We are sorry, but the presentation on this USB device was prepared on an IBM computer…it cannot be run on this Dell Comp

1. We are sorry, but the presentation on this USB device was prepared on an IBM computer…it cannot be run on this Dell Computer…

2. ISAC's perspective on data: standards, structure and analysis J. Paul Robinson SVM Professor of Cytomics Purdue University President, International Society for Analytical Cytology (ISAC)

3. The HTS rationale • The infinite monkey theorem defines the HTS rationale. It states that a monkey hitting keys at random on a typewriter keyboard will almost surely eventually type every book in France's Bibliothèque Nationale de France (National Library). • In the restatement of the theorem most popular among English speakers, the monkeys eventually type out the collected works of William Shakespeare. • The original image was presented in Émile Borel's 1913 book "Mécanique Statistique et Irréversibilité”. http://forum.swarthmore.edu/dr.math/problems/bridge8.5.98.html http://www.nutters.org/monkeys.html

4. One perspective… How many standards can you afford? BioIT Magazine 2002

5. Sssssssss Ssss Sss Sss Sss ss Monkey Business • Let’s look at the infinite monkey theorem again… • In 2003, scientists at Paignton Zoo and the University of Plymouth, in Devon in England reported that they had left a computer keyboard in the enclosure of six Sulawesi Crested Macaques for a month • Not only did the monkeys produce nothing but five pages consisting largely of the letter S, they started by attacking the keyboard with a stone, and continued by urinating and defecating on it.

6. Historical Picture • Began with flow cytometry • Invented in 1960s • 1970s started with single fluorescence signal and two laser scatter signals – total 3 variables • 1977 Herzenberg et al 2 color flow compensation • 1980s two fluorescence signals and two scatter signals • 1990s three to 11 fluorescence signals and two scatter signals • 2000s 32 fluorescence signals and 10-15 scatter signals

7. Fundamentals of Flow Cytometry PMT 5 4-50 variables/cell PMT 4 Sample PMT 3 Dichroic Filters Flow chamber PMT 2 PMT 1 Scatter Laser(s) Sensor Bandpass Filters 103 to 105 cells/sec

8. Advanced polychromatic cytometry From Mario Roederer

9. ISAC Standards Implementation 1984 – Introduction of FCS 1.0 (Flow Cytometry Standard) - Murphy & Chused

10. 1989 - FCS 2.0

11. 1997 - FCS 3.0 • Cytometry 28:118-122 (1997) 2008 - FCS 4.0 Proposed major update

12. Main challenges • HCS activities must leverage information sciences – integrating information from genomics, emerging protein interaction networks, and ongoing chemical-genetic studies into a public knowledge base of biological systems • Standards have to be defined and followed!

13. What is the issue? • There are thousands of analytical and diagnostic instrument in clinics and labs • These instruments are manufactured by 10-20 different companies • They use reagents from 1-200 companies for the same tests • Many tests used fluorescence as the reporter system • Imaging systems are not uniform – there are no accepted imaging standards • There are no algorithm evaluation standards • There is no way to take data sets into any single management engine • There are no organized databases that can evaluate the vast amount of research or clinical data collected

14. How Big a Problem is it? • 20,000 flow cytometers - 10 manufacturers • 5-8,000 confocal microscopes - 10 manufacturers • 20-50,000 other fluorescence systems – 20 manufacturers • 5-10,000 DNA microarray readers – 10 manufacturers • 500 Laser Scanning Microscopes – 2 manufacturers • 1000 plus HCS instruments – 20 manufacturers

15. How much data is enough? Flow assay • Standard 7 tube assay • Each tube 7 colors plus 2 scatter parameters • 50,000 cells per tube • 450,000 parameters per tube x 7 =3,150,000 • Run 25 patients/tests per day= 25x7=175 assays= 551,250,000 points per day • If you ran this assay 100 times in a year you would have 55,125,000,000 points

16. How much data is enough? HCS assay • 384 well plate assay • 6 images per well = 2304 per plate • Each image contains 100 cells (450k per image 600x800 pixels) (1,036,800k of image space = 1 Gbyte) • We collect 20 parameters per cell • We have 230,400 x 20 parameters = 4,608,000 • For a 10 plate assay we have 46,080,000 parameters • If we run 2 assays a day 5 days a week for 40 weeks • 46,080,000 x 2 x 5 x 40 = 18,432,000,000 parameters • Total storage space =1 Gbyte x 10 x 2 x 5 x 40 = 4 Tbytes

17. How many images are there… • Industry estimates indicate that 80 billion new images are created every year • 219,178,082 per day • 9,132,420 per hour • 152,207 per minute • 2,536 per second http://a06.cgpublisher.com/proposals/244/index_html

18. Calibration and Standards? • Very few real standards • Local calibration if at all • Standards processes must be created and implemented across several fields • Necessary to identify • Instrument standards • Reagent Standards • Analysis Standards • Data structure standards • Metadata standards • Algorithm identification (at least)

19. QSC Beads (Quantum Simply Cellular) Identical microbeads with various calibrated binding capacities of goat-anti-mouse IgG on their surface: • Antibody binding capacity (ABC) provided • by the manufacturer : • Blank. 0 MESF • 6851 MESF • 23379 MESF • 58333 MESF • 213369 MESF QSC, Cat. No. 815 Bangs Laboratories, Inc. www.bangslabs.com bead Ab site Events 3 4 1 2 Blank Mean fluorescence intensity (MFI) MESF=Molecules of equivalent soluble fluorochrome

20. Noise measurement with a standard R. M. Zuckerand O. Price, Cytometry 43 (2001) 273 - 294

21. QC- Optical Filters • Depending on location, filters can be placed under extreme stress • Environmental conditions (humidity)

22. Excitation Efficiency Profiles Management compensation of fluorescence overlap becomes crucial (note – there really isn’t a 545 nm line available!!)

23. Noise measurement in the images II raw image Wavelet transform 1 Wavelet transform 2 signal noise s/n parent s/n child scale corr. s/n filter wavelet shrinkage YES effect ? NO diff. denoised image (signal)

24. Light detector stability analysis I trend component: random walks (RW) periodic component: dynamic harmonic regression

25. What Standards are available? • Beads for size, intensity, color • No calibration tools available for high resolution optical microscope (Richardson slide no longer manufactured) • 1990 we created the Handbook of Flow Cytometry Methods to exactly define methods • 1997 we created Current Protocols in Cytometry

26. About original data… “It is crucially important to keep your original digital or analog data exactly as they were acquired and to record your instrument settings. This primary rule of good scientific practice will allow you or others to return to your original data to see whether any information was lost by the adjustments made to the images. In fact, some journal reviewers or editors request access to such primary data to ensure accuracy.” J Cell Biol. 166:11-15, 2004

27. Workshop on Standards and Calibration in Cytometry and Biological Imaging Modalities Jointly sponsored by International Society for Analytical Cytology (ISAC) & the Society for Biomolecular Sciences (SBS) Site and date not yet set To highlight the areas of cell analysis that need to be standardized To develop a series of recommendations on: Data file standards Imaging standards Archival/storage standards Compression modalities Algorithms and processing Analytical technologies

28. ISAC 21st Century • Flow and imaging are equally emphasized in ISAC • Standards and Calibration • Biosafety issue • Core managers support • Education • Public Policy

29. www.isac-net.org • www.cyto.purdue.edu • Cytometry web/email discussion • Educational materials, Tutorials, Lectures Some References R.A. Hoffman, Current Protocols in Cytometry, 1997 : 1.3.1-1.3.19 J.C.S. Wood, Current Protocols in Cytometry, 1997 : 1.4.1-1.4.12 Cytometry, Volume 33, Number 2, 1998 R. M. Zucker and O. Price, Cytometry 43 (2001) 273 - 294 Next ISAC Congress May 17-21, 2008, Budapest, Hungary