FlowCytometry

First Principles

Second edition

This page intentionally left blank

FlowCytometry

First Principles

Second editionflllce Longobardl Glvan

The Herbert C. Englert Cell Analysis Laboratoryof the Morris Cotton Cancer Center

and Department of PhysiologyDartmouth Medical SchoolLebanon, New Hampshire

-LISSNew York • Chichester

A John Wiley & Sons, Inc., Publication

Weinheim • Brisbane • Singapore • Toronto

This book is printed on acid-free paper. ©

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

Published simultaneously in Canada.

No part of this publication may be reproduced, stored in a retrieval system or transmitted inany form or by any means, electronic, mechanical, photocopying, recording, scanning orotherwise, except as permitted under Sections 107 or 108 of the 1976 United States CopyrightAct, without either the prior written permission of the Publisher, or authorization throughpayment of the appropriate per-copy fee to the Copyright Clearance Center, 222 RosewoodDrive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher forpermission should be addressed to the Permissions Department, John Wiley & Sons, Inc.,605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail:PERMREQ@WILEY.COM.

For ordering and customer service, call 1-800-CALL-WILEY.

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 0-471-38224-81. Flow cytometry. I. Title.[DNLM: 1. Flow Cytometry—methods. QH585.5.F56 G539f]

QH585.5.F56G58 1992574.87'028—dc20DNLM/DLCfor Library of Congress 92-5004

Printed in the United States of America.

1 0 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 toCurt, Ben, and Becky

(not only because I promised them that their turn would come).

This page intentionally left blank

Contents

Preface xi

Acknowledgments (First Edition) xv

Acknowledgments (Second Edition) xvii

1 The Past as Prologue 1

2 Setting the Scene 11

3 Instrumentation: Into the Black Box 15

Illumination of the Stream 16Centering Cells in the Illuminating Beam 18Detection of Signals from Cells 25Electronics 31

4 Information: Harnessing the Data 41

Data Storage 41Data Analysis 43

5 Seeing the Light: Lasers, Fluorochromes, andFilters 59

General Theory 59Lasers 61Fluorochromes 65Partitioning the Signal with Lenses, Filters, and

Mirrors 72Spectral Compensation 76

viii Flow Cytometry

6 Cells from Without: Leukocytes, Surface Proteins, andthe Strategy of Gating 81

Cells from Blood 82Staining for Surface Markers 87Controls 90Quantitation 95Sensitivity 98The Strategy of Gating 99Gating on Fluorescence 108

7 Cells from Within: Intracellular Proteins 115

Methods for Permeabilizing Cells 116Examples of Intracellular Staining 118

8 Cells from Within: DNA in Life and Death 123

Fluorochromes for DNA Analysis 123Ploidy 125Cell Cycle Analysis 131Two-Color Analysis for DNA and Another

Parameter 142Chromosomes 147Apoptosis 150Necrosis 154

9 The Sorting of Cells 159

Sorting Theory 159Characterization of Sorted Cells 166Alternative Methods for Sorting 1 70The Condition of Cells After Sorting 1 72

10 Disease and Diagnosis: The Clinical Laboratory 175

The Hematology Laboratory 178The Pathology Laboratory 186Solid Organ Transplantation 189Comments 192

Confenfs

11 Out of the Mainstream: Research Frontiers 195

Functional Assays 196The Aquatic Environment 202Reporter Molecules 207Microbiology 211Molecular Biology 213Multiplex Cytometry for Soluble Analytes 218Reproductive Technology 220

12 Flowing On: The Future 225

General References 229

Glossary 235

Figure Credits 257

Index 263

This page intentionally left blank

Preface

Although flow cytometry is simply a technique that is useful incertain fields of scientific endeavor, there is, at the same time, some-thing special about it. Few other techniques involve specialists fromso many different backgrounds. Anyone working with flow systemsfor any length of time will realize that computer buffs, electronicsexperts, mathematicians, optical and fluidics engineers, and organicchemists rub shoulders with biologists, physicians, and surgeonsaround the flow cytometer bench.

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

A third aspect of flow cytometry (known sometimes simply withthe acronym for fluorescence-activated cell sorter, FACS, or evenmore familiarly as just flow) that distinguishes it from many othertechniques is the way in which its wide and increasing usefulness hascontinued to surprise even those who consider themselves experts.What began as a clever technique for looking at a very limited rangeof problems is now being used in universities, in hospitals, withinindustry, at marine stations, on submersible buoys, and on boardships; plans have existed for use on board space ships as well. Theapplications of flow cytometry have proliferated (and continue toproliferate) rapidly both in the direction of theoretical science, with

XI

xii Flow Cytometry

ARTICLES IN THE MEDLINE DATABASEWITH REFERENCE TO "FLOW CYTOMETRY

Year of Publication

Fig. 1. Increasing reference to "flow cytometry" in the medical literature over thepast three decades. The development of flow cytometers antedates the use of the termitself.

botany, molecular biology, embryology, biochemistry, marine ecol-ogy, genetics, microbiology, and immunology, for example, all rep-resented; and in the direction of clinical diagnosis and medical prac-tice, with hematology, bacteriology, pathology, oncology, obstetrics,and surgery involved. We are, at present, living through what appearsto be a rapid phase in flow cytometry's growth curve (see Fig. 1).

Because flow cytometry is an unusual field, bringing togetherpeople with differing scientific backgrounds at meetings, on editorialboards, in hospital wards, on advisory panels, and at laboratorybenches and reaching increasing numbers of workers in new andunpredicted areas of endeavor, there is, as a result, a need to provideboth recent and potential entrants into this diverse community witha common basis of knowledge—so that we can all understand thevocabulary, the assumptions, the strengths, and the weaknesses of thetechnology involved. I have for many years taught new and futureusers of flow cytometers. My teaching attempts to present enoughtechnical background to enable students, scientists, technologists, and

Preface xiii

clinicians to read the literature critically, to evaluate the benefits ofthe technique realistically, and, if tempted, to design effective pro-tocols and interpret the results. I try to describe the theory of flowcytometry in a way that also provides a firm (and accurate) founda-tion for those few who will go on to study the technique in greaterdepth. Details of protocols are avoided, but my teaching attemptsto give enough information about applications to provide concreteexamples of general concepts and to allow some appreciation of therange of practical goals that the instruments are able to achieve. Withsome expansion (but with little change in style or objectives), myclassroom and workshop teaching is the basis for this book.

Notes on the Second Edition: This new edition, while similar to thefirst edition in style and scope, has been modified in many ways. Thearrangement of some material has been altered to present, in myopinion, a more coherent pedagogical sequence, reflecting my chang-ing thoughts on teaching. While all chapters have been re-written to asignificant extent, there are also some major expansions reflecting theprogress of the field. In particular, I have included more detail aboutthe cell as it passes through the laser beam; the laser/fluorochromechapter has been expanded to include recently developed fluoro-chromes and multilaser options; a new chapter has been added oncytoplasmic staining; a discussion of apoptosis has been added to thechapter on DNA; the section on sorting has been expanded to a fullchapter and includes high-speed sorting and alternative sortingmethods as well as traditional technology; the clinical and researchchapters have been updated and expanded considerably; the chapteron general references includes many of the recent excellent books inthe field; and the chapter on the future of flow cytometry is now asubjective glimpse into the new decade from the vantage point of theyear 2000.

This page intentionally left blank

Acknowledgments(First Edition)

Realizing how much I have learned and continue to learn fromothers, I hesitate to single out a few names for particular mention.However, with the disclaimer that any list of people to whom I amindebted is not meant to be and, indeed, could never be complete, Imust thank here the following friends, mentors, and colleagues whohave had a very direct impact on the writing of this book: GeorgeProud, for having had enough insight into the importance of a flowcytometer for transplantation surgery to want to have one; IvanJohnston and Ross Taylor, for hospitality (and the Marker BequestFund, the Northern Counties Kidney Research Fund, and the New-castle Health Authority, for financial assistance) within the De-partment of Surgery at Newcastle University; Brian Shenton, forintroducing me to the field of flow cy tome try and to the joys andhazards of clinical research; Mike White, for bailing me out (oftenfiguratively and once or twice literally) on so many occasions; PaulDunnigan, 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 beyondall reasonable expectation; Terry Godley, for being an extremelygood and (more importantly) a very communicative flow cytometryservice engineer; Ray Joyce, for considerable assistance with the designand drawing of many of the diagrams; the scientists and clinicians atNewcastle University and Durham University (many acknowledgedin the figure legends), for providing me with a continuous source ofinteresting and well-prepared cell material on which to practice myflow technique; Paul Guyre, for giving me a supportive and remark-

XV

xvi Flow Cytometry

ably enjoyable re-introduction to science in the New World; DaryllGreen, for giving me the benefit of his expertise in both physics andflow cytometry by reading and commenting about the chapters oninstrumentation and information; Brian Crawford, for being a literateand encouraging editor in the face of my rank inexperience; the lateRobert L. Conner, for providing me with that critical first of my still-continuing scientific apprenticeships; Curt Givan, for his unfailingloyalty and for his skill in reading the entire manuscript with twoeyes—one eye that of an old-fashioned grammarian who abhor dan-gling participles and the other that of a modern scientist who knowsnothing about flow cytometry; and Ben Givan, for the two draw-ings in Figure 8.1 [Fig. 10.1 in the Second Edition], Becky Givan,for organizational magic when it was very badly needed, and bothkids for lots of encouragement and some pretty funny suggestionsfor a title.

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

Acknowledgments(Second Edition)

I continue to be immensely grateful to the people whom I acknowl-edged in the First Edition of FCFP; their contributions are still cur-rent and form the foundation for this new edition. I have, however,during preparation of the new edition, received considerable addi-tional help. During the 9 years since the publication of the first edi-tion of this book, changes have taken place in my life as well as in thefield of flow cytometry. Now working at Dartmouth Medical School,I have learned much about clinical cytometry, flow analysis, andcomputers from Marc Langweiler; Marc taught me the acronym"RTFM," but has been restrained at using it in response to my naivequestions and I am grateful. In addition, Marc was a particular helpto me with suggestions for Chapter 10. Gary Ward, the flow sortingsupervisor in the Englert Cell Analysis Laboratory at Dartmouth,has, by his competence, made my sorting skills rusty, but he hasallowed me to pretend that I run "his" sorter when I need a dramaticphotograph, and I am grateful for his good humor. I am also gratefulto Ken Orndorff, who supervises the imaging service in the EnglertLab; he has been admirably tolerant of flow cytometrists (includingme) who have slowly begun to realize that a dot on a dot plot maynot 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 theEnglert Lab for their ability to provide me with continuing challenges.

First Colette Bean and then Luna Han at Wiley inherited editorialsupervision of this book from Brian Crawford and I owe them con-siderable thanks for their persistence and patience. At Dartmouth, Iam grateful for secretarial assistance from Mary Durand and artistic

XVII

xviii Flow Cytometry

help from Joan Thomson. Dean Gonzalez at Wiley, with artistry andstamina, is responsible for the great improvement in figure claritybetween the two editions of this book. I thank him.

During these last 9 years, I have had the opportunity to expand myacquaintance with members of the greater flow community. Amongmany from whom I have learned much, I would like especially tomention a few: Louis Kamentsky shared with me his lecture notes fora wonderful keynote address he gave at Dartmouth on the history offlow cytometry, and I am grateful to him. Howard Shapiro has beena source of limitless cytometric information cloaked seductively inoutrageous humor; I thank him for his knowledge, for his humor,and for his generosity. Paul Robinson has made many contributionsto flow cytometry, but I want particularly to mention his organiza-tion of the Purdue University flow network; I am grateful to him forthe information, the moral support, and the distance friendships thatthe network has given me. Over the years, I have had the privilegeof teaching workshops and courses with a group of people who aredevoted not just to flow technology (although they are talented flowcytometrists) but also to education of students in the correct use ofthe technique. Indeed, I also detect their devotion to the use of flowcytometry as a platform for teaching a rigorous approach to sciencein general. In thanks for all I have learned from these workshops, Iwish, particularly, to mention here Bruce Bagwell, Ben Hunsberger,James Jett, Kathy Muirhead, Carleton Stewart, Joe Trotter, and PaulWallace.

And—as ever—Curt Givan came through with unflappable proof-reading 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 firmlygrounded in history. In particular, flow technology finds intellectualantecedents in microscopy, in blood cell counting instruments, andin the ink jet technology that was, in the 1960s, being developed forcomputer printers. It was the coming together of these three strandsof endeavor that provided the basis for the development of the firstflow cytometers. Because thorough accounts of the history of flowcytometry have been written elsewhere (and make a fascinating storyfor those interested in the history of science), I cover past history herein just enough detail to give readers a perspective as to why currentinstruments have developed as they have.

Microscopes have, since the seventeenth century, been used toexamine cells and tissue sections. Particularly since the end of thenineteenth century, stains have been developed that make variouscellular constituents visible; in the 1940s and 1950s, fluorescence mi-croscopy began to be used in conjunction with fluorescent stains fornucleic acids in order to detect malignant cells. With the advent ofantibody technology and the work of Albert Coons in linking anti-bodies with fluorescent tags, the use of fluorescent stains gained widerand more specific applications. In particular, cell suspensions or tissuesections are now routinely stained with antibodies specific for anti-genie markers of cell type or function. The antibodies are either di-rectly or indirectly conjugated to fluorescent molecules (most usuallyfluorescein or rhodamine). The cellular material can then be exam-ined on a glass slide under a microscope fitted with an appropriatelamp and filters so that the fluorescence of the cells can be excitedand observed (Fig. 1.1). The fluorescence microscope allows us to seecells, to identify them in terms of both their physical structure and

Flow Cytometry

LIGHTSOURCE

1 first barrier filter: lets throughonly blue light with a wavelengthbetween 450 and 490 nm

3 second barrier filter: cuts outunwanted fluorescent signals passingthe specific green fluoresceinemission between 520 and 560 nm

2 beam-splitting mirror: reflectsdown light below 510 nm buttransmits light above 510 nm

objective lens

object

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

their orientation within tissues, and then to determine whether and inwhat pattern they fluoresce when stained with one or another of thespecific stains available. In addition, a microscope can also be fittedwith a camera or photodetector, which will then record the intensityof fluorescence arising from the field in view. The logical extension ofthis technique is image analysis cytometry, digitizing the output toallow precise quantitation of fluorescence intensity patterns in detail(pixel by pixel) within that field of view. The development of mono-clonal antibody technology (for which Kohler and Milstein wereawarded the Nobel Prize) led to a vast increase in the number ofcellular components that can be specifically stained and that can beused to classify cells. Whereas monoclonal antibody techniques arenot directly related to the development of flow technology, their inven-tion was a serendipitous event that had great impact on the poten-tial utility of flow cytometric systems.

In 1934, Andrew Moldavan in Montreal took a first step from staticmicroscopy toward a flowing system. He suggested the developmentof an apparatus to count red blood cells and neutral-red-stained

The Past as Prologue 3

yeast cells as they were forced through a capillary on a microscopestage. A photodetector attached to the microscope eyepiece wouldregister each passing cell. Although it is unclear from Moldavan'spaper whether he actually ever built this cytometer, the developmentof staining procedures over the next 30 years made it obvious that thetechnique he suggested could be useful not simply for counting thenumber of cells but also for quantitating their characteristics.

In the mid-1960s, Louis Kamentsky took his background in opticalcharacter recognition and applied it to the problem of automatedcervical cytology screening. He developed a microscope-based spec-trophotometer (on the pattern of the one suggested by Moldavan)that measured and recorded ultraviolet absorption and the scatter ofblue light ("as an alternative to mimicking the complex scanningmethods of the human microscopist") from cells flowing "at ratesexceeding 500 cells per second" past a microscope objective. Then, in1967, Kamentsky and Melamed elaborated this design into a sortinginstrument (Fig. 1.2) that provided for the electronic actuation of asyringe to pull cells with high absorption/scatter ratios out of theflow stream. These "suspicious" cells could then be subjected to de-tailed microscopic analysis. In 1969, Dittrich and Gohde in Minister,Germany, described a flow chamber for a microscope-based systemwhereby fluorescence intensity histograms could be generated based onthe ethidium bromide fluorescence from the DNA of alcohol-fixed cells.

During this period of advances in flow microscopy, so-calledCoulter technology had been developed by Wallace Coulter for anal-ysis of blood cells. In the 1950s, instruments were produced thatcounted cells as they flowed in a liquid stream; analysis was based onthe amount by which the cells increased electrical resistance as theydisplaced isotonic saline solution while flowing through an orifice.Cells were thereby classified more or less on the basis of their volumebecause larger cells have greater electrical resistance. These Coultercounters soon became essential equipment in hospital hematologylaboratories, allowing the rapid and automated counting of white andred blood cells. They actually incorporated many of the features ofanalysis that we now think of as being typical of flow cytometry: therapid flow of single cells in file through an orifice, the electronicdetection of signals from those cells, and the automated analysis ofthose signals.

At the same time as Kamentsky's work on cervical screening,

Flow Cytometry

Fig. 1.2. A diagram of Kamentsky's original flow sorter. From Kamentsky andMelamed (1967). Science 156:1364-1365. Copyright AAAS.

Mack Fulwyler at the Los Alamos Laboratory in New Mexico haddecided to investigate a problem well known to everyone looking atred blood cells in Coulter counters. Red cells were known to show abimodal distribution of their electrical resistance ("Coulter volume").Anyone looking at erythrocytes under the microscope cannot helpbut be impressed by the remarkable structural uniformity of thesecells; Fulwyler wondered if the bimodal Coulter volume distributionrepresented differences between two classes of these apparently veryuniform cells or, alternatively, whether the bimodal profile was simplyan artifact based on some quirky aspect of the electronic resistancemeasurements. The most direct way of testing these two alternatives

The Past as Prologue 5

was to separate erythrocytes according to their electronic resistancesignals and then to determine whether the separated classes remaineddistinct when they were re-analyzed.

The technique that Fulwyler developed for sorting the erythrocytescombined Coulter methodology with the ink jet technology beingdeveloped at Stanford University by RG Sweet for running computerprinters. Ink jet technology involves the vibration of a nozzle so asto generate a stream that breaks up into discrete drops and then thecharging and grounding of that stream at appropriate times so as toleave indicated drops, as they break off, carrying an electrical charge.For purposes of printing, those charged drops of ink can then bedeflected to positions on the paper as required by the computer printmessages. Fulwyler took the intellectual leap of combining thismethodology with Coulter flow technology; he developed an instru-ment that would charge drops containing suspended cells, therebyallowing deflection of the cells (within the drops) as dictated by sig-nals based on the cell's measured Coulter volume.

The data from this limited but pioneering experiment led to a con-clusion that with hindsight seems obvious: Erythrocytes are indeeduniform. When red cells are sorted according to their electrical resis-tance, the resulting cells from one class or the other still show abimodal distribution when re-analyzed for their electrical resistanceprofile. The bimodal "volume" signal from erythrocytes was there-fore artifactual—resulting in part from the discoid (nonspherical)shape of the cells. The technology developed for this landmark experi-ment is the essence of all the technology required for flow sorting aswe now know it. That experiment also, unwittingly, emphasized anaspect of flow cytometry that has remained with us to this day: Flowcytometrists still need to be continually vigilant (and know how touse a microscope) because signals from cells (particularly signals thatare related to cell volume) are subject to artifactual influences and maynot be what they seem. (Fulwyler's 1965 paper actually describes theseparation of mouse from human erythrocytes and the separation ofa large component from a population of mouse lymphoma cells; theexperiments on the bimodal signals from red cells have been relegatedto flow folk history.)

In 1953, PJ Crosland-Taylor, working at the Middlesex Hospitalin London, noted that attempts to count small particles suspended influid flowing through a tube had not hitherto been very successful.With particles such as red blood cells, the experimenter must choose

6 Flow Cytometry

between a wide tube that allows particles to pass two or more abreastacross a particular section or a narrow tube that makes microscopicalobservation of the contents of the tube difficult due to the differentrefractive 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 designof a flow system. A suspension of red blood cells was injected into thecenter of a faster flowing stream, thus allowing the cells to be alignedin a narrow central file within the core of the wider stream prepara-tory to electronic counting. This principle of hydrodynamic focusingwas pivotal for the further development of the field.

In 1969, Marvin Van Dilla and other members of the Los AlamosLaboratory group reported development of the first fluorescence-detection cytometer that utilized the principle of hydrodynamicfocusing and, unlike the microscope-based systems, had the axes offlow, illumination, and detection all orthogonal to each other; it alsoused an argon laser as a light source (Fig. 1.3). Indeed, the configu-ration of this instrument provided a framework that could supportboth the illumination and detection electronics of Kamentsky's deviceas 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 fluo-rescence from the Feulgen-DNA staining of Chinese hamster ovary

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

The Past as Prologue 7

cells and leukocytes as well as of their Coulter volume; however, theauthors "anticipated that extension of this method is possible and ofpotential value." Indeed, shortly thereafter, the Herzenberg groupat Stanford published a paper demonstrating the use of a similarcytometer to sort mouse spleen and Chinese hamster ovary cells onthe basis of their fluorescence due to accumulation of fluorescein di-acetate. These instruments thus led to systems for combining multi-parameter fluorescence, light scatter, and "Coulter volume" detectionwith cell sorting.

These sorting cytometers began to be used to look at ways of dis-tinguishing and separating white blood cells. By the end of the 1960s,they were able to sort lymphocytes and granulocytes into highlypurified states. The remaining history of flow cytometry involves theelaboration of this technology, the exploitation of flow cytometersfor varied applications, and the collaboration between scientists andindustry for the commercial production of cytometers as user-friendlytools (Fig. 1.4). At the same time that these instruments began to beseen as commercially marketable objects, research and developmentcontinued especially at the United States National Laboratories atLos Alamos (New Mexico) and Livermore (California), but also atsmaller centers around the world. At these centers, homemade in-

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

Flow Cytometry

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

struments continue to indicate the leading edge of flow technology.At the present time, this technology is moving simultaneously in twodirections: On the one hand, increasingly sophisticated instrumentsare being developed that can measure and analyze more aspects ofmore varied types of particles more and more sensitively and that cansort particles on the basis of these aspects at faster and faster rates.On the other hand, a different type of sophistication has streamlinedinstruments (Fig. 1.5) so that they have become user-friendly andessential equipment for many laboratory benches.

The Past as Prologue 9

FURTHER READING

Throughout this book, the "Further Reading" references at the endof each chapter, while not exhaustive, are intended to point the wayinto the specific literature related to the chapter in question. At theend of the book, "General References" will direct readers to globallyuseful literature. Titles in bold at the end of each chapter are textsthat are fully cited in the General References at the end.

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

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

Dittrich W, Gohde W (1969). Impulsfluorometrie dei Einzelzellen in Suspen-sionen. 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-activatedcell 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 intracellularfluorescence. Science 166:747-749.

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

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

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

Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR (1969). Cell micro-fluorimetry: A method for rapid fluorescence measurement. Science163:1213-1214.

Chapter 1 in Melamed et al., Chapter 3 in Shapiro, and Chapter 1 inDarzynkiewicz are good historical reviews of flow cytometry. AlbertoCambrosio and Peter Keating (2000) have used flow cytometry as a modelfor looking at historical changes in the way scientists use instrumentationto view the world: Of lymphocytes and pixels: The techno-visual productionof cell populations. Studies in History and Philosophy of Biological andBiomedical Sciences 31:233-270.

This page intentionally left blank

Setting the Scene

As mentioned in the previous chapter, flow cytometry has beenmoving in two directions at once. The earliest flow cytometers wereeither homemade Rube Goldberg (Heath Robinson, U.K.) monstersor, a few years later, equally complex, unwieldy commercial instru-ments. These were expensive; they were also unstable and thereforedifficult to operate and maintain. For these reasons, the cytometertended to collect around itself the trappings of what might be called aflow facility—that is, a group of scientists, technicians, students, andadministrators, as well as a collection of computers and printers, thatall revolved around the flow cytometer at the hub. If a scientist orclinician wanted the use of a flow cytometer to provide some requiredinformation, he or she would come to the flow facility, discuss theexperimental requirements, make a booking, and then return with theprepared samples at the allotted time. The samples would then be runthrough the cytometer by a dedicated and knowledgeable operator.Finally, depending on the operator's assessment of the skill of theend-user, a number, a computer print-out, or a computer disk wouldbe handed over for analysis.

Just at the moment when such flow facilities were beginning to seethemselves as essential components of modern research, the technol-ogy of flow cytometry began to move in a new direction. Users andmanufacturers both began to realize that different laboratories havedifferent instrumentation needs. A sophisticated sorter might be nec-essary for certain applications, but its demands on daily alignmentand skilled maintenance are time-consuming and its research capa-bilities 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