Flow Cytometry E-Book

Alice Longobardi Givan

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 373

Veröffentlichungsjahr: 2013

Beschreibung

Flow cytometry continually amazes scientists with its ever-expanding utility. Advances in flow cytometry have opened new directions in theoretical science, clinical diagnosis, and medical practice. The new edition of Flow Cytometry: First Principles provides a thorough update of this now classic text, reflecting innovations in the field while outlining the fundamental elements of instrumentation, sample preparation, and data analysis. Flow Cytometry: First Principles, Second Edition explains the basic principles of flow cytometry, surveying its primary scientific and clinical applications and highlighting state-of-the-art techniques at the frontiers of research. This edition contains extensive revisions of all chapters, including new discussions on fluorochrome and laser options for multicolor analysis, an additionalsection on apoptosis in the chapter on DNA, and new chapters onintracellular protein staining and cell sorting, including high-speed sorting and alternative sorting methods, as well as traditional technology. This essential resource: * Assumes no prior knowledge of flow cytometry * Progresses with an informal, engaging lecture style from simpleto more complex concepts * Offers a clear introduction to new vocabulary, principles of instrumentation, and strategies for data analysis * Emphasizes the theory relevant to all flow cytometry, with examples from a variety of clinical and scientific fields Flow Cytometry: First Principles, Second Edition provides scientists, clinicians, technologists, and students with the knowledge necessary for beginning the practice of flow cytometry and for understanding related literature.

Leseprobe

Contents

Preface

Acknowledgments (First Edition)

Acknowledgments (Second Edition)

1 The Past as Prologue

2 Setting the Scene

3 Instrumentation: Into the Black Box

ILLUMINATION OF THE STREAM

CENTERING CELLS IN THE ILLUMINATING BEAM

DETECTION OF SIGNALS FROM CELLS

ELECTRONICS

4 Information: Harnessing the Data

DATA STORAGE

DATA ANALYSIS

5 Seeing the Light: Lasers, Fluorochromes, and Filters

GENERAL THEORY

LASERS

FLUOROCHROMES

PARTITIONING THE SIGNAL WITH LENSES, FILTERS, AND MIRRORS

SPECTRAL COMPENSATION

6 Cells from Without: Leukocytes, Surface Proteins, and the Strategy of Gating

CELLS FROM BLOOD

STAINING FOR SURFACE MARKERS

CONTROLS

QUANTITATION

SENSITIVITY

THE STRATEGY OF GATING

GATING ON FLUORESCENCE

7 Cells from Within: Intracellular Proteins

METHODS FOR PERMEABILIZING CELLS

EXAMPLES OF INTRACELLULAR STAINING

8 Cells from Within: DNA in Life and Death

FLUOROCHROMES FOR DNA ANALYSIS

PLOIDY

CELL CYCLE ANALYSIS

TWO-COLOR ANALYSIS FOR DNA AND ANOTHER PARAMETER

CHROMOSOMES

APOPTOSIS

NECROSIS

9 The Sorting of Cells

SORTING THEORY

CHARACTERIZATION OF SORTED CELLS

ALTERNATIVE METHODS FOR SORTING

THE CONDITION OF CELLS AFTER SORTING

10 Disease and Diagnosis: The Clinical Laboratory

THE HEMATOLOGY LABORATORY

THE PATHOLOGY LABORATORY

SOLID ORGAN TRANSPLANTATION

COMMENTS

11 Out of the Mainstream: Research Frontiers

FUNCTIONAL ASSAYS

THE AQUATIC ENVIRONMENT

REPORTER MOLECULES

MICROBIOLOGY

MOLECULAR BIOLOGY

MULTIPLEX CYTOMETRY FOR SOLUBLE ANALYTES

REPRODUCTIVE TECHNOLOGY

12 Flowing On: The Future

General References

Glossary

Figure Credits

Index

This book is printed on acid-free paper.

Copyright © 2001 by Wiley-Liss, Inc. All rights reserved.

Published simultaneously in Canada.

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Library of Congress Cataloging-in-Publication Data:

Givan, Alice Longobardi.

Flow cytometry : first principles / Alice Longobardi Givan.

p. cm.

Includes bibliographical references and index.

ISBN 0-471-38224-8

1. Flow cytometry. I. Title.

[DNLM: 1. Flow Cytometry—methods. QH585.5.F56 G539f]

QH585.5.F56G58 1992

574.87′028—dc20

DNLM/DLC

for Library of Congress                             92-5004

10 9 8 7 6 5 4 3 2 1

The First Edition of this book was dedicated to my parents, Violet Litwin Longobardi and Vincent Longobardi, Jr., with gratitude for the example they set, with pride in their achievements, and with love.

The Second Edition is dedicated to Curt, Ben, and Becky (not only because I promised them that their turn would come).

Preface

Although flow cytometry is simply a technique that is useful in certain fields of scientific endeavor, there is, at the same time, something special about it. Few other techniques involve specialists from so many different backgrounds. Anyone working with flow systems for any length of time will realize that computer buffs, electronics experts, mathematicians, optical and fluidics engineers, and organic chemists rub shoulders with biologists, physicians, and surgeons around the flow cytometer bench.

And it is not just a casual rubbing of shoulders, in passing, so to speak. Many of the specialists involved in flow cytometry might, if asked, call themselves flow cytometrists because the second aspect of flow cytometry that distinguishes it from many other techniques is that flow cytometry has itself become a “field.” Indeed, it is a field of endeavor and of expertise that has captured the imaginations of many people. As a result, there exists a spirit of camaraderie; flow cytometry societies, groups, meetings, networks, websites, journals, courses, and books abound.

A third aspect of flow cytometry (known sometimes simply with the acronym for fluorescence-activated cell sorter, FACS, or even more familiarly as just flow) that distinguishes it from many other techniques is the way in which its wide and increasing usefulness has continued to surprise even those who consider themselves experts. What began as a clever technique for looking at a very limited range of problems is now being used in universities, in hospitals, within industry, at marine stations, on submersible buoys, and on board ships; plans have existed for use on board space ships as well. The applications of flow cytometry have proliferated (and continue to proliferate) rapidly both in the direction of theoretical science, with botany, molecular biology, embryology, biochemistry, marine ecology, genetics, microbiology, and immunology, for example, all represented; and in the direction of clinical diagnosis and medical practice, with hematology, bacteriology, pathology, oncology, obstetrics, and surgery involved. We are, at present, living through what appears to be a rapid phase in flow cytometry’s growth curve (see Fig. 1).

Fig. 1. Increasing reference to “flow cytometry” in the medical literature over the past three decades. The development of flow cytometers antedates the use of the term itself.

Because flow cytometry is an unusual field, bringing together people with differing scientific backgrounds at meetings, on editorial boards, in hospital wards, on advisory panels, and at laboratory benches and reaching increasing numbers of workers in new and unpredicted areas of endeavor, there is, as a result, a need to provide both recent and potential entrants into this diverse community with a common basis of knowledge—so that we can all understand the vocabulary, the assumptions, the strengths, and the weaknesses of the technology involved. I have for many years taught new and future users of flow cytometers. My teaching attempts to present enough technical background to enable students, scientists, technologists, and clinicians to read the literature critically, to evaluate the benefits of the technique realistically, and, if tempted, to design effective protocols and interpret the results. I try to describe the theory of flow cytometry in a way that also provides a firm (and accurate) foundation for those few who will go on to study the technique in greater depth. Details of protocols are avoided, but my teaching attempts to give enough information about applications to provide concrete examples of general concepts and to allow some appreciation of the range of practical goals that the instruments are able to achieve. With some expansion (but with little change in style or objectives), my classroom and workshop teaching is the basis for this book.

Notes on the Second Edition: This new edition, while similar to the first edition in style and scope, has been modified in many ways. The arrangement of some material has been altered to present, in my opinion, a more coherent pedagogical sequence, reflecting my changing thoughts on teaching. While all chapters have been re-written to a significant extent, there are also some major expansions reflecting the progress of the field. In particular, I have included more detail about the cell as it passes through the laser beam; the laser/fluorochrome chapter has been expanded to include recently developed fluorochromes and multilaser options; a new chapter has been added on cytoplasmic staining; a discussion of apoptosis has been added to the chapter on DNA; the section on sorting has been expanded to a full chapter and includes high-speed sorting and alternative sorting methods as well as traditional technology; the clinical and research chapters have been updated and expanded considerably; the chapter on general references includes many of the recent excellent books in the field; and the chapter on the future of flow cytometry is now a subjective glimpse into the new decade from the vantage point of the year 2000.

Acknowledgments (First Edition)

Realizing how much I have learned and continue to learn from others, I hesitate to single out a few names for particular mention. However, with the disclaimer that any list of people to whom I am indebted is not meant to be and, indeed, could never be complete, I must thank here the following friends, mentors, and colleagues who have had a very direct impact on the writing of this book: George Proud, for having had enough insight into the importance of a flow cytometer for transplantation surgery to want to have one; Ivan Johnston and Ross Taylor, for hospitality (and the Harker Bequest Fund, the Northern Counties Kidney Research Fund, and the Newcastle Health Authority, for financial assistance) within the Department of Surgery at Newcastle University; Brian Shenton, for introducing me to the field of flow cytometry and to the joys and hazards of clinical research; Mike White, for bailing me out (often figuratively and once or twice literally) on so many occasions; Paul Dunnigan, for teaching me about lasers (and also about fuses, relays, and loose wires); Ian Brotherick, for the animal amplification figure, and both he and Alison Mitcheson for good humor in the lab beyond all reasonable expectation; Terry Godley, for being an extremely good and (more importantly) a very communicative flow cytometry service engineer; Ray Joyce, for considerable assistance with the design and drawing of many of the diagrams; the scientists and clinicians at Newcastle University and Durham University (many acknowledged in the figure legends), for providing me with a continuous source of interesting and well-prepared cell material on which to practice my flow technique; Paul Guyre, for giving me a supportive and remarkably enjoyable re-introduction to science in the New World; Daryll Green, for giving me the benefit of his expertise in both physics and flow cytometry by reading and commenting about the chapters on instrumentation and information; Brian Crawford, for being a literate and encouraging editor in the face of my rank inexperience; the late Robert L. Conner, for providing me with that critical first of my still-continuing scientific apprenticeships; Curt Givan, for his unfailing loyalty and for his skill in reading the entire manuscript with two eyes—one eye that of an old-fashioned grammarian who abhor dangling participles and the other that of a modern scientist who knows nothing about flow cytometry; and Ben Givan, for the two drawings in Figure 8.1 [Fig. 10.1 in the Second Edition], Becky Givan, for organizational magic when it was very badly needed, and both kids for lots of encouragement and some pretty funny suggestions for a title.

A.L.G., 1991, Newcastle upon Tyne.

Acknowledgments (Second Edition)

I continue to be immensely grateful to the people whom I acknowledged in the First Edition of FCFP; their contributions are still current and form the foundation for this new edition. I have, however, during preparation of the new edition, received considerable additional help. During the 9 years since the publication of the first edition of this book, changes have taken place in my life as well as in the field of flow cytometry. Now working at Dartmouth Medical School, I have learned much about clinical cytometry, flow analysis, and computers from Marc Langweiler; Marc taught me the acronym “RTFM,” but has been restrained at using it in response to my naïve questions and I am grateful. In addition, Marc was a particular help to me with suggestions for Chapter 10. Gary Ward, the flow sorting supervisor in the Englert Cell Analysis Laboratory at Dartmouth, has, by his competence, made my sorting skills rusty, but he has allowed me to pretend that I run “his” sorter when I need a dramatic photograph, and I am grateful for his good humor. I am also grateful to Ken Orndorff, who supervises the imaging service in the Englert Lab; he has been admirably tolerant of flow cytometrists (including me) who have slowly begun to realize that a dot on a dot plot may not be enough and that they need to know what their cells look like. In addition, I want to thank the many users of the instruments in the Englert Lab for their ability to provide me with continuing challenges.

First Colette Bean and then Luna Han at Wiley inherited editorial supervision of this book from Brian Crawford and I owe them considerable thanks for their persistence and patience. At Dartmouth, I am grateful for secretarial assistance from Mary Durand and artistic help from Joan Thomson. Dean Gonzalez at Wiley, with artistry and stamina, is responsible for the great improvement in figure clarity between the two editions of this book. I thank him.

During these last 9 years, I have had the opportunity to expand my acquaintance with members of the greater flow community. Among many from whom I have learned much, I would like especially to mention a few: Louis Kamentsky shared with me his lecture notes for a wonderful keynote address he gave at Dartmouth on the history of flow cytometry, and I am grateful to him. Howard Shapiro has been a source of limitless cytometric information cloaked seductively in outrageous humor; I thank him for his knowledge, for his humor, and for his generosity. Paul Robinson has made many contributions to flow cytometry, but I want particularly to mention his organization of the Purdue University flow network; I am grateful to him for the information, the moral support, and the distance friendships that the network has given me. Over the years, I have had the privilege of teaching workshops and courses with a group of people who are devoted not just to flow technology (although they are talented flow cytometrists) but also to education of students in the correct use of the technique. Indeed, I also detect their devotion to the use of flow cytometry as a platform for teaching a rigorous approach to science in general. In thanks for all I have learned from these workshops, I wish, particularly, to mention here Bruce Bagwell, Ben Hunsberger, James Jett, Kathy Muirhead, Carleton Stewart, Joe Trotter, and Paul Wallace.

And—as ever—Curt Givan came through with unflappable proofreading skills and many cups of tea.

A.L.G., 2000, Lebanon, New Hampshire.

1

The Past as Prologue

Flow cytometry, like most scientific developments, has roots firmly grounded in history. In particular, flow technology finds intellectual antecedents in microscopy, in blood cell counting instruments, and in the ink jet technology that was, in the 1960s, being developed for computer printers. It was the coming together of these three strands of endeavor that provided the basis for the development of the first flow cytometers. Because thorough accounts of the history of flow cytometry have been written elsewhere (and make a fascinating story for those interested in the history of science), I cover past history here in just enough detail to give readers a perspective as to why current instruments have developed as they have.

Microscopes have, since the seventeenth century, been used to examine cells and tissue sections. Particularly since the end of the nineteenth century, stains have been developed that make various cellular constituents visible; in the 1940s and 1950s, fluorescence microscopy began to be used in conjunction with fluorescent stains for nucleic acids in order to detect malignant cells. With the advent of antibody technology and the work of Albert Coons in linking antibodies with fluorescent tags, the use of fluorescent stains gained wider and more specific applications. In particular, cell suspensions or tissue sections are now routinely stained with antibodies specific for antigenic markers of cell type or function. The antibodies are either directly or indirectly conjugated to fluorescent molecules (most usually fluorescein or rhodamine). The cellular material can then be examined on a glass slide under a microscope fitted with an appropriate lamp and filters so that the fluorescence of the cells can be excited and observed (Fig. 1.1). The fluorescence microscope allows us to see cells, to identify them in terms of both their physical structure and their orientation within tissues, and then to determine whether and in what pattern they fluoresce when stained with one or another of the specific stains available. In addition, a microscope can also be fitted with a camera or photodetector, which will then record the intensity of fluorescence arising from the field in view. The logical extension of this technique is image analysis cytometry, digitizing the output to allow precise quantitation of fluorescence intensity patterns in detail (pixel by pixel) within that field of view. The development of monoclonal antibody technology (for which Köhler and Milstein were awarded the Nobel Prize) led to a vast increase in the number of cellular components that can be specifically stained and that can be used to classify cells. Whereas monoclonal antibody techniques are not directly related to the development of flow technology, their invention was a serendipitous event that had great impact on the potential utility of flow cytometric systems.

Fig. 1.1. The optical path of a fluorescence microscope. In this example, the filters and mirrors are set for detection of fluorescein fluorescence. From Alberts et al. (1989).

In 1934, Andrew Moldavan in Montreal took a first step from static microscopy toward a flowing system. He suggested the development of an apparatus to count red blood cells and neutral-red-stained yeast cells as they were forced through a capillary on a microscope stage. A photodetector attached to the microscope eyepiece would register each passing cell. Although it is unclear from Moldavan’s paper whether he actually ever built this cytometer, the development of staining procedures over the next 30 years made it obvious that the technique he suggested could be useful not simply for counting the number of cells but also for quantitating their characteristics.

In the mid-1960s, Louis Kamentsky took his background in optical character recognition and applied it to the problem of automated cervical cytology screening. He developed a microscope-based spectrophotometer (on the pattern of the one suggested by Moldavan) that measured and recorded ultraviolet absorption and the scatter of blue light (“as an alternative to mimicking the complex scanning methods of the human microscopist”) from cells flowing “at rates exceeding 500 cells per second” past a microscope objective. Then, in 1967, Kamentsky and Melamed elaborated this design into a sorting instrument (Fig. 1.2) that provided for the electronic actuation of a syringe to pull cells with high absorption/scatter ratios out of the flow stream. These “suspicious” cells could then be subjected to detailed microscopic analysis. In 1969, Dittrich and Göhde in Münster, Germany, described a flow chamber for a microscope-based system whereby fluorescence intensity histograms could be generated based on the ethidium bromide fluorescence from the DNA of alcohol-fixed cells.

During this period of advances in flow microscopy, so-called Coulter technology had been developed by Wallace Coulter for analysis of blood cells. In the 1950s, instruments were produced that counted cells as they flowed in a liquid stream; analysis was based on the amount by which the cells increased electrical resistance as they displaced isotonic saline solution while flowing through an orifice. Cells were thereby classified more or less on the basis of their volume because larger cells have greater electrical resistance. These Coulter counters soon became essential equipment in hospital hematology laboratories, allowing the rapid and automated counting of white and red blood cells. They actually incorporated many of the features of analysis that we now think of as being typical of flow cytometry: the rapid flow of single cells in file through an orifice, the electronic detection of signals from those cells, and the automated analysis of those signals.

At the same time as Kamentsky’s work on cervical screening, Mack Fulwyler at the Los Alamos Laboratory in New Mexico had decided to investigate a problem well known to everyone looking at red blood cells in Coulter counters. Red cells were known to show a bimodal distribution of their electrical resistance (“Coulter volume”). Anyone looking at erythrocytes under the microscope cannot help but be impressed by the remarkable structural uniformity of these cells; Fulwyler wondered if the bimodal Coulter volume distribution represented differences between two classes of these apparently very uniform cells or, alternatively, whether the bimodal profile was simply an artifact based on some quirky aspect of the electronic resistance measurements. The most direct way of testing these two alternatives was to separate erythrocytes according to their electronic resistance signals and then to determine whether the separated classes remained distinct when they were re-analyzed.

Fig. 1.2. A diagram of Kamentsky’s original flow sorter. From Kamentsky and Melamed (1967). Science 156:1364–1365. Copyright AAAS.

The technique that Fulwyler developed for sorting the erythrocytes combined Coulter methodology with the ink jet technology being developed at Stanford University by RG Sweet for running computer printers. Ink jet technology involves the vibration of a nozzle so as to generate a stream that breaks up into discrete drops and then the charging and grounding of that stream at appropriate times so as to leave indicated drops, as they break off, carrying an electrical charge. For purposes of printing, those charged drops of ink can then be deflected to positions on the paper as required by the computer print messages. Fulwyler took the intellectual leap of combining this methodology with Coulter flow technology; he developed an instrument that would charge drops containing suspended cells, thereby allowing deflection of the cells (within the drops) as dictated by signals based on the cell’s measured Coulter volume.

The data from this limited but pioneering experiment led to a conclusion that with hindsight seems obvious: Erythrocytes are indeed uniform. When red cells are sorted according to their electrical resistance, the resulting cells from one class or the other still show a bimodal distribution when re-analyzed for their electrical resistance profile. The bimodal “volume” signal from erythrocytes was therefore artifactual—resulting in part from the discoid (nonspherical) shape of the cells. The technology developed for this landmark experiment is the essence of all the technology required for flow sorting as we now know it. That experiment also, unwittingly, emphasized an aspect of flow cytometry that has remained with us to this day: Flow cytometrists still need to be continually vigilant (and know how to use a microscope) because signals from cells (particularly signals that are related to cell volume) are subject to artifactual influences and may not be what they seem. (Fulwyler’s 1965 paper actually describes the separation of mouse from human erythrocytes and the separation of a large component from a population of mouse lymphoma cells; the experiments on the bimodal signals from red cells have been relegated to flow folk history.)

In 1953, PJ Crosland-Taylor, working at the Middlesex Hospital in London, noted that attempts to count small particles suspended in fluid flowing through a tube had not hitherto been very successful. With particles such as red blood cells, the experimenter must choose between a wide tube that allows particles to pass two or more abreast across a particular section or a narrow tube that makes microscopical observation of the contents of the tube difficult due to the different refractive indices of the tube and the suspending fluid. In addition, narrow tubes tend to block easily. In response to this dilemma, Crosland-Taylor applied the principles of laminar flow to the design of a flow system. A suspension of red blood cells was injected into the center of a faster flowing stream, thus allowing the cells to be aligned in a narrow central file within the core of the wider stream preparatory to electronic counting. This principle of hydrodynamic focusing was pivotal for the further development of the field.

In 1969, Marvin Van Dilla and other members of the Los Alamos Laboratory group reported development of the first fluorescencedetection cytometer that utilized the principle of hydrodynamic focusing and, unlike the microscope-based systems, had the axes of flow, illumination, and detection all orthogonal to each other; it also used an argon laser as a light source (Fig. 1.3). Indeed, the configuration of this instrument provided a framework that could support both the illumination and detection electronics of Kamentsky’s device as well as the rapid flow and vibrating fluid jet of Fulwyler’s sorter. In the initial report, the instrument was used for the detection of fluorescence from the Feulgen-DNA staining of Chinese hamster ovary cells and leukocytes as well as of their Coulter volume; however, the authors “anticipated that extension of this method is possible and of potential value.” Indeed, shortly thereafter, the Herzenberg group at Stanford published a paper demonstrating the use of a similar cytometer to sort mouse spleen and Chinese hamster ovary cells on the basis of their fluorescence due to accumulation of fluorescein diacetate. These instruments thus led to systems for combining multiparameter fluorescence, light scatter, and “Coulter volume” detection with cell sorting.

Fig. 1.3. Marvin Van Dilla and the Livermore flow sorter in 1973. Photograph courtesy of the Lawrence Livermore National Laboratory.

These sorting cytometers began to be used to look at ways of distinguishing and separating white blood cells. By the end of the 1960s, they were able to sort lymphocytes and granulocytes into highly purified states. The remaining history of flow cytometry involves the elaboration of this technology, the exploitation of flow cytometers for varied applications, and the collaboration between scientists and industry for the commercial production of cytometers as user-friendly tools (Fig. 1.4). At the same time that these instruments began to be seen as commercially marketable objects, research and development continued especially at the United States National Laboratories at Los Alamos (New Mexico) and Livermore (California), but also at smaller centers around the world. At these centers, homemade instruments continue to indicate the leading edge of flow technology. At the present time, this technology is moving simultaneously in two directions: On the one hand, increasingly sophisticated instruments are being developed that can measure and analyze more aspects of more varied types of particles more and more sensitively and that can sort particles on the basis of these aspects at faster and faster rates. On the other hand, a different type of sophistication has streamlined instruments (Fig. 1.5) so that they have become user-friendly and essential equipment for many laboratory benches.

Fig. 1.4. Bernard Shoor (left) and Leonard Herzenberg at Stanford University with one of the original Becton Dickinson flow cytometers as it was packed for shipment to the Smithsonian Museum. Photograph by Edward Souza, courtesy of the Stanford News Service.

Fig. 1.5. Three user-friendly benchtop cytometers (in alphabetical order). Top: A Beckman Coulter ®XLTM. Middle: A Becton Dickinson FACSCalibur. Bottom: A Dako Galaxy, manufactured for Dako by Partee.

FURTHER READING

Throughout this book, the “Further Reading” references at the end of each chapter, while not exhaustive, are intended to point the way into the specific literature related to the chapter in question. At the end of the book, “General References” will direct readers to globally useful literature. Titles in bold at the end of each chapter are texts that are fully cited in the General References at the end.

Coulter WH (1956). High speed automatic blood cell counter and size analyzer. Proc. Natl. Electronics Conf. 12:1034–1040.

Crosland-Taylor PJ (1953). A device for counting small particles suspended in a fluid through a tube. Nature 171:37–38.

Dittrich W, Göhde W (1969). Impulsfluorometrie dei Einzelzellen in Suspensionen. Z. Naturforsch. 24b:360–361.

Fulwyler MJ (1965). Electronic separation of biological cells by volume. Science 150:910–911.

Herzenberg LA, Sweet RG, Herzenberg LA (1976). Fluorescence-activated cell sorting. Sci. Am. 234:108–115.

Hulett HR, Bonner WA, Barrett J, Herzenberg LA (1969). Cell sorting: Automated separation of mammalian cells as a function of intracellular fluorescence. Science 166:747–749.

Kamentsky LA, Melamed MR (1967). Spectrophotometric cell sorter. Science 156:1364–1365.

Kamentsky LA, Melamed MR, Derman H (1965). Spectrophotometer: New instrument for ultrarapid cell analysis. Science 150:630–631.

Moldavan A (1934). Photo-electric technique for the counting of microscopical cells. Science 80:188–189.

Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR (1969). Cell microfluorimetry: A method for rapid fluorescence measurement. Science 163:1213–1214.

Chapter 1 in Melamed et al., Chapter 3 in Shapiro, and Chapter 1 in Darzynkiewicz are good historical reviews of flow cytometry. Alberto Cambrosio and Peter Keating (2000) have used flow cytometry as a model for looking at historical changes in the way scientists use instrumentation to view the world: Of lymphocytes and pixels: The techno-visual production of cell populations. Studies in History and Philosophy of Biological and Biomedical Sciences 31:233–270.

2

Setting the Scene

As mentioned in the previous chapter, flow cytometry has been moving in two directions at once. The earliest flow cytometers were either homemade Rube Goldberg (Heath Robinson, U.K.) monsters or, a few years later, equally complex, unwieldy commercial instruments. These were expensive; they were also unstable and therefore difficult to operate and maintain. For these reasons, the cytometer tended to collect around itself the trappings of what might be called a flow facility—that is, a group of scientists, technicians, students, and administrators, as well as a collection of computers and printers, that all revolved around the flow cytometer at the hub. If a scientist or clinician wanted the use of a flow cytometer to provide some required information, he or she would come to the flow facility, discuss the experimental requirements, make a booking, and then return with the prepared samples at the allotted time. The samples would then be run through the cytometer by a dedicated and knowledgeable operator. Finally, depending on the operator’s assessment of the skill of the end-user, a number, a computer print-out, or a computer disk would be handed over for analysis.

Just at the moment when such flow facilities were beginning to see themselves as essential components of modern research, the technology of flow cytometry began to move in a new direction. Users and manufacturers both began to realize that different laboratories have different instrumentation needs. A sophisticated sorter might be necessary for certain applications, but its demands on daily alignment and skilled maintenance are time-consuming and its research capabilities might be superfluous for routine processing of, for example, clinical samples. Instead of simply continuing to become larger, more expensive, more complicated, and more powerful, some flow cytometers started to become, in the late 1980s, smaller, less expensive, more accessible, and, although less flexible, remarkably stable. These instruments are called “benchtop” cytometers because they are self-contained and can be taken from their shipping crates, placed on a lab bench, plugged in, and (with luck) are ready to go.

The question arises as to how much training is required for use of these benchtop instruments. Pushing the buttons has become easy, but my prejudice on this issue should, of course, be obvious. If I believed that flow experiments could be designed and flow data could be acquired and analyzed appropriately by people with no awareness of the limitations and assumptions inherent in the technique, I would not have written this book. The actual operation of these small cytometers has been vastly simplified compared with that of the original research instruments. It certainly can be said that the new wave of cytometers has made flow analysis a great deal more accessible to the nonspecialist. A serious concern, however, is that the superficial simplicity of the instruments may lull users into a false sense of security about the ease of interpretation of the results. The basis for this concern is particularly clear in the medical community, where clinicians have been conditioned to expect that laboratory reports will contain unambiguous numbers; they may not be accustomed to the need for an intellectual framework in which to interpret those numbers. Anyone designing flow experiments or interpreting flow data needs some essential training in the technique; more training is required as the benchtop instruments (and the possible experiments) become more complex. At the high end, the operation of sorting instruments is best left to a dedicated operator; but, even here, the grass-roots user needs to understand flow cytometry in order to design an effective sorting protocol and communicate this protocol to the operator.

Although benchtop cytometers are less expensive than state-of-the-art instrumentation, they are still expensive. Therefore core facilities with shared instrumentation still provide for much of the current flow cytometric analysis. These shared facilities reflect a need by many for flow cytometric instrumentation, but also recognition of its high cost, its requirement for skilled maintenance and operation, and the fact that many users from many departments may each require less than full-time access. Such centralized facilities may have more than one cytometer. The trend now is to have one or more sophisticated instruments for specialized procedures accompanied by several bench-top cytometers as routine work horses. There are usually dedicated operators who run the sophisticated instruments, supervise use of the benchtop instruments, and ensure that all instruments are well-tuned for optimum performance. In addition, there may be a network of computers so that flow data can be analyzed and re-analyzed at leisure—away from the cytometer and possibly in the scientist’s own laboratory. A cytometry facility may also provide centrifuges, cell incubators, and fluorescence microscopes to aid in cell preparation. Increasingly, such institutional flow facilities are also becoming general cytometry facilities; the use of imaging microscopes for cell analysis has proved to be a technique that complements flow work. Similar stains are used in both flow and image analysis systems. Flow cytometry is based on analysis of light from a large number of cells as they individually pass a detector. Image analysis studies the distribution of light signals emanating from a large number of individual positions scanned over a single stationary cell.

The funding of such central facilities often involves a combination of institutional support, direct research grant support, and charges to users (or patients). The charges to users may vary from nominal to prohibitive and may need to support everything from consumables like computer paper and buffers to the major costs of laser replacement, service contracts, and staff salaries. With the increasing complexity and variety of instrumentation and with the need for data organization, booking systems, and financial accounting, the running of these flow facilities can become an administrative task in itself. Therefore a “corporate identity” often evolves, with logos designed, meetings organized, newsletters written, and training sessions provided.

Moreover, a funny thing has been happening. What originally appeared to be a rift in the field of flow cytometry, isolating the “high tech” from the “benchtop” users, has turned out to be, instead, a continuum. State-of-the-art instruments continue to improve, marking the advancing edge of technological and methodological development. However, benchtop flow cytometers have followed along behind, becoming increasingly powerful and combining the advantages of stable optics and fluidics with the new capabilities demonstrated on last year’s research instruments.

In this book, emphasis will be on the general principles of cytometry that apply equally to benchtop and state-of-the-art instrumentation. The chapter on sorting will, however, provide some insight into the ability of research cytometers to separate cells. In addition, the chapter on research frontiers provides a glimpse into the advanced capabilities of today’s research instruments (and, without doubt, many of these capabilities will soon appear on tomorrow’s benchtop cytometers).

3

Instrumentation: Into the Black Box

I want first to clear up some confusion that results simply from words. A flow cytometer, despite its name, does not necessarily deal with cells; it deals with cells quite often, but it can also deal with chromosomes or molecules or latex beads or with many other particles that can be suspended in a fluid. Although early flow cytometers were developed with the purpose of sorting particles, many cytometers today are in fact not capable of sorting. To add to the semantic confusion, the acronym FACS is even applied to some of these nonsorting cytometers. Flow cytometry might be broadly defined as a system for measuring and then analyzing the signals that result as particles flow in a liquid stream through a beam of light. Flow cytometry is, however, a changing technology. Defining it is something like capturing a greased pig; the more tightly it is grasped, the more likely it is to wriggle free. In this chapter, I describe the components that make up a flow cytometric system in such a way that we may not need a definition, but will know one when we see it.

The common elements in all flow cytometers are

A light source with a means to focus that light

Fluid lines and controls to direct a liquid stream containing particles through the focused light beam

An electronic network for detecting the light signals coming from the particles as they pass through the light beam and then converting the signals to numbers that are proportional to light intensity

A computer for recording the numbers derived from the electronic detectors and then analyzing them

In this chapter I describe the way a flow cytometer shapes the light beam from a laser to illuminate cells; the fluid system that brings the cells into the light beam; and the electronic network for detecting and processing the signals coming from the cells. The next chapter will cover computing and analysis strategies for converting flow cytometric data into useful information.

ILLUMINATION OF THE STREAM

A laser beam has a circular, radially symmetrical cross-sectional profile, with a diameter of approximately 1–2 mm. Lenses in the cytometer itself are used to shape the laser beam and to focus it to a smaller diameter as it illuminates the cells. Simple spherical lenses can provide a round spot about 60 μm in diameter, with its most intense region in the center and its brightness decreasing out toward the edge of the spot. The decrease in intensity follows a Gaussian profile; cells passing through the middle of the beam are much more brightly illuminated than those passing near the periphery. By convention, the nominal diameter of a Gaussian beam of light refers to the distance across the center of the beam at which the intensity drops to 13.5% of its maximal, central value. Therefore, if a cell is 30 μm from the center of a nominal 60 μm beam, it will receive only 13.5% of the light it would receive at the center of that beam. At 10 μm from the center, the intensity is about 78% and, at 3 μm from the center, the intensity is 98% of the central intensity. Because intensity falls off rapidly at even small distances from the center of the beam, cells need to be confined to a well-defined path through the beam if they are going, one by one, to receive identical illumination as they flow through a round beam of light.

To alleviate this stringency, the beam-focusing lenses most frequently used in today’s cytometers are a compromise; cylindrical lens combinations provide an elliptical spot, for example, 10–20 by 60 μm in size, with the short dimension parallel to the direction of cell flow and the longer dimension perpendicular to the flow (Fig. 3.1). By retaining a wide beam diameter across the direction of flow, an elliptical illumination spot can provide considerable side-to-side tolerance, thus illuminating cells more-or-less identically even if they stray from the exact center of the beam; but at the same time this elliptical profile can provide temporal resolution between cells, illuminating only one at a time as they pass one by one into and out of the beam in its narrow dimension. The narrower the beam is, the more quickly will a cell pass through it—giving opportunity for the signal from that cell to drop off before the start of the signal from the next cell in line and avoiding the coincidence of two cells in the beam simultaneously. In multilaser systems, each beam of light is focused in a similar way but at different points along the stream; a cell moves through each beam in sequence.

Fig. 3.1. Cells flowing past illuminating beams of different profiles. A beam with an elliptical profile (lower beam) allows cells to pass into and out of the beam quickly (avoiding the coincidence of two cells in the beam at the same time). In addition, it provides more equal illumination if cells stray from the center of the beam. The small circular beam at the top does not illuminate cells equally if they are at the edge of the stream core. The larger circular beam (in the middle) illuminates cells equally, but often includes multiple cells in the beam at the same time.

CENTERING CELLS IN THE ILLUMINATING BEAM

The fluidics in a flow cytometer are likely to be ignored until they go wrong. If they go wrong disastrously, they can make a terrible mess. If they go wrong with subtlety, they may turn a good experiment into artifactual nonsense without anyone ever noticing. On the assumption that the more disastrous problems can be solved by a combination of plumbing and mopping (both essential skills for flow cytometrists), I will concentrate on the more subtle aspects of fluid control. Nevertheless, the potential hazard of working at the same time with volumes of water and with a high-voltage source should never be far from the mind of anyone working with a water-cooled laser or with a sorting cytometer with high-voltage stream deflection plates.

The flow on a flow cytometer begins (Fig. 3.2) at a reservoir of liquid, called the sheath fluid. Sheath fluid provides the supporting vehicle for directing cells through the laser beam. The sheath fluid reservoir is pressurized, usually with pumped room air, to drive the sheath fluid through a filter to remove extraneous particles and then through plastic tubing to the illumination point. This sheath stream is usually buffer of a composition that is appropriate to the types of particles being analyzed. For leukocytes or other mammalian cells, this usually means some sort of phosphate-buffered saline solution. Other cells or other particles may have other preferences.

Different instruments employ different strategies for getting the sample with suspended cells into the sheath stream in the cytometer. Some instruments require cells to be in small test tubes that form a tight seal around an O-ring on a manifold. The manifold delivers air to the test tube, thus pushing the suspended cells up out of the test tube and through a plastic line to the sheath stream. Other instruments use a motor-driven syringe to remove a volume of sample and then inject it slowly into the cytometer. Depending on the instrument, there may be a greater or lesser degree of operator control over the rate of flow. The amount of pressure driving the sample through the system will affect the uniformity of alignment between the cells and the illuminating laser beam as the cells move through the cytometer. Low pressure is less likely to cause perturbation of the stream profile and of the position of the cells within that stream. Empirically, if increasing the pressure on the sample causes undue broadening or wavering of signals, the pressure is probably excessive.

Fig. 3.2. The fluidics system, with air pressure pushing both the sample (with suspended cells) and the sheath fluid into the flow cell.

If cells flow too slowly through the cytometer, people start to make bad jokes about how microscopes cost less and are quicker. Because increasing the pressure may not be possible and, even if it is, is probably only a good idea within reasonable limits, the best way of getting cells to flow at reasonably fast rates is simply to make up the original sample with cells at a reasonably high concentration. A million cells per milliliter is often about right; 105 cells per milliliter is beginning to be low enough to test one’s patience; 104 cells per milliliter is probably too low a concentration to be worth analyzing.

If you have few cells, make them up in a small volume (you will know how small a volume your system can handle). If the cells end up being too concentrated, they may flow too fast—but you can always dilute them on the spot and run the sample again. You may wonder why too rapid a flow is a source of problems. Faster seems as if it should always be better (especially around 5:00 PM). However, if cells are too close together as they flow through the laser beam, there may be difficulty separating their signals: A second cell may arrive in the illuminating beam before the preceding cell has emerged, and they will be measured together as if they were a single particle (with double the intensity). Figure 3.3 gives an indication of the probability of cells coinciding in the laser beam at different flow rates. Most cytometers seem to be quite happy looking at particles that are flowing at a rate of about 1000 particles per second.

Fig. 3.3. The probability of an event recorded by the flow cytometer as a single “cell” actually resulting from more than one cell coinciding in the laser beam. For this model, the laser beam was considered to be 30)μm high and the stream flowing at 10 m per second.