Flow Cytometry: Future & New Developments. BVAC/ABCA Workshop 5-6 May 2011, Mol, B. Dirk Van Bockstaele, Ph.D. Global Director Flow Cytometry

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1 BVAC/ABCA Workshop 5-6 May 2011, Mol, B. Dirk Van Bockstaele, Ph.D. Global Director Flow Cytometry & Cell Analysis Flow Cytometry: Future & New Developments

2 Evolution of Flow Cytometry 70-ies: first commercial flow cytometers Hardware driven (first PC (IBM) 1981!) Thousands of single cells per acquisition! Parameter distributions rather than average values of parameters in bulk measurements. FCS standard to store list mode data Immediately embraced by academic and clinical laboratories focused on hematology / immunology

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4 Research in Biomedicine When I graduated (1974) (pre-pc age) Basic sciences hypothesis driven it seeks to address a specific, measurable, and answerable question A well constructed hypothesis has several characteristics: it is clear, testable, falsifiable Medicine and Pharmacology Screening research brute force approach Normal reference range

5 Evolution of Flow Cytometry 70-ies: first commercial flow cytometers Hardware driven (first PC (IBM) 1981!) Thousands of single cells per acquisition! Parameter distributions rather than average values of parameters in bulk measurements. FCS standard to store list mode data Immediately embraced by academic and clinical laboratories focused on hematology / immunology Since then:

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7 Evolution of Flow Cytometry 70-ies: first commercial flow cytometers Hardware driven (first PC (IBM) 1981!) Thousands of single cells per acquisition! Parameter distributions rather than average values of parameters in bulk measurements. FCS standard to store list mode data Immediately embraced by academic and clinical laboratories focused on hematology / immunology Since then: Steered by software rather than hardware Steady increase in fluorescence parameters: limit is reached? Steady increase in ADC resolution / ADC moment from rear end to front end: real time sampling and digitalisation

8 Evolution of Flow Cytometry 70-ies: first commercial flow cytometers Hardware driven (first PC (IBM) 1981!) Thousands of single cells per acquisition! Parameter distributions rather than average values of parameters in bulk measurements. FCS standard to store list mode data Immediately embraced by academic and clinical laboratories focused on hematology / immunology Since then: Steered by software rather than hardware Steady increase in fluorescence parameters: limit is reached? Steady increase in ADC resolution / ADC moment from rear end to front end: real time sampling and digitalisation Fluorescence based FCM has reached its limits Some limits are not reached yet!

9 Research in Biomedicine When I graduated (1974) (pre-pc age) Basic sciences hypothesis driven it seeks to address a specific, measurable, and answerable question A well constructed hypothesis has several characteristics: it is clear, testable, falsifiable Medicine and Pharmacology Now: 2011 Screening research brute force approach Normal reference range

10 Research in Biomedicine When I graduated (1974) (pre-pc age) Basic sciences hypothesis driven it seeks to address a specific, measurable, and answerable question A well constructed hypothesis has several characteristics: it is clear, testable, falsifiable Medicine and Pharmacology Screening research brute force approach Normal reference range Now: 2011(computers and robots are all around) Merging of Sciences (BioMedicine) Data acquisition is no problem anymore (albeit FCM??) Data mining is the problem now Hypothesis driven research is the poor man s (scientist s) research It becomes criticised It creates self fulfilling prophecies and is biased Experiments are constructed to prove the hypothesis rather than to test it Screening research has become Discovery research The results extend beyond the bounds of the investigator s imagination It delivers new links, connections and thus new hypothesis to test

11 Has fluorescence based FCM reached its limits? Yes: No: the number of fluorescence parameters is at its maximum Limited by fluorescence compensation background broadening May only be expanded by the development of fluorescent probes with no overlapping fluorescence spectra (Qdots,...) High troughput data ACQUISITION High troughput data ANALYSIS Are there alternatives? Yes...

12 FCM: improvements and for whom Pharmacology research: High throughput screens More and more cell-based FCM is underutilized Limitation in handling large number of samples Because of lack of fast sample loading technology # acquisitions per minute!! Because of lack of suitable analysis software Allow for multiplexing (bar code type of detection) Data deconvolution by software Scoring and presenting hits : heat maps representation. Alternatives: very few choices available... IntelliCyt, Nolan s group...

13 Intellicyt

14 HyperCyt sampling process Black and al, Assay and Drug Development Technologies, vol 9: 13-20, 2011

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16 Fluorescent cell barcoding: Nolan et al Many cellular conditions: Not stimulated, stimulated no drug stimulated + drug cellular barcode them Amine-reactive fluorescent dye(s) at different concentrations Throw them together Run as a single acquisition One informative read-out: phosporylation status of a signal transduction molecule

17 96 well plate, 70 compound screen

18 Barcode cells 3 different amine reactive stains at 4, 4 and 6 concentrations resp. 96 combinations

19 Stain for read-out and deconvolution Mix cells from all wells Incubate for your read-out marker eg pstat1 alexa 647 and perk PE Run as one single acquisition Deconvolute different well cell populations by software Color code read-out results: Hits: Y/N % inhibition: heat map

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21 FCM: improvements and for whom Pharmacology research: High throughput screens More and more cell-based FCM is underutilized Limitation in handling large number of samples Because of lack of fast sample loading technology # acquisitions per minute!! Because of lack of suitable analysis software Allow for multiplexing (bar code type of detection) Data deconvolution by software Scoring and presenting hits : heat maps representation. Alternatives: very few choices available... IntelliCyt, Nolan s group...

22 FCM: improvements and for whom Pharmacology research: High throughput screens More and more cell-based FCM is underutilized Limitation in handling large number of samples Because of lack of fast sample loading technology # acquisitions per minute!! Because of lack of suitable analysis software Allow for multiplexing (bar code type of detection) Data deconvolution by software Scoring and presenting hits : heat maps representation. Alternatives: very few choices available... IntelliCyt, Nolans s group Clinical research, clinical laboratory, clinical trials setting: magnificent instrument that delivers all what is needed and all what is feasible If there is a need for more simultaneous measured parameters... Start thinking about alternatives Mass Spectrometry flow cytometry,...

23 Organic chromophores: Limited by spectral overlap, compensation induces background broadening Nanocristals / quantum dots, Alivisatos et al, Science 281: , 1998 Rare earth isotopes: Lanthanides Atomic mass (daltons)

24 Scott Tanner s group, Toronto

25 Ionisation source: ICP (inductively coupled plasma) Cell ion cloud enlarges by diffusion transient signal of 200 µs

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27 Mass separation: TOF-MS (time of flight)

28 Mass separation: TOF-MS (time of flight) Typical values: 200 µs transient signal form single cell generated ion cloud sequential mass spectra recorded at 13 µs intervals Ion signals that cluster for appr. 20 scans correspond to a full individual cell ionization Maximum cell introduction rate 1000 per second

29 Use of Rh or Ir containing DNA-intercalators

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31 Scott Tanner s group: Toronto (L to R): Xudong Lou, Alexei Antonov, Olga Ornatsky, Vladimir Baranov, Scott Tanner, Robert Kinach, Adrienne Halupa, Sergey Vorobiev, and Dmitry Bandura.

32 DVS Sciences, Inc COMPANY OVERVIEW DVS Sciences, Inc. is an analytical equipment and reagents development company that produces and markets globally the CyTOF, a high throughput mass cytometer for individual cell analysis based on a novel elemental massspectrometry detection technology, and the MAXPAR system of novel reagents related to massively multi-parametric biological assays. Both are covered by company patents.

33 Literature Intellicyt group Black et al, Assay and Drug Development Technologies 2011, 9: Garry Nolan s group Krutzik et al, Clinical Immunology 2001, 110: Krutzik et al, Nature Methods 2006, 3: Hammer et al, Assay and Drug Development Technologies 2009, 7: Maecker et al, Current opinion in Endocrinology, diabetes & Obesity 2010, 17: Scott Tanner s group Tanner et al, Spectrochimica Acta Part B 62 (2007) Tanner et al, Pure Appl chem 2008, 80: Ornatsky et al, Journal of Immunological Methods 2010, 361: Quantum dots: Paul Alivisatos s group Bruchez et al, Science 1998, 281: Quantum dots: Shuming Nie s group Chan et al, Science 1998, 281: Chan et al, Current Opinion in Biotechnology 2002, 13: 40-46