Molecular- and Flow Cytometry-based Diagnosis of Primary Immunodeficiency Disorders Joao B. Oliveira & Thomas A. Fleisher Published online: 4 August 2010 # US Government 2010 Abstract Primary immunodeficiencies are an expanding group of genetic disorders resulting in recurrent and/or severe infections, autoimmunity, or autoinflammation. The laboratory plays a critical role in the diagnosis of these conditions given their frequently overlapping signs and symptoms. We discuss here advances in flow cytometry and molecular techniques applied to the study of primary immunodeficiencies. Keywords Flow cytometry . Genetics . Primary immunodeficiency . Diagnosis . Molecular Introduction Flow cytometry provides a clinical tool for evaluating the immune system that can detect the absence of a specific cell population or subpopulation, screen for altered expression of a specific extracellular or intracellular protein, assess for biological changes associated with specific immune defects, and evaluate certain functional immune characteristics. Applied in the setting of possible immune deficiency, this technology can in some cases clarify a diagnosis and in other settings help direct additional testing to establish a diagnosis. More recently, molecular methods have emerged as important diagnostic tools in the evaluation of patients with possible immune deficiency. In this review, very basic principles of flow cytometry are outlined together with a discussion of disease-specific applications. This is followed by a brief review of current methods used for mutation analysis as well as a discussion of T-cell receptor excision circle (TREC) quantitation and T-cell repertoire/diversity evaluation, all in the setting of applications for evaluating possible immune deficiency. Flow Cytometry The design specifics of a flow cytometer are beyond the scope of this article, but briefly, the instrument has four major elements: optics, fluidics, electronics, and a computer (with specific software) [1]. The optical system uses monochromatic light sources (typically lasers) that provide the excitation energy. The optical bench collects light derived directly from each cell as it passes through the laser beam(s). Each cell emits nonfluorescent (forward and side scatter) as well as fluorescent signals if one or more fluorochrome conjugated monoclonal antibodies are bound to the cell. The two nonfluorescent parameters collected provide an index of cell size (forward light scatter) and a measure of cell granularity/regularity (side-angle light scatter). The combination of these two parameters allows for a “three-part leukocyte differential,” which in nonma- lignant settings easily distinguishes among lymphocytes, monocytes, and granulocytes in a whole blood sample following red blood cell lysis [2]. The fluorescent signals result from cell surface or intracellular binding of specific monoclonal antibodies conjugated directly to fluorochromes that, following exci- tation by a specific wavelength, emit light of lower energy J. B. Oliveira : T. A. Fleisher (*) Department of Laboratory Medicine, Clinical Center, National Institutes of Health, NIH Building 10, Room 2C306, 10 Center Drive, MSC 1508, Bethesda, MD 20892, USA e-mail: tfleishe@mail.nih.gov J. B. Oliveira e-mail: oliveirajb@cc.nih.gov Curr Allergy Asthma Rep (2010) 10:460–467 DOI 10.1007/s11882-010-0137-8