Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. EDITORIAL COMMENT Applications of flow cytometry for the study of primary immune deficiencies Joao B. Oliveira a , Luigi D. Notarangelo b and Thomas A. Fleisher a Introduction The application of flow cytometry now plays a central role in the evaluation of patients with suspected primary immune deficiencies (PIDs). The initial use of this tech- nology was for determining changes in lymphocyte popu- lations or subpopulations associated with specific inherited immune disorders. Additional applications have emerged that have proven useful in clarifying the diagnosis of PIDs by focusing on the presence or absence of specific proteins that are associated with immune deficiency states. This now consists of not only evaluating cell surface proteins but also intracellular proteins. The latter approach requires cell fixation and permeabilization and has as its primary limita- tion the availability of appropriate monoclonal reagents directed to the proteins of interest. In addition, this method has the same caveat as any immunoassay in which the absence of protein is generally diagnostic, whereas presence of protein can be associated with the production of a dysfunctional protein associated with disease or indicate nondisease. More recently, flow cytometry has evolved as a tool to evaluate specific aspects of immune cell function and these applications have also proven to be useful in establishing the diagnosis of certain PIDs. Finally, this method is proving to be useful in assessing immunologic recovery and monitoring immune status in immunodeficient patients following therapeutic hemato- poietic stem cell and thymic transplantation. We provide below some examples of the applications of flow cytometry for the study of PID. a Immunology Service, Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland and b Division of Immunology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, USA Correspondence to Thomas A. Fleisher, MD, Immunology Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bldg. 10, Room 2C306, 10 Center Drive, MSC 1508, Bethesda, MD 20892-1508, USA Tel: +1 301 496 5668; e-mail: tfleishe@mail.nih.gov Current Opinion in Allergy and Clinical Immunology 2008, 8:499–509 Purpose of review This review focuses on the current applications of flow cytometry for the diagnosis and evaluation of primary immune deficiencies (PIDs). Recent findings The immunophenotypic evaluation of selected PIDs provides diagnostic clues as well as information useful to classify patients and predict clinical outcome. In addition, the evaluation of intracellular proteins associated with selected PIDs has evolved as a useful diagnostic screening method. Finally, functional flow cytometry can now help to clarify possible sites of genetic defects associated with specific PIDs. Summary The range of PIDs in which flow cytometry has proven to be useful from a clinical and diagnostic purpose has significantly expanded. This now includes not only patients presenting with clinical histories consistent with classical antibody deficiencies and severe combined immune deficiency, but also patients with more limited infectious histories. Included among these are patients with genetic defects associated with Mendelian susceptibility to mycobacterial disease focusing the evaluation on specific surface protein expression and cell function analysis. In addition, flow cytometry appears to provide a useful screening approach to evaluate for possible toll-like receptor- pathway defects. Furthermore, immunophenotyping and intracellular flow cytometry have proven to be valuable discriminators in the evaluation of patients with immune dysregulation syndromes including immune dysregulation, polyendocrinopathy, enteropathy, X-linked, and autoimmune lymphoproliferative syndrome. Finally, flow cytometry has been shown to be useful to screen patients with possible X-linked lymphoproliferative syndrome and familial hemophagocytic lymphohistiocytosis. Keywords flow cytometry, immunophenotype, intracellular protein, primary immune deficiency, protein phosphorylation Curr Opin Allergy Clin Immunol 8:499–509 ß 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins 1528-4050 1528-4050 ß 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI:10.1097/ACI.0b013e328312c790