Sigrid J. Stewart

Four color compensation

Four-color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument is used, the fluorochromes evaluated are usually FITC, PE, PE-TR and PE-CY5 (or PerCP). For two-laser excitation APC is generally used in place of PE-TR. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether different batches from the same supplier or conjugates from different suppliers all require unique compensation. This inconsistency results in erroneous data, negates the use of single labeled particles as a method for providing adequate compensation and requires dual and triple labeled cells of known pattern to verify compensation. It is also shown that improper compensation can reduce or eliminate completely the detection of fluorescence emission from PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugation to antibodies is not sufficiently controlled to produce reagents with uniform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by using the same second step tandem reagent to an appropriate first step antibody or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tandem conjugate.

Chapter 4 Multiparameter Analysis of Leukocytes by Flow Cytometry

Publisher Summary Multiparameter analysis is complex and time consuming because populations that are labeled with more than one antibody must be individually identified. Each file must be analyzed separately because current software is not capable of analyzing data automatically in a reliable fashion. This is because the relative positions of populations resolved by multiple antibodies can move relative to one another when the same panel is used with different subjects. Multiple antibodies provide the opportunity to resolve unique cell populations that cannot otherwise be identified. These populations may be most important in understanding normal and disease processes. This chapter discusses the strategies for analyzing this type of data and the pitfalls encountered in multiparameter data acquisition and analysis. The chapter aims to illustrate the procedures that will lead to good flow cytometry data acquisition and analysis and to illustrate problematic areas. The most important rule of all is to recognize when there is a problem. The principles discussed in the chapter can be applied to any type of cell population.

Multiparameter data acquisition and analysis of leukocytes by flow cytometry

Each laboratory has to establish its own experience base and standard operating procedures. The intent of this discussion has been to illustrate the procedures that will lead to good flow cytometry data acquisition and analysis and to illustrate problematic areas. The most important rule of all is to recognize when there is a problem and find the correct solution. It is hoped the information provided herein will be of help in the recognition process.

A Software Method for Color Compensation

When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome. Spectral compensation is the process of removing the undesired overlap of signal. Although very successful for two and three fluorochromes, the general practice of adjusting instrument compensation becomes increasingly inadequate and unforgiving as the number of fluorochromes increases. When data are collected uncompensated, software compensation provides the flexibility of setting correct compensation every time for every sample. Software methods do have problems. The linearization assumptions made by the software algorithms may be more or less in error. Binning effects become more of a problem with increasing numbers of compensated parameters. This explanatory unit also contains protocols that illustrate the process of software compensation utilizing matrix algebra that provides for elements of all possible PMT detection combinations. Although details are limited to four colors, the principles described can be applied to any desired number. When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome.

Multiparameter Data Acquisition and Analysis of Leukocytes

For data acquisition, each supplier provides the software necessary and unique to its instrument. For data analysis, the same software may be used. In addition, several second party vendors provide software often with more capabilities than that provided by the instrument companies. Because of the increase in multiparameter data acquisition, we describe one method for validating instrument performance prior to data acquisition. The fundamentals of data analysis leading to a generic strategy for analysis of any number of parameters are described. This generic approach is designed to simplify the increasing complexity of multiparameter data analysis.

Flow Cytometric Immunophenotyping Using Cluster Analysis And Cluster Editing

Flow cytometry is the best method for classifying leukemias and lymphomas (Braylan, 1989; Braylan, 1993; Deegan, 1989; Drexler, 1987; Duque, 1993; Foon, 1986; Neame, 1986; Stewart, 1989, 1990A, 1990B, Terstappen, 1988; Verwer, 1993; Weber, 1990; Wilman, 1989). In a flow cytometer, a distinct group of cells is identified by its repertoire of cell surface markers or epitopes. An epitope is labeled using a monoclonal antibody with an attached fluorochrome. A flow cytometer is used to detect the monoclonal antibody through its attached fluorochrome, which, in turn, identifies an epitope on a cell. Appropriate combinations of monoclonal antibodies provide a set of probes for labeling all important epitopes. This process enables the identification of cell types in a specimen. For three-color immunophenotyping, the set of three different probes is referred to as a panel. Each panel is designed to label a specific set of cell types. A panel of three probes provides more information about specific groups of cells than do panels of one or two probes. This additional information requires the use of more complex data analysis methods than are now available.

UNIT 4.1 Titering Antibodies

Flow cytometry using fluorochrome‐conjugated antibodies has emerged as a major approach to automated cellular identification. One of the most important issues in immunophenotyping is using the correct amount of antibody. This unit presents techniques for ascertaining the optimal titer for individual, dual, and multiple antibodies used for simultaneous phenotyping, stressing the importance of quality control in making batches of antibody for routine use. Keywords: flow cytometry; monoclonal antibodies; antibody titration; immunophenotyping.