Robert A. Hoffman

Determination of CD4 antigen density on cells: Role of antibody valency, avidity, clones, and conjugation†

The number of R-phycoerythrin (R-PE)-conjugated antibodies bound to a cell can be quantitated on a flow cytometer by using beads with known numbers of attached R-PE molecules (QuantiBRITE PE). Using these reference beads, we have observed that a number of factors affect the accuracy of the quantitation and conclusions about epitope density. These factors include valence of antibody binding, the use of antibody fragments (Fabs) versus intact monoclonal antibodies (mAbs), fixation, the purity of the conjugate (i.e., percentage of 1:1 ratios), dissociation rate, the use of washed versus unwashed preparations, and the location of epitope on target antigen. We used CD4 on T cells as a model to explore these challenges in detail. We conclude that CD4+ T cells bind approximately 49,000 CD4 (Leu 3a) antibody molecules, that this binding is bivalent, and therefore that there are approximately 98,000 CD4 antigen molecules on the surface of these cells.

EVALUATING FLUORESCENCE SENSITIVITY ON FLOW CYTOMETERS : AN OVERVIEW

The current paradigms for assessing fluorescence sensitivity on flow cytometers do not provide an adequate assessment of an instruments ability to detect and measure weak fluorescence on stained particles. The capability to resolve dimly stained populations depends on two factors: the background noise (B), and the efficiency (Q) with which the fluorescence from the fluorochrome molecules are converted to photoelectrons. Any single statistical measure of fluorescence histogram distributions will be unable to uniquely characterize an instrument. Therefore, neither of the routinely used methods (detection threshold and delta channel) measure sensitivity completely and unambiguously. We show the limitations of these methods and propose that instrument sensitivity be characterized in terms of both background noise and detection efficiency in order to determine better the capability to detect and resolve weakly fluorescent particles.

Characterization of Flow Cytometer Instrument Sensitivity

Fluorescence sensitivity, measured in terms of resolution, allows the researcher to more accurately answer the question of how dim a cell can be and still be resolvable from another population. This measure focuses on the width, i.e., the standard deviation, of the population distributions and not on the location, i.e., the mean intensity, of populations on a histogram scale relative to the background. To determine the fluorescence sensitivity in terms of the resolution, both the detection efficiency, Q, and optical background, B, need to be measured. Both factors affect the ability to resolve dimly fluorescent subpopulations, and both factors are required to uniquely characterize the performance of a flow cytometer. Using Q and B, it is possible to determine the minimum fluorescence intensity that is resolvable from the background or from another population. This unit describes a practical and robust approach to measure the critical factors that determine the ability of a flow cytometer to analyze dimly fluorescent particles.

Standardization, Calibration, and Control in Flow Cytometry

Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions are provided for common terms to alert the reader to critical distinctions in meaning. The unit presents extensive background on particle types and cautions and goes on to describe practical aspects of methods to standardize and calibrate instruments, in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity. Finally, suggestions follow for analyzing particles used for calibration.

Pulse Width for Particle Sizing

The widths of optical pulses in flow cytometry contain information about the size of particles. This size information is independent of many of the factors that affect light scatter as a measure of particle size, and any light scatter or fluorescence signal can be used to measure pulse width. For fluorescence signals, the pulse width can be predicted theoretically for many particle shapes, and quantitative size calibration is possible. To be a meaningful independent parameter, the pulse‐width measurement must be independent of the pulse amplitude. This unit provides protocols for determining the signal range over which amplitude independent pulse‐width measurements can be made and methods for calibrating the pulse‐width measurements to particle diameter. Calibration and application examples are provided and briefly discussed. Curr. Protoc. Cytom. 50:1.23.1‐1.23.17.

Overview of Flow Cytometry Instrumentation

This unit describes the technology in general, to give a feel for the interplay between the various parts of a flow cytometer. Topics include cell preparation, detectors, analysis, and sorting. A brief chronology of flow instrumentation development illustrates the long history of the field.

Evaluating flow cytometer performance with weighted quadratic least squares analysis of LED and multi‐level bead data

We developed a fully automated procedure for analyzing data from LED pulses and multilevel bead sets to evaluate backgrounds and photoelectron scales of cytometer fluorescence channels. The method improves on previous formulations by fitting a full quadratic model with appropriate weighting and by providing standard errors and peak residuals as well as the fitted parameters themselves. Here we describe the details of the methods and procedures involved and present a set of illustrations and test cases that demonstrate the consistency and reliability of the results. The automated analysis and fitting procedure is generally quite successful in providing good estimates of the Spe (statistical photoelectron) scales and backgrounds for all the fluorescence channels on instruments with good linearity. The precision of the results obtained from LED data is almost always better than that from multilevel bead data, but the bead procedure is easy to carry out and provides results good enough for most purposes. Including standard errors on the fitted parameters is important for understanding the uncertainty in the values of interest. The weighted residuals give information about how well the data fits the model, and particularly high residuals indicate bad data points. Known photoelectron scales and measurement channel backgrounds make it possible to estimate the precision of measurements at different signal levels and the effects of compensated spectral overlap on measurement quality. Combining this information with measurements of standard samples carrying dyes of biological interest, we can make accurate comparisons of dye sensitivity among different instruments. Our method is freely available through the R/Bioconductor package flowQB.

Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference.

Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet produced results that are of wide clinical interest or are instrument‐independent across all fluorescence channels. This unit details a procedure for quantifying surface and intracellular biomarkers by calibrating the output of a multicolor flow cytometer in units of antibody bound per cell (ABC). The procedure includes (1) quality control of the flow cytometer, (2) fluorescence intensity calibration using hard dyed microspheres assigned with fluorescence intensity values, (3) compensation for fluorescence spillover between adjacent fluorescence channels, and (4) application of a biological reference calibrator to establish an ABC scale. The unit also points out current efforts for quantifying biomarkers in a manner that is independent of instrument platforms and reagent differences.

Flow Cytometry Instrumentation

T he purpose of this chapter is to acquaint readers with the instrumentation utilized in flow cytometry by describing various elements of the technology, and to provide detailed information on specific methods of using the instruments. Introducing the chapter is UNIT 1.1, an overview of the instrumentation involved in flow cytometry. This unit describes the various parts of a flow cytometer, explains the basics of how each component works, and presents a brief history of the development of flow cytometry instrumentation.

UNIT 1.1 Overview of Flow Cytometry Instrumentation

This unit describes the technology in general, to give a feel for the interplay between the various parts of a flow cytometer. Topics include cell preparation, detectors, analysis, and sorting. A brief chronology of flow instrumentation development illustrates the long history of the field.