The Art and Science of Flow Cytometry

1. The Art and Science of Flow Cytometry Life in a Parallel Universe John Daley Director Flow Cytometry Hematologic NeoPlasia Dana-Farber Cancer Institute

2. Science The classification of knowledge gained by observation and experimentation Definitions T R T H TRUTH ART The seeking for an Ideal truth

3. What makes a Good Sort • Purity • Recovery • Integrity

4. What makes a Good Sorter • Reliability • Reproducibility • Responsiveness • Stability

5. The Other Part of the Picture • People Operator: Investigator: Supporters: Places: Environment makes it work

6. Critical Mass • Multi application exposure • Large user base generates support and application development • Equilibrium dynamics coexist between instrument, operators, support, and users • Interaction dynamics between Experiment, cells , instrument, researchers, and operators • Active Focus on priority at hand: Sort in Progress • Not too Big not too small

7. When things go Right • Keep going, don’t look back • If it works: Duplicate, duplicate, duplicate • Be on the lookout for hidden hisses and the sounds of sparks • Stay calm • Keep a mindful eye on the relevant features • Trust your senses! • Be humble

8. When things go not so Right • Stay calm • Think what are the components of the issue • Try the easiest thing first • Remember past experiences • Employ back up system if possible • Start from beginning • Replace the o ring • Reboot the computer

9. The Rituals of Flow • Startup • Shutdown • Keeping control • Sort Set up • Active Sort • Post Sort • Sterilization

10. When the Sort works well • Everybody is happy • Still need to follow cells down the data path • The Proof is in the Pudding • Faith in the System, Operator and Protocol • Creates confidence for the next experiment • Proves Preparation was worth it

11. How we did it :1996-2007 • Open door policy as much as possible • Educate ourselves and users about the science and technology • Segregate analysis and sorting • Maintain as sterile as an environment as possible • Tried to keep equipment as up to date as possible • Constant form is maintained by constant growth • Created a long range Plan • Created a concept of supported self service system • Always had a back up instrument system in place • Always placed experiments on appropriate instruments • Learned from our mistakes in trusting others

12. Time for Expansion • 1999: need for high speed and space reorganization • Room consolidation : infrastructure • Due diligence for instrument best suited for our needs: • Critical mass established: need created a confidence that more powerful equipment would be utilized and self supported

13. Facility Structure • Organize User priority • Create a common information system: web page/ scheduler • Expand tentacles of facility via extended analysis • Creating an internal accounting system that could serve as a check in rectifying discrepancies between vendors and administration

14. Don’t be an Operator Be a Scientist • Educate • Information resource • Never say No (most of the time) • Try not to get taken advantage of • Mutual Respect is key • Above all Maintain a sense of Humor

15. The Good the Bad and the Ugly • When private flow went core • When operators were considered paying employees • When the bottom line of cost recovery creates an atmosphere of greed • Is flow a business or a science?

16. How to make a sort work • It’s in the set up • What's needed: operator decides, let user be part of the design , operator maintain transparency • Always do a final pre check right before liftoff • Transfer a sense of calm to the responsible parties • Be part of the sort