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Chapter 18 : Flow Cytometry-Based Immunophenotyping Method and Applications
This chapter provides a broad description of the principles of flow cytometric immunophenotyping, provides specific methods for lymphocyte enumeration with particular emphasis on CD4 T-cell counting, and reviews quality assurance issues that improve the accuracy and reproducibility of overall immunophenotyping results. Samples for immunophenotyping are prepared by incubation with fluorochrome-labeled monoclonal antibodies (MAbs), and red blood cells are removed by lysis to prepare the sample for analysis on the flow cytometer. MAbs are created by the fusion of B-cell tumors and primary B cells previously selected to make antibodies to only one epitope of a specific antigen. The lyse/no-wash (LNW) method permits single-platform (SPT) absolute counting. A second protocol is provided for laboratories that perform more advanced immunophenotyping, using MAbs that must be used in lyse/wash (LW) sample procedures prior to running on the cytometer. The use of a sample handling device which can be loaded with sample tubes inside the biosafety cabinet and attached to the flow cytometer for automated processing reduces sample handling and samples can be safely vortexed throughout sample acquisition. CD4 T-cell levels are important measures for staging human immunodeficiency virus (HIV)-1 disease and predicting disease progression. In HIV-1-infected adults the CD4 T-cell absolute count is the most useful measure of disease progression, while in children CD4 T-cell percentage is preferred because of high variability in lymphocyte counts.