FLOW CYTOMETRYIN DRUG DISCOVERYAND DEVELOPMENT

Edited by

VIRGINIA LITWIN

Covance Central Laboratory Services, Inc.

PHILIP MARDER

Redram Consulting, LLC

FLOW CYTOMETRY IN DRUG DISCOVERYAND DEVELOPMENT

FLOW CYTOMETRYIN DRUG DISCOVERYAND DEVELOPMENT

Edited by

VIRGINIA LITWIN

Covance Central Laboratory Services, Inc.

PHILIP MARDER

Redram Consulting, LLC

Copyright � 2011 by John Wiley & Sons, Inc. All rights reserved.

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ISBN: 978-0-470-43356-0

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1 0 9 8 7 6 5 4 3 2 1

PHILIP MARDER (1948–2010)

DEDICATION

This book is dedicated to my dear friend and coeditor, Philip Marder.

Phil was one of the early leaders in the use of flow cytometry pertaining to drug

discovery and development.He participated in ISAC,GLIFCA, IndyFlow, andAAPS,

and was a key contributor to both the flow cytometry and the Pharma communities.

In 1973, he joined Eli Lilly where he went on to build a strong team of research

scientists: biomarker assays developed by this team supported the full spectrum of

the drug development process, including in vitro studies, toxicology, and clinical

trials.

Midway through this editing project, Phil was diagnosed with glioblastoma

multiforme. To say that he approached this illness with grace, intelligence, and

courage would be a considerable understatement. Those of us who followed his blog

were impressed by his ability to continue to enjoy so many different aspects of his life

throughout the treatment process. Among these would be the arrival of a much loved

puppy by the name of Chaim, and the professional quality reviews he provided for

many of the fine (and not so fine) restaurants from Indianapolis toChicagowithmany

stops in between. Most impressive of all, however, was the connection he maintained

with his strong and loving family, and, of course, they with him.

Completely in character, Phil continued to be involved with the details of this

project until November 2009. It was truly a pleasure to collaborate with him on this

book that should be seen as a fitting tribute to his substantial contributions to flow

cytometry and drug development.

Our very best wishes to his family and friends.

CONTENTS

PREFACE xi

FOREWORD xiii

ACKNOWLEDGMENTS xv

CONTRIBUTORS xvii

PART I INTRODUCTION 1

Philip Marder and Virginia Litwin

1 Introduction to Flow Cytometry 3

Elizabeth Raveche, Fatima Abbasi, Yao Yuan, Erica Salerno, Siddha Kasar,

and Gerald E. Marti

2 Recent Advances in Flow Cytometry: Platforms, Tools,

and Challenges for Data Analysis 23

Paul J. Smith, Roy Edward, and Rachel J. Errington

3 Introduction to Biomarkers 55

Ole Vesterqvist and Manjula P. Reddy

vii

PART II FLOW CYTOMETRY IN THE DRUG DEVELOPMENT

PROCESS 69

Virginia Litwin and Philip Marder

4 HTS Flow Cytometry, Small-Molecule Discovery,

and the NIH Molecular Libraries Initiative 71

Larry A. Sklar and Bruce S. Edwards

5 A Multiparameter Approach to Cell Cycle Analysis

as a Standard Tool in Oncology Drug Discovery 99

Carmen Raventos-Suarez and Byron H. Long

6 Flow Cytometry in Preclinical Toxicology/Safety Assessment 123

David McFarland and Kristi R. Harkins

7 Use of Flow Cytometry to Study Drug Target Inhibition

in Laboratory Animals and in Early-Phase Clinical Trials 151

David W. Hedley

8 CD4 T Cell Assessments in Evaluation of HIV Therapeutics 169

Thomas N. Denny, Raul Louzao, John Wong, and Brooke Walker

9 Monitoring the Cellular Components of the Immune

System During Clinical Trials: A Translational

Medicine Approach 189

Virginia Litwin and James Andahazy

10 Immunogenicity Testing Using Flow Cytometry 205

Denise M. O�Hara and Valerie Theobald

11 Pharmacokinetics by Flow Cytometry: Recommendations for

Development and Validation of Flow Cytometric Method

for Pharmacokinetic Studies 225

Yuanxin Xu and Susan M. Richards

PART III VALIDATION AND REGULATORY COMPLIANCE 241

Virginia Litwin and Philip Marder

12 Regulatory Compliance and Method Validation 243

Carla G. Hill, Dianna Y. Wu, John Ferbas, Virginia Litwin,

and Manjula P. Reddy

viii CONTENTS

13 Instrument Validation for Regulated Studies 267

John Ferbas and Michelle J. Schroeder

PART IV FUTURE DIRECTIONS 279

Virginia Litwin and Philip Marder

14 Probability State Modeling: A New Paradigm for

Cytometric Analysis 281

C. Bruce Bagwell

15 Phospho Flow Cytometry: Single-Cell Signaling Networks in

Next-Generation Drug Discovery and Patient Stratification 303

Peter O. Krutzik, Sean C. Bendall, Matthew B. Hale,

Jonathan M. Irish, and Garry P. Nolan

INDEX 335

CONTENTS ix

PREFACE

The recent contraction within the pharmaceutical sector and the push to decrease the

timelines and costs associated with drug development have, fortuitously, coincided

with a marked advancement in the field of flow cytometry. As a result, flow cytometry

is now a critically important analytical tool for drug development. Flow Cytometry in

Drug Discovery and Development chronicles the unique application of flow cyto-

metry in drug development and provides examples of how it can be applied along the

drug development pathway from drug discovery to clinical testing.

The Part I of this book provides the reader with essential background information

regarding both flow cytometry and drug development. In the chapters in Part II, the

focus is on theway inwhich flowcytometry serves as an essential tool for each stage of

the drug development life cycle. The organization of this part follows along the lines

of the drug development process: drug discovery/preclinical toxicology/clinical

testing. In addition, the reader will be introduced to distinct therapeutic areas such

as oncology, anti-infectives, and autoimmunity/inflammation that are highlighted in

several chapters.

During the drug discovery phase, the life cycle of a new drug begins with the

identification of a therapeutic need and potential molecular targets that may interfere

with the disease process. The next steps in the long and costly process of bringing a

new drug to market are target validation, high-throughput compound screening, and

multiple rounds of compound selection followed by characterization. With each

successive round of selection and characterization, more rigorous standards are

applied until, ultimately, a small pool of lead compounds, or potential drug candi-

dates, is identified from the initial compound library. Chapters 4–7 address the

application of flow cytometry in these early stages of drug development. The

development of high-throughput flow cytometry and its application in compound

xi

screening are addressed in Chapter 4. During the earlier stages of compound

selection and characterization in oncology drug discovery, cell cycle analysis using

flow cytometry plays a critical role, as illustrated in Chapter 5. Awider variety of flow

cytometric methods are critical during the later stages of preclinical compound

selection and characterization as described in Chapter 6. The process of conducting

formal GLP toxicology studies with a small pool of highly characterized potential

drug candidates is also introduced in Chapter 6. The transition from preclinical

testing to clinical testing is highlighted in Chapter 7 that emphasizes the use of panels

of phosphospecific antibodies in cancer therapeutics.

Clinical trial testing of novel therapeutic compounds includes biomarker mea-

surements for efficacy and pharmacodynamic (PD) effect, safety assessment, and

pharmacokinetic (PK) evaluation. Chapters 8–11 describe howflow cytometry is used

to achieve each of these objectives. Chapter 8 reviews the historical and continued

importance of CD4 T-cell monitoring as an efficacy marker in the evaluation of

antiretroviral therapy for HIV infections. Chapter 9 outlines the translational med-

icine approach to PD biomarker evaluation with an emphasis on inflammatory and

autoimmune disorders. Chapter 10 focuses on safety concerns associated with the

clinical administration of biotherapeutic compounds and the advantages of flow

cytometric analysis in determining antidrug antibody responses. Flow cytometric

methods can also be used for PK analysis of protein- and peptide-based therapeutics as

described in Chapter 11.

The importance of validation and regulatory compliance in drug development

is highlighted by the fact that an entire section of the book is dedicated to this topic

(Part III, Validation and Regulatory Compliance). Different stages of the drug

development life cycle are subjected to different regulatory requirements. For data

to be acceptable for submission to regulatory agencies, laboratory instrumentation

and analytical methods must be validated according to applicable standards.

The last, Part IV, focuses on how multiparametric flow cytometry data will be

analyzed and applied in near future. At present, one of the most pressing needs in flow

cytometry is for advanced data analysis tools, capable of rapidly evaluating the large,

complex data sets generated by multiparametric methods. Chapter 14 presents a new

paradigm for data analysis, called probability state modeling, which is capable of

visualizing and analyzing multiparametric files. The last chapter brings together one

of the newest, potentiallymost valuable applications offlowcytometry, phospho-flow,

with one of the most exciting and potentially most valuable aspects of patient

treatment, personalized medicine.

The contributors to the book include both flow cytometry thought leaders from

academia and industry and flow cytometry users from the pharmaceutical sector.

This diverse mix of expertise is typical of the collaboration among disciplines that

increasingly has been a hallmark of the flow cytometry community, and part of

what makes working in this challenging and exciting discipline a rewarding

experience.

xii PREFACE

FOREWORD

Almost 10 years ago, Phil Marder asked me how ISAC could expand its interest in

high-content screening and in particular the role that flow cytometry could play in this

emerging area. Over the next decade, PhilMarder was responsible for developing and

advancing high-content screening tools within the pharma industry. This book is the

result of that drive of Phil Marder to show howmuch impact flow cytometry can have

on this arena. It is with great sadness that Phil did not live to see this volume published.

Without doubt, it reflects the knowledge, skills, and foresight of Phil Marder who saw

the impact of flow cytometry on screening well before anyone else.

What this book does effectively is to establish a well-defined set of assays,

processes, and regulations that must be considered in the performance of robust

screening by flow cytometry. Specific assays are identified and discussed with

sufficient details to be reproduced in screening mode. The variety of screens

presented covers a wide spectrum from cell culture assays, to human and animal

studies and to the role that management and manipulation of data play in today�sscientific world.

Flow cytometry has been used for over 40 years to define intricate pathways and

responses from single cells. Its power as the leader in single cell analysis is

unquestioned by most, but many scientists fail to see its capability at the systems

level. Indeed, it is this capacity that is now opening up new opportunities that were

previously not possible. The ability to multiplex both cells and beads has made it to

possible to track cellular phenotype and function in multiple dimensions simulta-

neously. In drug screening, very complex assays can be automated and analyzed in

xiii

minutes rather than days. The need now is to advance the analytical tools required for

the very complex opportunities.

This book brings out an entirely new set of opportunities for this mature

technology. It opens up a Pandora�s box of assay systems and approaches in a way

that may well expand the field well beyond what was imaginable a few years ago.

J. Paul Robinson

xiv FOREWORD

ACKNOWLEDGMENTS

The editors wish to offer sincere thanks to all the contributors for the expertise they

provided during the writing and compilation of this book. Thanks also to Jonathan

Rose at John Wiley & Sons, Inc. for approaching us about the project, to the artist

Glenn Vilppu for the drawing of Phil Marder, and Ira Schieren for the cover design.

xv

CONTRIBUTORS

Fatima Abbasi, CBER, FDA, NIH, Bethesda, MD, USA

James Andahazy, Partec North America, Swedesboro, NJ, USA

C. Bruce Bagwell, Verity Software House, Topsham, ME, USA

Sean C. Bendall, Stanford University, Stanford, CA, USA

Thomas N. Denny, Duke University Medical Center, Durham, NC, USA

Roy Edward, Biostatus Ltd., Shepshed, Leicester, UK

Bruce S Edwards, University of New Mexico Center for Molecular Discovery,

Albuquerque, NM, USA

Rachel J. Errington, Cardiff University, Cardiff, UK

John Ferbas, Amgen Inc., Thousand Oaks, CA, USA

Matthew B. Hale, Stanford University, Stanford, CA, USA

Kristi R. Harkins, Harkins Strategic Consulting, LLC, Madrid, IA, USA

David W. Hedley, Princess Margaret Hospital/Ontario Cancer Institute, Toronto,

ON, Canada

Carla G Hill, ICON Central Laboratories, Farmingdale, NY, USA

Jonathan M. Irish, Stanford University, Stanford, CA, USA

Siddha Kasar, NJMS/UMDNJ, Newark, NJ, USA

Peter O. Krutzik, Stanford University, Stanford, CA, USA

xvii

Virginia Litwin, Covance Central Laboratory Services, Indianapolis, IN, USA

Byron H. Long, Doylestown, PA, USA

Raul Louzao, Duke University Medical Center, Durham, NC, USA

Philip Marder, Redram Consulting, LLC, Indianapolis, IN, USA

Gerald E. Marti, CBER, FDA, NIH, Bethesda, MD, USA

David McFartand, iCyt, a Sony Company, Champaign, IL, USA

Garry P. Nolan, Stanford University, Stanford, CA, USA

Denise M. O’Hara, Pfizer (formerly Wyeth), Andover, MA, USA

Elizabeth Raveche, NJMS/UMDNJ, Newark, NJ, USA

Carmen Raventos-Suarez, Bristol-Myers Squibb Company, Princeton, NJ, USA

Manjula P. Reddy, Ortho Biotech, Unit of Centocor R&D, Johnson and Johnson,

Radnor, PA, USA

Susan M. Richards, Genzyme, Framingham, MA, USA

Erica Salerno, NJMS/UMDNJ, Newark, NJ, USA

Larry A. Sklar, University of New Mexico, Albuquerque, NM, USA

Paul J. Smith, Cardiff University, Cardiff, UK

Michelle J. Schroeder, Amgen Inc., Thousand Oaks, CA, USA

Valerie Theobald, Genzyme, Framingham, MA, USA

Ole Vesterqvist, Covance Central Laboratory Services Inc., Indianapolis, IN, USA

Brooke Walker, Duke University Medical Center, Durham, NC, USA

John Wong, Duke University Medical Center, Durham, NC, USA

Dianna Y. Wu, Bristol-Myers Squibb Company, Pennington, NJ, USA

Yuanxin Xu, Genzyme, Framingham, MA, USA

Yao Yuan, NJMS/UMDNJ, Newark, NJ, USA

xviii CONTRIBUTORS

PART I

INTRODUCTION

PHILIP MARDER AND VIRGINIA LITWIN

The discovery and development of novel therapeutic compounds is a lengthy, difficult,

and expensive process with recent estimates of more than 1.2 billion dollars required

for each new drug brought to market. As a result, the standard processes of the

pharmaceutical industry are being reevaluated and modified in order to increase

efficiencies in the drug development process. One approach in process transformation

is to promote more informed decision making by incorporating advanced technol-

ogies such as flow cytometry.

Awide variety of flow cytometric methods are employed during various stages of

the drug development life cycle. This book explores many of the benefits and

complexities associated with this unique application of the technology. Part I is

intended to provide the reader with essential background information regarding both

flow cytometry (Chapters 1 and 2) and drug development (Chapter 3).

Flow Cytometry in Drug Discovery and Development, Edited by Virginia M. Litwin and Philip MarderCopyright � 2011 John Wiley & Sons, Inc.

1

1INTRODUCTION TO FLOWCYTOMETRY

ELIZABETH RAVECHE, FATIMA ABBASI, YAO YUAN, ERICA SALERNO,SIDDHA KASAR, AND GERALD E. MARTI

1.1 INTRODUCTION

This chapter presents, in basic terms, the concepts and principles of flow cytometry.

Numerous books and articles describing flowcytometers and their use in a clinical and

biomedical research setting have been published [1–7]. In this chapter, flow

cytometerswill be discussed from their infancy arriving at the current instrumentation

that allows for detection of numerous features of individual cells or particles,

including determination of size and granularity, surface marker expression, DNA

content, intracellular protein expression, and function. The key to flow cytometers is

that the analysis is done on cells in suspension [8–10]. The analysis of individual cells

(or particles) rather than the whole population allows for detection of multiple

properties measured on the same cell. The detection is rapid (as fast as the cell in

the fluid sheath passes through the laser beam). In addition to analysis of individual

cells, some types of flow cytometers can physically sort cells based on signals

associated with the parameters being detected. The term fluorescence-activated cell

sorter or FACS has been adopted to refer to this type of analysis [11]. Flow cytometry

is a very useful tool for both clinical diagnosis and scientific research. The history of

flow cytometers has been the subject of numerous reviews [12–20]. The first flow

cytometers were introduced in the mid-1970s and first used for DNA analysis and

leukemia immunophenotyping [7, 21–25]. A further impetus to bring flow cytometers

to the forefront of clinical labs came in the early 1980s with the discovery that

individuals infected with the HIV virus developed AIDS, which could be monitored

Flow Cytometry in Drug Discovery and Development, Edited by Virginia Litwin and Philip MarderCopyright � 2011 John Wiley & Sons, Inc.

3

by enumerating the number of CD4þ T cells by flow cytometric analysis [26–30].

Currently, there are emerging areas with flow cytometric applications including the

enumeration of CD34þ hematopoietic stem cells [29, 31, 32], detection of circulating

metastatic tumor cells [33–37], determination of antigen-specific T cells [38–40], and

identification of pathogens [41–45], to list a few. Combination of sorting with

molecular analysis represents an important use of the sorting aspects of flow

cytometers. There are over 100,000 flow cytometers in use and the employment of

this instrument in clinical diagnostics has increased dramatically, particularlywith the

increase in FDA-approved fluorochrome reagents for in vitro diagnostics (fluoro-

chrome-conjugated antibodies). However, in third-world countries, access to clinical

flow cytometers is not optimal [46, 47]. The use of flow cytometers and the impact of

this instrument on biomedical and clinical studies can be appreciated by looking at the

increase in publications in which the word “flow cytometry” appeared in the abstract

or title with time (Figure 1.1).

Improvements in instrumentation and computer-assisted analysis have made the

flow cytometer a critical instrument in biomedical research, clinical diagnostics, and

drug discovery. Herzenberg was honored for his work in flow cytometry by the

American Association for Clinical Chemistry with the Ullman Award in 2002 and

some of the history described in this chapter comes from his lecture and the

accompanying article [19]. The original description of the first flow cytometer was

provided in Scientific American [48]. This instrument consisted of one laser and two

light detectors, one for forward scatter to measure cell size and the other for

fluorescence. This meant that one was restricted to measuring a single marker. When

one of the authors of this article used that prototype instrument, the LASL, we were

measuring the DNA content of individual cells. This was one of the first uses of these

FIGURE 1.1 A bar graph showing the number of publications having “flow cytometry” in

their title/abstract since 1970 to present. There is almost a 150% increase since 1980–1989.

4 INTRODUCTION TO FLOW CYTOMETRY

early flow cytometers since reagents were available that not only bound specifically to

DNA (e.g., ethidium bromide developed by Dittrich and Gohde in 1969 [49]) but also

emitted fluorescencewhen excited with a laser. Much of the essentials of the modern-

day FACS are the same as those in the early flow cytometers. However, these early

flow cytometers were cumbersome and required an on-site engineer. The laser was

water cooled and alignment issues were critical. In addition, no computer was

attached to these early flow cytometers, nor were programs available for data

analysis [50]. At one point, we took Polaroid pictures of oscilloscopes and sent data

to a DEC10 supercomputer and wrote our own programs for cell cycle analysis.

Although the development of FACS depended on many advances in various

disciplines including dye chemistry, electronics, and computers, one important

breakthrough that was critical for the development of flow cytometers was the

principle of measuring cells or particles in liquid suspension. Advances in the flow

principle began in 1940withCrosland-Taylor using the flowprinciple and light scatter

to measure blood cells [51]. The breakthrough technology was first developed by

Coulter and the Coulter principle describes changes in the electrical conductivity of a

small saline-filled orifice as a cell passes through it. In 1953, Wallace Coulter and his

brother Joe obtained a U.S. patent for the Coulter counter that automated counting of

particles, particularly cells in the blood [52]. The use of a liquid stream (or a sheath) to

which a sample is introduced allows individual cells to be distributed in the sheath that

then passes through a nozzle (detecting electrical conductivity changes) to generate a

trigger, which indicates the presence of a signal that exceeds the threshold level.

Many of the applications for FACS analysis involve the identification ofmembrane

markers via the use of fluorochrome-tagged antibodies, which recognize these

markers. Many of these membrane markers are surface proteins or surface antigens,

which help to define the cell. These antigens are used to classify the cells and are often

assigned a cluster of differentiation number or a CD number. Antibodies (which are

normally produced by B lymphocytes) can be made that specifically bind to these CD

molecules. There are more than 200 CD molecules that have been identified and

specific antibodies have been produced that recognized these CDmarkers [53–55]. In

addition, many of these antibodies are commercially available as labeled antibodies

with different fluorochromes.

1.2 BASIC PRINCIPLES OF HOW A FLOW CYTOMETER WORKS

The basic components of a flow cytometer (Figure 1.2) consist of (1) a flow cell that

forces single cells into the middle of a fluidic sheath, (2) a laser source of light, (3)

optical components to focus light of different wavelengths (colors) onto a detector, (4)

a photomultiplier to amplify the signal, and (5) a computer.

In a basic flow cytometer, the sample (containing the cells tagged with fluor-

ochromes in a liquid) is drawn up and pumped into the flow cell through tubing. The

cells flow through the flow chamber rapidly and singly and are passed through one or

more laser light beams. As the laser beam hits the cells, the light beam is scattered in a

forward direction and a side direction. Fluorescence emission can also be detected.

BASIC PRINCIPLES OF HOWA FLOW CYTOMETER WORKS 5

Scatter or fluorescence is captured, filtered (based on thewavelength), and directed to

the appropriate photodetectors for conversion to electronic signals. The electronics in

the flow cytometer amplify the signal and convert the analog data to digital data,

which can then be analyzed by computer software programs.

1.3 FLUIDICS

1.3.1 Flow Cells

In order to perform flow cytometric analysis, the sample must be in a suspension and

the cell in the sample streammust be centered in the laminar flow [49]. Hydrodynamic

focusing induces cells to orient with their long axis parallel to the flow. The end result

is that the introduced sample passes by the laser with each cell oriented in the center of

the sample stream in a particular manner in three dimensions.

FIGURE 1.2 Diagrammatic representation of a basic flow cytometer. The fluorescently

labeled cells are hydrodynamically focused into a single file in the flow cell. Individual cells are

excited by the laser light source and the fluorescence emissions, FSC, and SSC are detected. The

cells can then be given a particular charge based on their fluorescence profile and deflected

toward the oppositely charged plates. In the figure, light grey cells and dark grey cells are given

negative and positive charges, respectively, and are thus deflected toward two different tubes.

6 INTRODUCTION TO FLOW CYTOMETRY

1.4 OPTICS

Flow cytometers depend on the laws of optics, such as reflection, refraction, and other

principles, which are not new but based on works established centuries ago [56].

Optics are present on both the excitation and the emission side. The excitation optics

encompass the lasers and the lenses that focus the laser beam. The emission optics are

involved in collecting the emission following excitation. These involve lenses to

collect emitted light and mirrors and filters to route specified wavelengths of the

collected light to designated optical detectors. Light coming out of a laser may be

considered a beam but fluorescence must be considered as a photon.

1.4.1 Light Scatter

Due to differences between the refractive indices of cells and the surrounding sheath

fluid, light impinging upon the cells is scattered. The forward light scatter (FSC)

provides empirical information on cell size. Light scattered in an orthogonal direction

or side scatter (SSC), which is collected by a different detector, provides information

about granularity.

1.4.2 Types of Lasers

Laser stands for light amplification by stimulated emission of radiation. Gas lasers

havemirrors at each end of a cylinder or plasma tube filledwith an inert gas. The gas is

ionized to a higher energy state by a high-voltage electric current.When these excited

atoms return to the ground state, they give off photons of a characteristic wavelength.

The photons can be reflected by the mirrors and the excitation of the atoms in the

plasma can be amplified but thewavelengths of the emission still are the characteristic

wavelengths for that gas [57]. In the front of the laser there is a small optic that allows

the transmitted light to form a laser beam of desired output wavelengths. The light

from lasers is a stimulated emission and it has uniform characteristics. For current

stream-in-air instrumentation, it is desirable to have at least 50mWof power for each

laser line in use, since the fluorescence signal (and thus sensitivity) increases with

laser power. Cytometers use multiple lasers that are positioned spatially such that

there is a time delay for each laser beam intercept with the cell. Newer solid-state

diode lasers [58–60] are becoming prevalent and these are significantly cheaper than

the older gas ion lasers. Diode lasers are pumped by input of electric current. A partial

list of different lasers is presented in Table 1.1.

The most common lasers for flow cytometers are the argon ion lasers that run at

488 nm. The lasing medium in an ion laser is plasma. A high-voltage pulse is used to

ionize the gas to start the plasma. Ion lasers require a high current to maintain the

plasma discharge. In addition to the 488 nm emission, argon ion lasers also emit at

515 nm (green) and 457 nm (violet-blue). Other emissions can be obtained using

specially coatedmirrors. The new low-power, air-cooled argon laser gives out 25mW

at 488 nm. To obtain other lines of emission, large lasers capable of giving 100mW in

UV must be used.

OPTICS 7

Krypton lasers can give out strong blue-green lines and UV and violet lines.

Krypton lasers need to be water cooled and optimized and the alignment is very

difficult. Another type of laser is a dye laser and the lasing medium in a dye laser is a

fluorescent dye. The selection of dye depends on the wavelength at which the

operation is desired. Helium–neon (He–Ne) lasers are also small, air cooled, and

stable. The most common lasers emit at 633 nm and have power outputs ranging

from 1 to 50mW. He–Ne lasers are available at 633, 543, 594, and 611 nm.

Helium–cadmium (He–Cd) lasers emit 5–200mW in blue (441 nm) and 1–50mW

in UV (325 nm). They plug into the wall and do not require water cooling.

1.4.3 Filters for Emission

All signals that are emitted from fluorochromes that are excited as the cells to which

they are bound are interrogated by the laser beams are routed to detectors via a system

of mirrors and optical filters. In addition, beam splitters direct light of different

wavelengths in different directions. The most commonly used filters are short-pass

filters (which transmit wavelengths of light equal to or shorter than the specified

wavelength), long-pass filters (which transmit wavelengths of light equal to or larger

than the specified wavelength), and band-pass filters (which allow a narrow range of

wavelengths to reach the detector). An example of these types of filters is presented in

Figure 1.3. Because each fluorochrome has an emission spectrum, the choice of filters

optimizes detection of the specific fluorochrome by one detector or photomultiplier

tube (PMT).

Detection of fluorochromes requires selection of appropriate filters that are placed

before each detector or PMT. The type of filter selected must collect as much emitted

light from the primary fluorochrome for high sensitivity, but as little as possible from

other fluorochromes to reduce the compensation required. A partial list of filters is

presented in Table 1.2.

TABLE 1.1 Partial List of Laser with Their Excitation Wavelength Line and the

Fluorochromes which Can be Detected

Laser Excitation Line (nm) Fluorochrome

UV 355 Hoescht 33342, 33250

He–Cd 325 DAPI, ELF-97, AMCA (AlexaFluor 350),

INDO-1

Mercury lamp

Violet 405 Pacific Blue, CasB

Krypton ion 435 CasY, AlexaFluor 405 (AF405)

Blue (argon) 488 PE-TR, 6FP, FITC, PE,AF488, PE-Cy7, PerCP,

PE-Cy5, SYTO 9, PerCP-Cy5.5

Red (solid state) 640 APC, APC-Cy7, SYTO 59–61

He–Ne 633 AF647, APC-Cy7

Red diode 635 APC-Cy5.5, AF700

Yellow/green 561 PE-Texas Red, PerCP, PE

8 INTRODUCTION TO FLOW CYTOMETRY

1.5 TYPES AND CHOICE OF FLUOROCHROMES

Afluorochrome is a fluorescentmarker that emits a particularwavelengthwhen a laser

light hits it. Fluorescence occurs when a molecule, which is excited by light from a

laser at one wavelength, loses its energy and emits light of a longer wavelength. The

emitted wavelength is what is detected. The excited and emitted light are of different

wavelengths. The fluorescence intensity that is emitted is proportional to the quantity

of binding sites for the fluorescent compound on the cell. Therefore, the more the

fluorescence that is emitted the more the binding sites on the cell. For instance, for an

antibody tagged with FITC (fluorescein isothiocyanate, which is excited by a 488 nm

FIGURE 1.3 An example of fluorescence emission of various wavelengths (top) as it passes

through different types of optical filters (bottom).

TABLE 1.2 Partial List of Filters Typically Employed with

Various Fluorochromes

Fluorochrome Filter

Pacific Blue, BD Horizon V450, CasB 440/40

AmCyan 525/40

FITC, AlexaFluor 488 (AF488) 530/30

CasY, AF430 545/90

PE 585/40

PE-Texas Red, AF595 625/40

APC, AF647 660/20

PE-Cy5, PerCP-Cy5.5, PerCP 695/40

APC-Cy5.5 705/50

AF700 720/45

PE-Cy7, BD-APC H7 780/60

TYPES AND CHOICE OF FLUOROCHROMES 9

argon laser but emits in the 520 nm (green) range) that recognizes and binds to CD4,

the more the 520 nm emission the more the CD4 on the cell (Figure 1.4).

The fluorochrome label for a reagent depends on instrument configuration (type

and number of lasers and type of optical filters and detectors), which determines if a

given instrument can excite a given fluorochrome and detect the emission. While it is

not possible to uniformly state the best fluorochrome combination, there are a few

guidelines that can help in this choice. The first issue is to determine what is

the reagent brightness, which takes into account the resolvable signal associated

with the presence of themarker being detected by comparing a negative and a positive

sample. The negative population emission is the background emission. Background is

signal (emission) due to electronic noise (dark current), cell autofluorescence,

nonspecific staining, and background emission that is a spillover from another

fluorochrome [61, 62]. The rule of thumb is to use the brightest reagents possible [63,

64]. There is a caveat to this statement. The spillover problems increase as the number

of colors to be resolved (different emissions) increases. Compensation canhelp prevent

the spillover contribution, but as a rule of thumb, one should use fluorochromes whose

emissions have the least amount of spectral overlap [65, 66]. In addition, logically the

FIGURE 1.4 Excitation and emission spectra of FITC and phycoerythrin (PE). Fluorescent

molecules absorb light of a characteristic wavelength and emit light of a longer wavelength.

FITC and PE that are commonly used for flow cytometry absorb at 488 and 488–560 nm,

respectively, but emit at 520 and 590 nm, respectively. Thus, they can be excited by the same

laser line and used together in the same tube [10].

10 INTRODUCTION TO FLOW CYTOMETRY