We are sorry, but the
presentation on this USB device
was prepared on an IBM
computer…it cannot be run on
this Dell Computer…
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)
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
One perspective…
How many standards can you afford?
BioIT Magazine
2002
Monkey Business
• Let’s look at the infinite monkey theorem again…
Sssssssss
Ssss
Sss
Sss
Sss
ss
• 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.
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
Fundamentals of Flow Cytometry
PMT 5
4-50 variables/cell
PMT 4
Sample
PMT 3
Flow chamber
Dichroic
Filters
Scatter
Sensor
103 to 105 cells/sec
PMT 2
PMT 1
Laser(s)
Bandpass
Filters
Advanced polychromatic cytometry
From Mario Roederer
ISAC Standards Implementation
1984 – Introduction of FCS 1.0 (Flow Cytometry Standard)
- Murphy & Chused
1989 - FCS 2.0
1997 - FCS 3.0
• Cytometry 28:118-122 (1997)
2008 - FCS 4.0
Proposed major update
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!
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
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
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
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
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
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)
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 :
0 MESF
1.
6851 MESF
2.
23379 MESF
3.
58333 MESF
4.
213369 MESF
Ab site
QSC, Cat. No. 815
Bangs Laboratories, Inc.
www.bangslabs.com
Events
bead
Blank.
34
1 2
Blank
Mean fluorescence intensity (MFI)
MESF=Molecules of equivalent soluble fluorochrome
Noise measurement with a standard
R. M. Zucker and O. Price, Cytometry 43 (2001) 273 - 294
QC- Optical Filters
• Depending on location, filters can be placed
under extreme stress
• Environmental conditions (humidity)
Excitation Efficiency Profiles
Management compensation of fluorescence overlap becomes crucial
(note – there really isn’t a 545 nm line available!!)
Noise measurement in the images II
raw image
Wavelet
Wavelet
transform 1
transform 2
signal
noise
s/n parent
s/n child
scale corr.
s/n filter
wavelet shrinkage
YES
effect ?
NO
diff.
denoised
image (signal)
Light detector stability analysis I
trend component:
random walks (RW)
b t  b t s  t s
It  b t  e t
periodic component:
dynamic harmonic
regression
It   {a jt cos( j t )  b jt sin( j t )}  e t
j
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
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
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
ISAC 21st Century
• Flow and imaging are equally
emphasized in ISAC
• Standards and Calibration
• Biosafety issue
• Core managers support
• Education
• Public Policy
• 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