Register for CYTO U's latest webinar: Predicting the Best Resolution & Sensitivity in Panel Development & Reducing Inter-instrument Variability in Flow Cytometry by Steve Perfetto and Jim Wood on April 28!

[Pulliam, Kanika F.]

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Predicting the Best Resolution and Sensitivity in Panel Development and Reducing Inter-Instrument Variability in Flow Cytometry



Presented by

Steve Perfetto

Chief, Flow Cytometry Core Section, Vaccine Research Center (VRC), NIH



Jim Wood

Manager, Flow Cytometry Shared Resource, Wake Forest School of Medicine



April 28, 2015

12:00 pm EST US/Canada
5:00 pm GMT/UK/Portugal

Register: http://cytou.peachnewmedia.com
Cost: FREE

Stephen P. Perfetto received a B.S. degree in Medical Technology from the West Virginia University in 1977 and completed his M.S. degree in 1981 from West Virginia University. He studied and worked in the clinical Blood Bank and clinical immunology laboratories until 1988, when he joined the EPICS Division of Coulter Corporation. In 1990, he was recruited to the Walter Reed Army Institute of Research (WRAIR) and was the manager of the core flow cytometry facility. While at WRAIR, he was involved in large HIV vaccine trials to study the immune system of infected individuals.  In 2000 he joined the NIH in the department of the Vaccine Research Center (VRC) as a staff scientist and manager of the flow core facility. This facility is the world-leader in multicolor flow cytometry and continues to actively develop this technology on a number of different fronts; one focus is on hardware development and reagent and analysis development.

James Wood obtained his BA in Physics from Gettysburg College; however, he also availed himself to a broad range of general and advanced biology, and chemistry courses during his undergraduate years.  He completed both his MS and PhD degrees in Biophysics from The Pennsylvania State University studying the effects on the life cycle of cells after radiation exposure.  In graduate school, he also developed the first microprocessor based two-parameter flow cytometer data acquisition system.  After completing a postgraduate position in the lab of Dr. Leon Wheeless at the University of Rochester Medical Center where he contributed to the development of a 3D slit-scan flow cytometer, he moved to Florida accepting a position with the EPICS Division of Coulter Corporation.  He was manager of the New Products Research and Applications Laboratory during most of his tenure at Coulter and Beckman-Coulter.  He currently consults for flow cytometry and pharmaceutical companies, and manages the flow cytometry shared resource at Wake Forest University School of Medicine Comprehensive Cancer Center (WFUCCC).

Webinar Summary:
Successful quantitative flow cytometry requires an understanding of the characteristics of a flow cytometer instrument that affect the measurement and analysis of the acquired photometric data.  Flow cytometer instruments must be characterized before being used for critical work.  The immediate goals include achieving better reproducibility of data, reducing variation and to facilitate intra/inter-lab comparisons of cytometry data.  Characterization  includes the determination of the instrument linearity, dynamic range, precision, accuracy, detection efficiency (Q), and electronic and optical noise (B).  In particular, Q and B affect how well cellular receptors with low expression can be measured.  For example, staining panels may be inadvertantly designed to avoid measuring dim markers on PMTs with poor resolution.  Unfortunately, current methods using bead sets to assess PMT resolution only provide rough estimates of Q and B.  Thus, staining panel design and similar protocols remain a largely empirical process, requiring time and intimate experience with an instrument's performance. Ascertaining these instrument characteristics are the first steps toward assessing how instruments can be standardized and calibrated. Ultimately, this may be part of the validation process to certify that a flow cytometer used for critical clinical and/or GMP work is performing at the required level needed to obtain accurate and precise results.  Additionally, as part of this process we need to move from the common use of "relative intensity units (channel numbers)" to data measured and presented in physically relavent optical units (photons, photoelectrons, dye molecules, antibody binding sites).

Recently, James Wood (Wake Forest University) developed a new device, known as an LED Pulser, to measure B and Q directly and accurately.  This device delivers consistent, uniform broad-spectrum light pulses, the amplitude of which can be adjusted with an electronic and/or an optical attenuator.  Our presentation will demonstrate how this device is used to calculate Q and B values.  In the past, data from multilevel bead sets have been used to facilitate comparisons of instrument sensitivity with Q and B; however, it has proved difficult to manufacture bead sets that have closely matched intrinsic CVs.  In practice, the LED Pulser, along with calibrated fluorochrome-loaded beads, can used to determine the relationship between fluorescence channel numbers and the number of photoelectrons generated at the PMT photocathode i.e. the statistical photoelectron estimate (Spe) using a weighted quadratic fitting of pulsed LED series data.  This information, along with estimates of receptor density, can be used to calculate index values that provide quantitative guidance for panel design.  Our presentation will describe this process step-by-step, with examples.  Additionally, we will show how the metrics can be used for inter-laboratory comparisons.

The first part of the webinar will present the principles behind the LED Pulser.  The appropriate use of the LED Pulser and how it compares to using multilevel bead sets for the calculation of Q and B.

The second part of the webinar will introduce how the LED Pulser, along with calibrated fluorochrome-loaded beads lead to better estimates of the four facets of predictive panel design (i) accurate Q and B values for each detector, (ii) optimal voltage settings, (iii) spillover/spreading error calculations, and (iv) estimates of receptor density and contribute to more quantitative determinations for optimizing panel design.

CYTO U Webinar Recordings
A recording of this webinar will be posted online at CYTO U within 24-48 hours after the live event for free viewing by all.  You may also want to watch the recordings of CYTO U's last three webinars Forensic Flow Cytometry by Jennifer Wilshire, Standardization of Flow Cytometry Assays: What for? How? Where does it lead us? by Tomáš Kalina, and Growing a Successful and Fruitful Core: You Reap What You Sow by Joanne Lannigan at CYTO U.  For more details, visit: http://cytou.peachnewmedia.com.

CYTO University
CYTO University is an online educational resource created by ISAC for its members and the wider cytometry community. In addition to free webinars, CYTO U presents recorded courses and tutorials from the CYTO Conference, and interactive online courses on a variety of cytometry topics. These are available at no cost to ISAC members and for a nominal charge for non-members. Webinars are free for all. Learn more at http://cytou.peachnewmedia.com

CYTO U Access for ISAC Members
For current ISAC members, your user login and password to CYTO U are the same user login and password you have for the members-only section of the ISAC Web site. If you remember your user log in to the ISAC Web site but cannot remember your password, you can obtain your password from the ISAC Web site by clicking on the link "forgot your password?".  If you cannot remember your username, you can contact [hidden email] for your login information.

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Kanika F. Pulliam, Ph.D.
Education Manager
International Society for Advancement of Cytometry (ISAC)
9650 Rockville Pike
Bethesda, MD 20814
[phone]
[hidden email]<mailto:[hidden email]>