J Clin Pathol 1997;50:275-277 275

Origins of... J Clin Pathol: first published as 10.1136/jcp.50.4.275 on 1 April 1997. Downloaded from and applications in oncology

Walter Giaretti

Flow cytometry dates back to the synthesis of and often automatic analysis and final reports. dyes in the late 1800s and to the fundamental Earlier instruments required manual interven- work done in Stockholm by T Caspersson and tion of skilled people at many steps of the colleagues."1 They demonstrated, in 1938, that measuring process. DNA content, measured by ultraviolet and vis- The heart ofa flow cytometer is a chamber in ible light absorption in unstained cells, doubled which the suspension fluid, commonly during the .4 Feulgen and Papanicolau coaxial to an external fluid (sheath fluid), is staining techniques were then in their infancy, driven to reach a laminar flow so that the cells and a few fluorescent dyes had been proposed are hydrodynamically focused to the centre of for detection of cells in cytology. How- the stream. Incident light sources were origi- ever, it was not until 1950, when Coons and nally arc lamps; the vast majority of flow Kaplan reported on the improved detection of cytometers today use laser light. One ofthe first antigens using fluorescence antibody flow cytometers designed to measure DNA methods,5 that it was realised that fluorescence content was based on a mercury arc lamp that measurements could offer more advantages provided good ultraviolet illumination. This than absorption. Since then, fluorescein re- choice remains most suitable after cell or nuclei mains the most common label for quantitative staining with 4',6-diamidino-2-phenylindole- immunofluorescence analysis, and now for 2HC1 (DAPI).78 Arc lamp based flow cyto- routine flow cytometric applications in haema- meters commonly have an optical scheme tology and immunology. similar to that of a In 1956 there was a breakthrough in flow under epi-illumination conditions to keep the cytometry when an apparatus was built by WH illumination homogeneous at the focus

Coulter in which blood cells, in saline suspen- through which the cells flow. These instru- http://jcp.bmj.com/ sions, passed one by one through a small orifice ments use oil immersion optics with a numeri- and were detected by changes of electrical cal aperture of about 1.3 and are insensitive to impedance at the orifice.6 This instrument was the defocusing effect ofthe liquid stream or the the progenitor of the modern flow cytometers shape and orientation of the cells in the flow. developed almost simultaneously in United Most recent laser based flow cytometers offer States and Europe between 1965 and 1970.' the advantage ofmeasuring scatter and absorp- Concomitant driving forces at the origin of tion as well as fluorescence. In particular, on October 1, 2021 by guest. Protected copyright. such new instruments were the established intensities of scattered light in the forward and principle of Caspersson and colleagues that perpendicular directions, that correlate with cancer cells had higher DNA content than nor- size and internal structure of the cells or cell mal cells, the Coon's principle of cell recogni- subunits, are commonly measured. Fluores- tion by immunofluorescent antibodies, and the cent light intensity of five or more colours can immense increase in the use of computers in be measured simultaneously. Fluorescence the early 1960s. emission at 900 with respect to the incident Flow cytometry was based mainly on light light is measured through an optical system of scattering and fluorescence and the first appli- lenses, filters, and dichroic mirrors. Light cations were measurement of nuclear DNA signals are converted by photomultipliers into (using stoichiometric DNA stains) and cell electronic signals which are amplified for surface antigens (using immunofluorescence). analysis of area, peak, and duration. Each ana- Laboratory of Methodologies and applications since then logue parameter is then converted into an inte- Biophysics and have increased tremendously.' ger (analogue-digital conversion). Cytometry, National Monoparametric and multiparametric data Institute for Cancer Research, Genoa, Italy Basic characteristics of instrumentation are supervised in real time by a dedicated The evolution of the instrumentation was not microcomputer, which also controls cell "gat- Correspondence to: as fast as for the methods and applications. ing" and "sorting". These two important prop- Dr W Giaretti, Early and most recent flow cytometers are erties are based on a predefined set ofthreshold Laboratory of Biophysics and Cytometry, National quite similar, except for the fact that they have criteria applied to the acquired real-time Institute for Cancer Research become increasingly computer controlled. parameters to select and physically separate (1st), Largo R Bensi n. 10, Powerful general purpose computers assist specific cell subpopulations. Cell sorting, in 16132, Genova, Italy. today in optimising nearly all measurement particular, is commonly obtained in laser based Accepted for publication conditions and allow automatic data storage, systems by breaking the liquid stream into 11 September 1996 list-mode acquisition, graphic representations, droplets containing single cells, by selectively 276 Giaretti

charging the droplets with negative and posi- rospective studies of paraffin wax embedded tive charges according to the "sorting" criteria, material did not provide enough useful data and by deflecting them into separate cell because of the poor quality of measurements. collectors. Sorting rates of 5000 cells per New and more strict guidelines were necessary, second with a purity up to 99% can be using fresh-frozen material in large prospective J Clin Pathol: first published as 10.1136/jcp.50.4.275 on 1 April 1997. Downloaded from obtained. Suitable cell viability after cell studies and multiparameter flow cytometry sorting for subculturing and functional studies and these were developed by the various have been reported. groups.'4"-8 Overall, more significant biological insight and clinically useful information ap- Early and late applications peared necessary before more widespread The earliest flow cytometric applications- application of DNA content flow cytometry measurement of DNA and surface antigens in could be considered appropriate or warranted. haematology and immunology-remain the To achieve a better understanding of the most common applications. However, the flow biological significance of DNA aneuploidy, cytometric measurements of cell components several studies have recently tried to investigate other than DNA and the recent important sec- flow cytometric DNA content in combination tor of applications of cell function measure- with molecular biology analysis of oncogenes ments are becoming increasingly important. In and tumour suppressor genes.20.26 general, flow cytometers can potentially pro- vide rapid, sensitive, and quantitative cyto- Evaluation ofproliferation, cell cycle chemical measurements of any cell component kinetics, and apoptosis in human cancer that is specifically stained by specific fluoro- The fraction of cells in the cell cycle S phase chromes and monoclonal antibodies. In addi- (SPF) is a common parameter extracted from tion, they may exploit properties of autofluo- DNA histograms obtained by flow cytometry. rescence, scattering, and absorption. A great SPF values may be evaluated according to dif- potential offlow cytometry is the simultaneous ferent cell cycle models.27 28 For DNA aneu- acquisition of multiple parameters correspond- ploid tumours, SPF may be evaluated relative ing to different cell compartments of the same to the DNA aneuploid cells. However, for cells. Suspensions of cells may enable-for DNA diploid tumours, SPF is commonly and example, simultaneous measurement of nu- imprecisely evaluated from total number of clear DNA, RNA, and cytoplasmic or nuclear cells. The clinical usefulness of SPF was , as well as membrane antigens. The addressed by the same consensus conference main aim of acquiring multiple parameters in referred to previously.""'8 Overall, it was found flow cytometry is optimally to characterise het- that a large body of literature supported an erogeneous cell populations. Cell sorting, in important association between SPF values and addition, enables purification and enrichment increased risk of recurrence and mortality for of homogenous cell populations which can patients with cancer at the various sites exam-

then be investigated by other techniques. ined. However, methodological problems for http://jcp.bmj.com/ assessing SPF, because of the type of analysis, Measurement of DNA content in human paraffin wax embedded material, and especially cancer in DNA diploid and DNA aneuploid near- Benign and malignant human neoplasms often diploid subclones, were clearly recognised. contain subclones with abnormal DNA content. Multiparameter flow cytometric analysis, in- Models of DNA content aneuploidisation and cluding coupling with potentially relevant pro-

evolution have been proposed,9 ' but the precise liferation and differentiation associated anti- on October 1, 2021 by guest. Protected copyright. mechanisms are poorly understood. Protocols gens was recommended.'1'8 for specific DNA content measurement in nuclei A relevant multiparameter flow cytometric or cell suspensions by flow cytometry have been analysis of proliferation was introduced after extensively reviewed.-3 8 11 12 The DNA index the development of a monoclonal antibody (DI) that represents the degree of DNA aneu- against bromodeoxyuridine (BrdUrd).29.32 Re- ploidy relative to DNA diploid cells is a cent studies have demonstrated the feasibility common parameter extracted from DNA of making routine flow cytometric measure- histograms.'3 Quality controls are extremely ments ofdetailed cell kinetics in human cancer, important; a recent consensus conference including the potential doubling time (Tpot) aimed to evaluate critical quality controls and after administration of BrdUrd solutions to clinical usefulness of flow cytometry cancer patients...... Tpot evaluation" appears measurement of DNA content in bladder promising in predicting local control in head cancer,'4 breast cancer,15 colorectal cancer,'6 and neck squamous cell carcinomas and to neoplastic haematopathology,'7 and prostate select radiotherapy regimens.37 Tpot includes cancer. 18 information about the quiescent cell cycle Before and during this consensus confer- compartment but does not take into account ence, several North American and European the cell loss factor. Cell loss information in groups examined published studies. Contro- cancer, due to exfoliation, metastasis, necrosis, versies, such as those concerning the use of and apoptosis would most valuable for a paraffin wax embedded material, according to better understanding oftumour biology and for the method of Hedley et al,'9 were analysed. optimising therapies. The usefulness of flow Overall, the methodological adequacy of most cytometry for the assessment of apoptosis in published studies (adequate criteria for patient human cancer needs to be evaluated. Several and clinical parameters, adequate flow cyto- flow cytometric techniques have been metric guidelines) was considered critical. Ret- proposed.39"" It is likely that the combination Origins of . . Flow cytometry and applications in oncology 277

of flow cytometry and image cytometry using aneuploidy to molecular genetic alterations in colorectal carcinoma. Gastroenterology 1993;102: 1612-19. the "Tunel" apoptosis method39 42 and others 22 Carder P, Wyllie AH, Purdie CA, Morris RG, White S, Piris may be relevant. Similarly, image and flow J, et al. Stabilised p53 facilitates aneuploid clonal divergence in colorectal cancer. Oncogene 1993;8: 1397- cytometry coupling may be relevant in the 401. future with use of histological type specific 23 Meling GI, Lothe RA, Borresen AL, Graue C, Hauge S, J Clin Pathol: first published as 10.1136/jcp.50.4.275 on 1 April 1997. Downloaded from proliferation and differentiation markers. Clausen OPF, et al. The TP53 tumour suppressor gene in colorectal carcinomas. II. Relation to DNA ploidy pattern and clinicopathological variables. BrJ Cancer 1993;67:93- Thanks are due to Drs A DiVinci and E Geido for reviewing the 8. manuscript. I also acknowledge the financial support of the 24 Giaretti W, Pujic N, Rapallo A, Nigro S, Di Vinci A, Geido National Research Council (grant n. 95.00512.PF39) and the E, et al. K-ras-2 G-C and G-T transversions correlate with Italian Association for Cancer Research (grant TE to Dr FP DNA aneuploidy in colorectal adenomas. Gastroenterology Rossini), and Mr Roger Munford for English editing. 1995;108: 1040-7. 25 Giaretti W, Monaco R, Pujic N, Rapallo A, Nigro S, Geido E. Intratumor heterogeneity of K-ras2 mutations 1 Shapiro HM. Practical flow cytometry. New York: Alan R in colo- Liss, 1988. rectal adenocarcinomas. Association with degree of DNA 2 Melamed MR, Lindmo T, Mendelsohn ML. Flow sytometry aneuploidy and S-phase fraction. AmJPathology 1996;149: and sorting. Wiley-Liss, 1990. 237-45. 3 Darzynkiewicz Z, Robinson JP, Crissman HA. Flow 26 Ried T, Knutzen R, Steinbeck R, Blegen H, Schrock E, cytometry. Methods in . Vol. 41-42, 2nd edn. San Heselmeyer K, et al. Comparative genomic hybridization Diego: Academic Press, 1994. reveals a specific pattern of chromosomal gains and losses 4 Caspersson T, Schultz J. Nucleic acid metabolism of the during the genesis of colorectal tumors. Genes Chromosomes chromosomes in relation to gene reproduction. Nature Cancer 1996;15:234-45. 1938;142:294-7. 27 Baisch H, Gohde W, Linden W. Analysis of PCP-data to 5 Coons AH, Kaplan MH. Localization of antigen in tissue determine the fraction of cells in the various phases of the cells. II. Improvements in a method for the detection of cycle. Radiat Environ Biophys 1975;12:31-7. antigen by means of fluorescent antibody. J Exp Med 1950; 28 Dean PN, Jett JH. Mathematical analysis of DNA distribu- 91:1-4. tions derived from flow microfluorometry. J7 Cell Biol 1974; 6 Coulter WH. High speed automatic blood cell counter and 60:523-7. cell size analyzer. Proc Natl Electronics Conf 1956;12:1034- 29 Gratzner H. Monoclonal antibody against 5-bromo-and 40. 5-iododeoxyuridine. A new reagent for detection of DNA 7 Gohde W, Schuman J, Zante J. The use of DAPI in pulse replication. Science 1982;218:474-5. cytophotometry. In: Lutz D, ed. Pulse cytophotometry. 30 Dolbeare FA, Gratzner HG, Pallavicini MG, Gray JW. Flow Ghent: European Press, 1978:229-32. cytometric measurement of total DNA content and incor- 8 Otto F. Dapi staining of fixed cells for high-resolution flow porated bromodeoxyuridine. Proc NatlAcadSci USA 1983; cytometry of nuclear DNA. Cell Biol 1990;33:105-19. 80:5573-7. 9 Shackney SE, Smith CA, Miller BW, Burholt DR, Murtha 31 Dean Dolbeare K, Giles HR, et al. Model for genetic evolution of human PN, F, Gratzner H, Rice G, Gray JW. Cell- solid tumors. Cancer Res 1989;49:3344-54. cycle analysis using a monoclonal antibody to BrdU. Cell 10 Giaretti W. A model of DNA aneuploidization and evolution Tissue Kinet 1984;17:427-36. in colorectal cancer. Lab Invest 1994;71:904-10. 32 Dolbeare F, Beisker W, Pallavicini MG, Vanderlaan M, Gray 11 Vindelov LL, Christensen IJ, Jensen G, Nissen NI. Limits of JW. Cytochemistry for BrdU/DNA analysis: Stoichiometry detection of nuclear DNA abnormalities by flow cytomet- and sensitivity. Cytometry 1985;6:521-30. ric DNA analysis. Results obtained from a set of methods 33 Begg AC, McNally NJ, Shrieve DC, Kaercher H. A method for sample-storage, staining and internal standardization. to measure the duration of DNA synthesis and the poten- Cytometry 1983;3:332-9. tial doubling time from a single sample. Cytometry 1985;6: 12 Vindelov LL, Christensen IJ. A review of techniques and 620-6. results obtained in one laboratory by an integrated system 34 Riccardi A, Danova M, Wilson G, Ucci G, Dormer P, Maz- of methods designed for routine clinical flow cytometric zini G, et al. Cell kinetics in human malignancies studied DNA analysis. Cytometry 1990;11:753-70. with in vivo administration of bromodeoxyuridine and flow 13 Hiddemann W, Schumann J, Andreeff M, Barlogie B, Her- cytometry. Cancer Res 1988;48:6238-45. man CJ, Leif RC, et al. Convention on nomenclature for 35 Wilson GD, McNally NJ, Dische S, Saunders MI, Des DNA cytometry. Cancer Genet Cytogenet 1984;13: 181-3. Rochers C, Lewis AA, et al. Measurement of cell kinetics in http://jcp.bmj.com/ 14 Wheeless LL, Badalament RA, de Vere White RW, Fradet Y, human tumours in vivo using bromodeoxyuridine incorpo- Tribukait B. Consensus review of the clinical utility of ration and flow cytometry. BrJCancer 1988;58:423-31. DNA cytometry in bladder cancer. Cytometry 1993;14: 36 Terry NHA, Peters LJ. The predictive value of tumor-cell 478-81. kinetic parameters in radiotherapy: considerations regard- 15 Hedley DW, Clark GM, Cornelisse CJ, Killander D, Kute T, ing data production and analysis. J7 Clin Oncol 1995;13: Merkel D. Consensus review of the clinical utility of DNA 1833-6. cytometry in carcinoma of the breast. Cytometry 1993;14: 482-5. 37 Corvo R, Giaretti W, Sanguineti G, Geido E, Orecchia R, 16 Bauer KD, Bagwell GB, Giaretti W, Melamed M, Zarbo RJ, Guenzi M, et al. In vivo cell kinetics in head and neck Witzig TE, et al. Consensus review of the clinical utility squamous cell carcinomas predicts local control and helps

of on October 1, 2021 by guest. Protected copyright. DNA flow cytometry in colorectal cancer. Cytometry 1993; guide radiotherapy regimen. Jf Clin Oncol 1995;13: 1843- 14:486-91. 50. 17 Duque RE, Andreeff M, Braylan RC, Diamond LW, Peiper 38 KerrJFR, Wyllie AH, Currie AR. Apoptosis: basic biological SC. Consensus review of the clinical utility of DNA flow phenomenon with wide-ranging implications in tissue cytometry in neoplastic hematopathology. Cytometry 1993; kinetics. BrJ Cancer 1972;26:239-57. 14:492-6. 39 Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz 18 Shankey TV, Kallioniemi OP, Koslowski JM, Lieber ML, MA, Lassota P, et al. Features of apoptotic cells measured Mayall BH, Miller G, et al. Consensus review of the clinical by flow cytometry. Cytometry 1992;13:795-808. utility of DNA content cytometry in prostate cancer. 40 Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Cytometry 1993;14:497-500. Wyllie AH. Analysis and discrimination of necrosis and 19 Hedley DW, Friealander HL, Taylor IW, Rugg CA, apoptosis (programmed cell death) by multiparameter flow Musgrove EA. Method for analysis of cellular DNA cytometry. Biochim BiophysActa 1992;1133:275-85. content of paraffin embedded archival material using flow 41 Ormerod MG, Collins MKL, Rodriguez-Tarduchy G, Rob- cytometry. J Histochem Cytochem 1983;31:1333-5. ertson D. Apoptosis in interleukin-3-dependent haemopoi- 20 Burmer GC, Loeb LA. Mutations in the KRAS2 oncogene etic cells. Jf Immunol Meth 1992;153:57-65. during progressive stages of human colon carcinoma. Proc 42 Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of Natl Acad Sci USA 1989;86:2403-7. programmed cell death in situ via specific labeling of 21 Offerhaus GJ, De Feyter EP, Cornelisse CJ, Tersmette KW, nuclear DNA fragmentation. Jf Cell Biol 1992;119:493- Floyd J, Kern SE, et al. The relationship of DNA 501.