Predicting the best resolution and sensitivity in panel development and reducing inter-instrument variability in Flow Cytometry

Presented by Steve Perfetto and Jim Wood. Moderated by Pratip Chattopadhyay

 Steve Perfetto

Stephen P. 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 and was the manager of the core flow facility. While at WRAIR, he was involved in large HIV vaccine trials and developed functional 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. For several years, this lab has collaborated to develop Quantum Dots for use in immunophenotyping experiments. The advent of these fluorochromes provided an enormous advance in multicolor technology, allowing us to proceed from 12-color to 18-color very quickly. This group is actively working on new data analysis techniques - one major focus is the analysis and presentation of meta-data (for example, summarizing our functional analysis in which they have broken down each single response into hundreds of categories defined by the expression patterns of individual cytokines or other functional measurements). Recently, the Flow Cytometry Core broke the 18 color detection barrier by the discovery of 30 parameter flow cytometry and soon this will be further advanced to the world’s first 40 parameter instrument. Another focus is on particular aspects of immune function or viral dynamics, within the context of the major research efforts. (i) Detailed assays to an in vivo system to explore the role of CTL in lymphocyte dynamics during viral replication. (ii) Identification of viral reservoirs in CD4 T cell subsets. Sequence analysis of viruses isolated from specific CD4 T cell subsets give us an understanding of the spread of virus through the CD4 compartment, and the contribution of different CD4 subsets to both active viral production and latent reservoirs. (iii) Analysis and sorting of HIV Ag specific B cells to study neutralizing antibodies and to correlate the immunophenotyping of these cells using index sorting.

James Wood


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). 

At the WFUCCC, he serves as a resource to the researchers and students.  He helps to design experiments and give advice on data interpretation.  He has worked to make flow cytometry an easily accessible technology for the Cancer Center users.  His knowledge of the technology as well as the associated biology of the applications enables him to guide the researchers and students in the successful application of the technology by explaining how to best probe the biological application with the flow cytometry technology.  He has a particular interest in the application of flow cytometry to the study of cell cycle kinetics and pursues this as time permits.  He enjoys sharing his passion for the flow cytometry technology and encouraging others to use it to its fullest extent.

He is a long time member and contributor to International Society for the Advancement of Cytometry (ISAC) and, in the past, has served as chairperson and continues to be a member of the ISAC Data File Standards Task Force.  He has engaged in the research and development of the optical, fluidic, digital and analog electronics, and mechanical components of flow cytometers and cell sorters.  He has developed mathematical models to predict instrument performance, and has used the models to identify the critical features of a flow cytometer that limit the instrumentation’s performance. He has participated in the development of data analysis software for flow cytometry data and has worked on the development of mathematical models for cell life cycle analysis.  Currently, his interests have centered on (i) how to improve the instrument’s sensitivity and resolution of dim populations, (ii) optimizing the instrument’s ability to detect emissions from multiple fluorescent dyes, (iii) standardization of instrumentation setup and (iv) methods of data presentation and analysis. For these and other applications, he has been called upon as an expert resource to speak at users’ meetings and international courses.  He is actively developing standards for characterizing flow cytometer performance and is currently developing an LED based technology to optimize and standardize flow cytometry instrument setup.


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.


This continuing medical laboratory education activity is recognized by the American Society for Clinical Pathology for 1 CMLE credit.  ASCP CMLE credits are acceptable for the ASCP Board of Registry Certification Maintenance Program.

Seminar Information
Date Presented:
April 28, 2015 12:00 PM Eastern
1 hour
Predicting the best resolution and sensitivity in panel development and reducing inter-instrument variability in Flow Cytometry
Speaker Information
Steve Perfetto   [ view bio ]
Jim Wood
Individual topic purchase: Selected
American Society for Clinical Pathology
CMLE: 1.00
This continuing medical laboratory education activity is recognized by the American Society for Clinical Pathology for 1 CMLE credit. ASCP CMLE credits are acceptable for the ASCP Board of Registry Certification Maintenance Program.
ISAC Member Price: $0.00
Non-Member Price:$0.00