
6.4.3.5 Pulsed Collinear Lasers
6.4.4 Special Solutions
6.4.4.1 Pie-Shaped Design
6.4.4.2 Benches for (De)Polarized Signals
6.4.4.3 Spectrally Enhanced Optical Benches
References
Chapter 7: Detectors and Electronics
7.1 Photodetectors
7.1.1 Photodiodes (PDs)
7.1.2 Avalanche Photodiodes (APDs)
7.1.3 Photomultipliers (PMTs)
7.1.4 Multi-anode Photomultipliers
7.1.5 Silicon Photomultipliers (SiPMs)
7.1.6 Charged-Coupled Devices (CCDs)
7.1.7 Trans-impedance Amplifiers (TIAs)
7.2 Circuitry
7.2.1 Analog Model
7.2.1.1 Baseline Restorers
7.2.1.2 Comparators and Threshold
7.2.1.3 Accessory Circuits
7.2.1.4 Amplifiers
Linear Amplifiers
Logarithmic Amplifiers
7.2.1.5 DC Restorers
7.2.1.6 Peak Detectors and Integrators
Measurement of H (Peak Detectors)
Measurement of A (Integrators)
Measurement of W
Pulse Processing Synchronization
7.2.1.7 Analog-to-Digital Converters (ADCs)
Bit Number
Clock Number
7.2.2 Digital Model
7.2.2.1 Data Acquisition Boards (DAQs)
Analog-to-Digital Converters (ADCs)
Field-Programmable Gate Arrays (FPGAs)
Digital Signal Processors (DSPs)
7.2.2.2 General Considerations on the Digital Model
7.2.3 Hybrid Model
References
Chapter 8: Signal Analysis
8.1 The Background
8.1.1 Instrumental Background (BCAL)
8.1.2 Experimental Background (Bsos)
8.2 The Pulse
8.2.1 Pulse Analysis in Analog Systems
8.2.2 Pulse Analysis in Digital Systems
8.2.2.1 Window´s Gate and Window´s Extension
8.2.2.2 Area Scaling
8.2.3 Practical Applications of Pulse Analysis
8.2.3.1 FSC-A vs. FSC-H vs. FSC-W
8.2.3.2 FL-A vs. FL-W
8.2.3.3 FL-H vs. FL-W
8.3 Dynamic Range of the Signal
8.3.1 Effective Resolution
8.3.2 Picket Fence Phenomenon
References
Chapter 9: The Cytometric File
9.1 FCS Format
9.2 Segments
9.2.1 Header Segment
9.2.2 Text Segment
9.2.3 Data Segment
9.2.4 Analysis Segment
9.2.5 Optional Segments
9.3 Keywords
9.3.1 Standard Keywords
9.3.1.1 Required Keywords
9.3.1.2 Optional Keywords
9.3.2 Non-standard Keywords
9.3.3 Relationships Between Keywords and Compensation Procedures
References
Chapter 10: Data Transformation
10.1 Logarithmic Transformation
10.2 Log-Like Transformations
10.3 Polynomial Transformation
References
Chapter 11: Data Representation
11.1 Histogram
11.1.1 Histograms of Lin Amplified Data
11.1.2 Histograms of Log Amplified/Transformed Data
11.1.3 Histograms of Log-Like Transformed Data
11.2 Cytograms
11.2.1 Representation by Dots (Dot Plot)
11.2.2 Representation by Contours (Contour Plot)
11.2.2.1 Logarithmic Density
11.2.2.2 Probability
11.2.3 Representation by False Colors or Gray Tones
11.2.4 Pseudo-Three-Dimensional Representation
11.2.5 Three-Dimensional Representation
References
Chapter 12: Data Analysis
12.1 Immunofluorescence Measurements
12.1.1 The Vexed Question of the Negative Control
12.1.1.1 Isotype Control
12.1.1.2 Isoclonic Control
12.1.1.3 Fluorescence-Minus-One (FMO) Control
12.1.1.4 Unstained Control
12.1.2 Histograms
12.1.2.1 MFI (Mean Fluorescence Intensity)
12.1.2.2 RFI (Relative Fluorescence Intensity)
12.1.3 Cytograms
12.1.4 Weak Positivity in Immunofluorescence
12.1.4.1 Weak Positivity with a Negative Component
12.1.4.2 Weak Positivity Without an Apparent Negative Component
12.2 DNA Content Measurements
12.2.1 DNA Content versus BrdU Incorporation
12.3 Concept of Gate and Concept of Region
12.3.1 Combined (Boolean) Use of Regions and Gates
12.3.1.1 In the Determination of Hematopoietic Stem Cells (HSCs)
ISHAGE Protocol
12.3.1.2 In the Determination of the Minimal Residual Disease (MRD)
12.3.1.3 In the Augmentation of the Dimensionality in Cell Subset Analysis
12.4 Advanced Tools and Future Perspectives
12.4.1 Pre-processing Programs
12.4.1.1 Quality Assessment
12.4.1.2 Data Normalization
12.4.1.3 Data Compensation
12.4.1.4 Data Transformation
12.4.2 Data Processing Programs
12.4.2.1 Dimensionality Reduction Based Programs
12.4.2.2 Clustering-Based Programs
References
Chapter 13: Standards, Setup, Calibration, and Control Techniques
13.1 Standards in Flow Cytometry
13.1.1 Natural Standards
13.1.2 Artificial Standards
13.1.2.1 Type 0 Artificial Standards
13.1.2.2 Type I Artificial Standards
13.1.2.3 Type II Artificial Standards
Type IIa
Type IIb
Type IIc
13.1.2.4 Type III Artificial Standards
Type IIIa
Type IIIb
Type IIIc Standards in Daily Practice
13.1.2.5 Primary Performance Parameters (PPP)
13.1.3 Standard Use in Quality Procedures
13.1.3.1 Internal Quality Controls (IQCs)
13.1.3.2 External Quality Assessments (EQAs)
13.2 Optical Bench Setup
13.3 Photodetectors´ Setup
13.3.1 SDen (Electronic Noise Standard Deviation)
13.3.2 PMT Setup
13.3.3 APD Setup
13.4 Calibration
13.4.1 Calibration in ERF
13.4.2 Calibration in MESF
13.4.3 Calibration in ABC
13.4.3.1 With Conjugated Antibodies
13.4.3.2 With Unconjugated Antibodies
13.4.4 Calibration in FLU
13.4.5 Calibration in Nanometers
13.5 Instrument Performance and Its Control
13.5.1 Linearity
13.5.2 Accuracy
13.5.2.1 Carry-over
13.5.2.2 Count Inaccuracy at High Speed
13.5.3 Resolution
13.5.4 Sensitivity
13.5.4.1 Q, Qr, Stain Index, and Other Indexes
13.5.4.2 Antibody Titration
13.5.5 Limits of Blank (LOB), Detection (LOD), and Quantification (LOQ)
13.5.5.1 LOB, LOD, and LOQ in the Detection of Weak Signals
LOB (Limit of Blank)
LOD (Limit of Detection)
LOQ (Limit of Quantification)
13.5.5.2 LOB, LOD, and LOQ in Rare Event Analysis
LOB (Limit of Blank)
LOD (Limit of Detection)
LOQ (Limit of Quantification)
LOD and LOQ and Minimal Residual Disease (MRD)
13.5.6 Precision
13.5.7 Specificity
References
Chapter 14: Fluorochromes: Overview
14.1 Spectral Behavior of Fluorescent Molecules
14.2 Relationships with the Environment
14.2.1 Spectral Effects
14.2.1.1 Bathochromic Effect
14.2.1.2 Hypsochromic Effect
14.2.1.3 Hyperchromic Effect
14.2.1.4 Hypochromic Effect
14.2.1.5 Solvatochromic Effect
14.2.2 Other Effects
14.2.2.1 Extinction or Quenching
14.2.2.2 Photodestruction or Photobleaching
14.2.2.3 Non-radiative Transfer of Energy (FRET)
14.3 Accessory Groups
References
Chapter 15: Fluorochromes Suitable for Antibody Conjugation
15.1 Large Protein Molecules
15.1.1 Phycobiliproteins
15.1.1.1 R-Phycoerythrin (R-PE)
15.1.1.2 Allophycocyanin (APC)
15.1.1.3 B-Phycoerythrin (B-PE)
15.1.1.4 Phycocyanin-C (PC)
15.1.1.5 Cryptofluor Molecules
15.1.2 Peridinin-Chlorophyll-Protein (PerCP)
15.1.3 AmCyan and AmCyan 100
15.2 Small Organic Molecules
15.2.1 Pyrene Derivatives
15.2.2 Pyrydil-Oxazole Derivatives
15.2.3 Coumarin Derivatives
15.2.4 Xanthene Derivatives
15.2.4.1 Blue Excited Xanthenes (FITC and Others)
15.2.4.2 Green and Yellow-Green Excited Xanthenes (TRITC and Others)
15.2.4.3 Red and NIR Excited Xanthenes (Vita Blue and Others)
15.2.5 Cyanines
15.2.5.1 Cyanine 2 (CY2) and Cyanine CY2-Like
15.2.5.2 Cyanine 3 (CY3) and Cyanine CY3-Like
15.2.5.3 Cyanine 3.5 (CY3.5) and Cyanine CY3.5-Like
15.2.5.4 Cyanine 5 (CY5) and Cyanine CY5-Like
15.2.5.5 Cyanine 5.5 (CY5.5) and Cyanine CY5.5-Like
15.2.5.6 Cyanine 7 (CY7) and Cyanine CY7-Like
15.2.5.7 Other Near-Infrared (NIR) Emitting Cyanines
15.2.6 Proprietary Molecules
15.2.6.1 Alexa Series
15.2.6.2 Other Series
15.2.6.3 Miscellany
15.3 π-Conjugated Organic Polymers (Brilliant Violet and Others)
15.3.1 Organic Polymers-Based Tandems
15.3.2 Narrow-Band Emissive Chromoforic Polymer Dots (Pdots)
15.4 Nanocrystals
15.4.1 Quantum Dots (Qdots)
15.4.2 Upconverting Nanoparticles (UCNPs)
15.5 Tandem Fluorochromes
15.5.1 UV-Excited Tandem
15.5.2 Violet-Excited Tandem
15.5.2.1 BV421-Based Tandems
15.5.2.2 Super Bright 436-Based Tandems
15.5.2.3 Supernova V428-Based Tandems
15.5.3 Blue-Excited Tandems
15.5.3.1 PE-CF594
15.5.3.2 PE-Dazzle 594
15.5.3.3 PE-Texas Red (PE-TR)
15.5.3.4 PE-CY5
15.5.3.5 PE-AL647
15.5.3.6 PE-CY5.5
15.5.3.7 PerCP-CY5.5
15.5.3.8 PE-CY7
15.5.3.9 Other PE-Based NIR-Emitting Tandems
15.5.3.10 BB515-Based Tandems
15.5.4 Green-Yellow-Excited Tandems
15.5.5 Red-Excited Tandems
15.5.5.1 APC-CY5.5
15.5.5.2 APC-AF700
15.5.5.3 APC-CY7 and APC-H7
15.5.5.4 Other APC-Based NIR-Emitting Tandems
15.6 New Solutions
15.6.1 Nanoparticle-Encapsulated Fluorophores
15.6.2 Backbone-Linked Fluorophores (Kiravia, NovaFluor)
References
Chapter 16: Fluorochromes That Bind Nucleic Acids
16.1 Triaryl Derivatives
16.2 Stylbene Derivatives
16.3 Phenylindole Derivatives (DAPI, DIPI)
16.4 Bisbenzimidazole Derivatives
16.4.1 HO33258
16.4.1.1 HO33258 in Chromosome Analysis
16.4.2 HO33342
16.4.2.1 HO33342 in ``Side Population´´ Analysis
16.4.3 HO34580
16.4.4 Dyecycle Violet (DCV)
16.5 Phenanthridine Derivatives
16.5.1 Propidium Iodide (PI)
16.5.1.1 PI in DNA Content Evaluation
16.5.1.2 PI in Membrane Permeability Evaluation
16.5.2 Ethidium Bromide
16.5.3 Benzophenanthridine Alkaloids
16.6 Cyanines
16.6.1 TOTO Series Molecules
16.6.2 TO-PRO Series Molecules
16.6.2.1 YO-PRO-1
16.6.2.2 TO-PRO-3
16.6.2.3 TO-PRO-5
16.6.3 SYTOX Series Molecules
16.6.4 SYTO Series Molecules
16.6.5 SYBR Series Molecules
16.7 Aromatic Molecules, Heterocyclic
16.7.1 Acridines
16.7.1.1 Acridine Orange
16.7.1.2 Proflavine
16.7.1.3 Quinacrine
16.7.1.4 Coriphosphine O
16.7.2 Oxazines
16.7.3 Thiazole Orange (TO) and Its Analogs (DETC)
16.7.4 7-Amino-Actinomycin D
16.7.5 Pyronin Y
16.7.6 LDS-751
16.7.7 Berberine (BRB)
16.8 Aromatic Molecules, Non-heterocyclic
16.8.1 Tricyclic Antibiotics
16.8.2 Anthraquinones
16.8.2.1 DRAQ Molecules
16.8.2.2 CyTRAK Orange
References
Chapter 17: Fluorochromes for the Study of the Cell Features
17.1 Protein Content
17.2 Nucleic Acid Content and Chromatin Organization
17.3 Cell Viability
17.3.1 DNA Impermeant Probes
17.3.2 Amine Reactive Dyes
17.3.3 Fluorescein Derivatives
17.3.3.1 Carboxyfluorescein Esters
17.3.3.2 Calcein
17.3.4 Calcofluor White
17.3.5 Trypan Blue
17.4 Membrane Potential
17.4.1 Carbocyanines
17.4.2 Oxonol Derivatives
17.4.3 Xanthene Derivatives
17.5 Mitochondrial Membrane Potential
17.5.1 JC-1
17.6 Mitochondrial Mass
17.6.1 Nonyl Acridine Orange (NAO)
17.6.2 Mitotracker Molecules
17.6.3 Mitofluor Molecules
17.7 Intracellular pH
17.7.1 DCH
17.7.2 Bcecf-Acetoxymethyl Ester
17.7.3 SNARF-1 Acetoxymethyl Ester
17.8 Lysosomal Mass and Lysosomal pH
17.8.1 Lysotracker and Lysohunt Molecules
17.8.2 Lysosensor Molecules
17.8.3 Acridine Orange (AO)
17.9 Free Radicals (Oxidative Burst)
17.9.1 Dihydroethidium (DHE)
17.9.2 Dichlorofluorescin Diacetate (DCF-DA)
17.9.3 Dihydrorhodamine 123 (DHR123)
17.9.4 Tetrazolium Derivatives
17.10 Calcium Content
17.10.1 INDO-1 Acetoxymethyl Ester
17.10.2 Xanthene Derivatives (Fluo Molecules and Others)
17.10.3 Fura Molecules
17.10.4 BTC
17.11 Sodium Content
17.11.1 SBFI
17.11.2 Sodium Green
17.12 Potassium Content
17.12.1 PBFI
17.12.2 BCECF
17.13 Chloride Content
17.14 Magnesium Content
17.15 Glutathione Content
17.16 Heavy Metals Content
17.17 Cell Proliferation
17.17.1 Fluorescein Esters (CFSE)
17.17.2 Lipophilic Carbocyanines
17.17.2.1 DiO, DiI, DiD, AND DiR Molecules
17.17.2.2 PKH Molecules
PKH2
PKH26
PKH67
17.17.3 CellVue Series Molecules
17.18 Multidrug Resistance
17.18.1 Rhodamine 123 (RH123)
17.18.2 Calcein Acetoxymethyl Ester
17.19 Membrane Fluidity
17.20 Lipid Content
17.20.1 NILE RED
17.20.2 Bodipy and Its Derivatives
17.21 Lipid Peroxidation
17.21.1 Cis-Parinaric Acid
17.21.2 C11-BODIPY581/591
17.22 Endoplasmic Reticulum (ER) Labeling
References
Chapter 18: Fluorescent Proteins
18.1 Fluorescent Proteins and Flow Cytometry
18.1.1 Green Fluorescent Proteins (GFPs)
18.1.2 Blue Fluorescent Proteins (BFPs)
18.1.3 Cyan Fluorescent Proteins (CFPs)
18.1.4 Yellow Fluorescent Proteins (YFPs)
18.1.5 Orange Fluorescent Proteins (OFPs)
18.1.6 Red Fluorescent Proteins (RFP)
18.1.7 Infrared Fluorescent Proteins (iRFPs)
References
Chapter 19: Spillover and Compensation
19.1 Spillover
19.1.1 Intra-Laser Spillover
19.1.1.1 Intra-Laser Spillover to the Right
19.1.1.2 Intra-Laser Spillover to the Left
19.1.2 Inter-Laser Spillover
19.1.3 Spillover Matrix and Compensation Matrix
19.2 Compensation
19.2.1 Paradoxical Effects
19.2.1.1 Spillover Spreading (SS)
19.2.1.2 Perturbation of the Negative Clusters Distribution
19.2.2 Negative Values and their Management
19.2.3 Compensation by Hardware
19.2.4 Compensation by Software
19.2.4.1 In Digital Instruments
19.2.4.2 In Analog Instruments
References
Chapter 20: Artifacts
20.1 Escapees
20.2 Debris
20.3 Other Artifacts
20.3.1 Due to Anticoagulants
20.3.2 Due to Fluorochromes
20.3.2.1 Influence on Mab Binding Capacity
20.3.2.2 Interaction with Fc Receptors
20.3.2.3 Interaction with Antigen Receptors
20.3.2.4 Interaction with Antigens Other Than those Considered in the Previous Points
20.3.2.5 Unknown Mechanisms
20.3.3 Due to Serum Factors
20.3.4 Anecdotal Reports
References
Chapter 21: Cell Sorting
21.1 Fluid Switching Sorters
21.1.1 Pros and Cons of Fluid Switching Sorters
21.2 Electrostatic Sorters
21.2.1 Sorting Procedures
21.2.2 Sorting Modalities
21.2.2.1 Purity Mode
21.2.2.2 Recovery Mode
21.2.2.3 Single-Cell Mode
21.2.3 High-Pressure Systems
21.3 Pneumatic Sorters
References
Chapter 22: Non-Conventional Flow Cytometry
22.1 Imaging Flow Cytometry
22.1.1 CCD-Based Imaging Flow Cytometry
22.1.1.1 Optical Bench in CCD Based Imaging Flow Cytometry
22.1.1.2 Compensation Issues in CCD-Based Imaging Flow Cytometry
22.1.1.3 Format Issues in CCD-Based Imaging Flow Cytometry
22.1.1.4 Pros and Cons in CCD-Based Imaging Flow Cytometry
22.1.2 PMT-Based Imaging Flow Cytometry
22.1.2.1 Compensation and Format Issues in PMT-Based Imaging Flow Cytometry
22.2 Spectral Flow Cytometry (SFC)
22.2.1 Optical Bench in Spectral Flow Cytometry
22.2.2 Spectral Unmixing in Spectral Flow Cytometry
22.2.3 Format Issues in Spectral Flow Cytometry
22.2.4 PROs and CONs in Spectral Flow Cytometry
22.3 Mass Cytometry (MC)
22.3.1 Format Issues in Mass Cytometry
22.3.2 PROs and CONs in Mass Cytometry
22.3.2.1 Pros
22.3.2.2 Cons
22.4 Lifetime Cytometry
22.5 Raman Cytometry
22.6 Microfluidic Devices (Labs on Chips)
22.6.1 Hydraulic Issues in Microfluidic Devices
22.6.2 Sorting Procedures in Microfluidic Devices
References
Chapter 23: Statistics: A Cytometric Point of View
23.1 Concept of Distribution
23.1.1 Log-Normal Distribution
23.1.2 Normal (Gaussian) Distribution
23.1.3 Poisson Distribution
23.2 Location Measurements
23.2.1 Mean (Am)
23.2.2 Geometric Mean (gM)
23.2.3 Truncated Mean
23.2.4 Mode (v0)
23.2.5 Median (Me)
23.3 Spread Measurements
23.3.1 Variance (Var)
23.3.2 Standard Deviation (SD)
23.3.3 Median Absolute Deviation (MAD)
23.3.4 Robust Standard Deviation (rSD)
23.3.5 Coefficient of Variation (CV)
23.3.6 Robust Coefficient of Variation (rCV)
References
Index