Three-color supplement to the NIAID DAIDS guideline for flow cytometric immunophenotyping.

Since the publication of the ‘‘NIAID DAIDS Guideline for Flow Cytometric Immunophenotyping’’ (1) in 1993, significant scientific and technological advances in the development and production of reagents, instrumentation, and software have permitted widespread access to multicolor flow cytometry in both research and clinical laboratories. As is often the case with complex new technologies, each advance, while opening up exciting new capabilities, can also bring new sources of variability and a commensurate increase in the requirements for quality assurance at all levels. The purpose of this document is to update the 1993 NIAID DAIDS Guideline with new recommendations designed to help standardize the methodology and minimize measurement variability in determining patients’ CD4 and CD8 T-cell counts using 3-color flow cytometry. Issues pertaining to specimen collection and handling, problematic specimens, hematology, and laboratory performance assessment were addressed in the previous guideline.1 In assembling this supplement, factors such as reduced technician time, decreased need for isotype controls, fewer assay tubes, and the potential for diminished cost were considered advantages of 3-color (vs. 2-color) flow cytometry. In contrast, the increased complexity of factors such as spectral compensation, instrument set up, and data collection/analysis were considered disadvantages. In addition, some antibody combinations are not commercially available, and certain third-color fluorochromes may not be optimal with all flow cytometers. The specifications and recommendations contained herein were developed for use in laboratories that support clinical trials and epidemiologic studies done under the auspices of the National Institute of Allergy and Infectious Diseases, Division of AIDS (NIAID DAIDS). Efforts currently underway by the Centers for Disease Control and Prevention and by the National Committee for Clinical Laboratory Standards (NCCLS) will likely provide additional guidance in this rapidly changing arena. 5.1 3-COLOR IMMUNOPHENOTYPING 5.01. 3-Color Monoclonal Antibody Panel