Official reprint from UpToDate®
www.uptodate.com ©2017 UpToDate, Inc. and/or its affiliates. All Rights Reserved.

Flow cytometry for the diagnosis of primary immunodeficiencies

James Verbsky, MD, PhD
John M Routes, MD
Section Editor
Luigi D Notarangelo, MD
Deputy Editor
Elizabeth TePas, MD, MS


Flow cytometry is a powerful technique for the measurement of multiple characteristics of individual cells within heterogeneous populations. This topic review gives an overview of the technical aspects of flow cytometry and highlights some of its uses in the diagnosis of primary immunodeficiencies (PIDs). Each of these immunodeficiencies is discussed in greater detail separately in specific topic reviews.


A basic flow cytometer consists of five main components: a flow cell (through which cells flow), a laser, optical components, detectors to amplify signals, and an electronics/computer system. With these five components, the flow cytometer is capable of performing instantaneous measurements by passing thousands of cells per second through a laser beam and capturing the emerging light from each cell as it passes through the interrogation point. Any suspended cell or particle ranging from 0.2 to 150 micrometers in size is suitable for analysis. Analyses to determine cellular characteristics, such as size, granularity, viability, and immunophenotyping, are the most common types of studies done.

Theoretically, any biologic sample can be analyzed by flow cytometry, although peripheral blood is the most common sample analyzed. Depending upon the specific assay, whole blood may be used, or peripheral blood mononuclear cells (PBMCs) may be isolated and used for analysis. Bone marrow samples are used during the workup of suspected leukemia. Other biologic sources have been used for flow cytometry, such as cerebral spinal fluid or bronchoalveolar lavage, although the lack of published normals from these biologic sites can make interpretation difficult.

Immunophenotyping — Immunophenotyping is a technique used to characterize the makeup of cell populations by detecting cellular protein expression. Immunophenotyping uses an antibody specific for the antigen of interest that is conjugated to a fluorescent compound known as a fluorophore or fluorochrome (figure 1). These fluorescent compounds absorb energy from the laser source, causing an electron to be raised to a higher energy level. The excited electron quickly returns to its ground state, emitting the excess energy as a photon of light of a characteristic wavelength that is detected by the flow cytometer. Different fluorochromes are excited by different wavelengths of light and emit light at different wavelengths. Thus, it is possible to simultaneously detect several different antigens on a cell by using lasers of different wavelengths and filters of specific wavelengths to detect the fluorescent emission. As an example, flow cytometers currently in use may have as many as five lasers of different wavelengths. Each laser may be paired with three to four different filters, allowing for an analysis of up to 20 different proteins simultaneously.

Data collection and analysis — Each cell is analyzed by the following parameters as the cell suspension passes through the flow cytometer:

To continue reading this article, you must log in with your personal, hospital, or group practice subscription. For more information on subscription options, click below on the option that best describes you:

Subscribers log in here

Literature review current through: Nov 2017. | This topic last updated: Feb 22, 2017.
The content on the UpToDate website is not intended nor recommended as a substitute for medical advice, diagnosis, or treatment. Always seek the advice of your own physician or other qualified health care professional regarding any medical questions or conditions. The use of this website is governed by the UpToDate Terms of Use ©2017 UpToDate, Inc.
  1. International Union of Immunological Societies Expert Committee on Primary Immunodeficiencies, Notarangelo LD, Fischer A, et al. Primary immunodeficiencies: 2009 update. J Allergy Clin Immunol 2009; 124:1161.
  2. Eberle P, Berger C, Junge S, et al. Persistent low thymic activity and non-cardiac mortality in children with chromosome 22q11.2 microdeletion and partial DiGeorge syndrome. Clin Exp Immunol 2009; 155:189.
  3. Chatila TA. Role of regulatory T cells in human diseases. J Allergy Clin Immunol 2005; 116:949.
  4. Liu W, Putnam AL, Xu-Yu Z, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 2006; 203:1701.
  5. Caudy AA, Reddy ST, Chatila T, et al. CD25 deficiency causes an immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome, and defective IL-10 expression from CD4 lymphocytes. J Allergy Clin Immunol 2007; 119:482.
  6. Cohen AC, Nadeau KC, Tu W, et al. Cutting edge: Decreased accumulation and regulatory function of CD4+ CD25(high) T cells in human STAT5b deficiency. J Immunol 2006; 177:2770.
  7. Breitfeld D, Ohl L, Kremmer E, et al. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J Exp Med 2000; 192:1545.
  8. Chevalier N, Jarrossay D, Ho E, et al. CXCR5 expressing human central memory CD4 T cells and their relevance for humoral immune responses. J Immunol 2011; 186:5556.
  9. Linterman MA, Rigby RJ, Wong RK, et al. Follicular helper T cells are required for systemic autoimmunity. J Exp Med 2009; 206:561.
  10. Mazerolles F, Picard C, Kracker S, et al. Blood CD4+CD45RO+CXCR5+ T cells are decreased but partially functional in signal transducer and activator of transcription 3 deficiency. J Allergy Clin Immunol 2013; 131:1146.
  11. Kanegane H, Futatani T, Wang Y, et al. Clinical and mutational characteristics of X-linked agammaglobulinemia and its carrier identified by flow cytometric assessment combined with genetic analysis. J Allergy Clin Immunol 2001; 108:1012.
  12. Futatani T, Miyawaki T, Tsukada S, et al. Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection. Blood 1998; 91:595.
  13. Conley ME. Early defects in B cell development. Curr Opin Allergy Clin Immunol 2002; 2:517.
  14. Bernard F, Picard C, Cormier-Daire V, et al. A novel developmental and immunodeficiency syndrome associated with intrauterine growth retardation and a lack of natural killer cells. Pediatrics 2004; 113:136.
  15. Bigley V, Haniffa M, Doulatov S, et al. The human syndrome of dendritic cell, monocyte, B and NK lymphoid deficiency. J Exp Med 2011; 208:227.
  16. Grier JT, Forbes LR, Monaco-Shawver L, et al. Human immunodeficiency-causing mutation defines CD16 in spontaneous NK cell cytotoxicity. J Clin Invest 2012; 122:3769.
  17. Jawahar S, Moody C, Chan M, et al. Natural Killer (NK) cell deficiency associated with an epitope-deficient Fc receptor type IIIA (CD16-II). Clin Exp Immunol 1996; 103:408.
  18. Bonilla FA, Barlan I, Chapel H, et al. International Consensus Document (ICON): Common Variable Immunodeficiency Disorders. J Allergy Clin Immunol Pract 2016; 4:38.
  19. Park MA, Li JT, Hagan JB, et al. Common variable immunodeficiency: a new look at an old disease. Lancet 2008; 372:489.
  20. Wehr C, Kivioja T, Schmitt C, et al. The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood 2008; 111:77.
  21. Warnatz K, Schlesier M. Flowcytometric phenotyping of common variable immunodeficiency. Cytometry B Clin Cytom 2008; 74:261.
  22. O'Gorman MR, Zaas D, Paniagua M, et al. Development of a rapid whole blood flow cytometry procedure for the diagnosis of X-linked hyper-IgM syndrome patients and carriers. Clin Immunol Immunopathol 1997; 85:172.
  23. Gallagher J, Adams J, Hintermeyer M, et al. X-linked Hyper IgM Syndrome Presenting as Pulmonary Alveolar Proteinosis. J Clin Immunol 2016; 36:564.
  24. Bleesing JJ, Brown MR, Straus SE, et al. Immunophenotypic profiles in families with autoimmune lymphoproliferative syndrome. Blood 2001; 98:2466.
  25. Teachey DT, Manno CS, Axsom KM, et al. Unmasking Evans syndrome: T-cell phenotype and apoptotic response reveal autoimmune lymphoproliferative syndrome (ALPS). Blood 2005; 105:2443.
  26. Ma CS, Chew GY, Simpson N, et al. Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3. J Exp Med 2008; 205:1551.
  27. Milner JD, Brenchley JM, Laurence A, et al. Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome. Nature 2008; 452:773.
  28. Tabata Y, Villanueva J, Lee SM, et al. Rapid detection of intracellular SH2D1A protein in cytotoxic lymphocytes from patients with X-linked lymphoproliferative disease and their family members. Blood 2005; 105:3066.
  29. Marsh RA, Villanueva J, Zhang K, et al. A rapid flow cytometric screening test for X-linked lymphoproliferative disease due to XIAP deficiency. Cytometry B Clin Cytom 2009; 76:334.
  30. Marsh RA, Bleesing JJ, Filipovich AH. Using flow cytometry to screen patients for X-linked lymphoproliferative disease due to SAP deficiency and XIAP deficiency. J Immunol Methods 2010; 362:1.
  31. Ochs HD. Mutations of the Wiskott-Aldrich Syndrome Protein affect protein expression and dictate the clinical phenotypes. Immunol Res 2009; 44:84.
  32. Kawai S, Minegishi M, Ohashi Y, et al. Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations. J Immunol Methods 2002; 260:195.
  33. Yamada M, Ariga T, Kawamura N, et al. Determination of carrier status for the Wiskott-Aldrich syndrome by flow cytometric analysis of Wiskott-Aldrich syndrome protein expression in peripheral blood mononuclear cells. J Immunol 2000; 165:1119.
  34. Filipovich AH. Hemophagocytic lymphohistiocytosis and related disorders. Curr Opin Allergy Clin Immunol 2006; 6:410.
  35. Weren A, Bonnekoh B, Schraven B, et al. A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes. J Immunol Methods 2004; 289:17.
  36. Godoy-Ramirez K, Franck K, Gaines H. A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry. J Immunol Methods 2000; 239:35.
  37. Verbsky JW, Grossman WJ. Hemophagocytic lymphohistiocytosis: diagnosis, pathophysiology, treatment, and future perspectives. Ann Med 2006; 38:20.
  38. Accetta D, Syverson G, Bonacci B, et al. Human phagocyte defect caused by a Rac2 mutation detected by means of neonatal screening for T-cell lymphopenia. J Allergy Clin Immunol 2011; 127:535.
  39. Roesler J, Hecht M, Freihorst J, et al. Diagnosis of chronic granulomatous disease and of its mode of inheritance by dihydrorhodamine 123 and flow microcytofluorometry. Eur J Pediatr 1991; 150:161.
  40. Fleisher TA, Dorman SE, Anderson JA, et al. Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin Immunol 1999; 90:425.
  41. Uzel G, Frucht DM, Fleisher TA, Holland SM. Detection of intracellular phosphorylated STAT-4 by flow cytometry. Clin Immunol 2001; 100:270.
  42. Casrouge A, Zhang SY, Eidenschenk C, et al. Herpes simplex virus encephalitis in human UNC-93B deficiency. Science 2006; 314:308.
  43. Picard C, Puel A, Bonnet M, et al. Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 2003; 299:2076.
  44. Hirschfeld AF, Bettinger JA, Victor RE, et al. Prevalence of Toll-like receptor signalling defects in apparently healthy children who developed invasive pneumococcal infection. Clin Immunol 2007; 122:271.
  45. Deering RP, Orange JS. Development of a clinical assay to evaluate toll-like receptor function. Clin Vaccine Immunol 2006; 13:68.
  46. Depner M, Fuchs S, Raabe J, et al. The Extended Clinical Phenotype of 26 Patients with Chronic Mucocutaneous Candidiasis due to Gain-of-Function Mutations in STAT1. J Clin Immunol 2016; 36:73.
  47. Milner JD, Vogel TP, Forbes L, et al. Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations. Blood 2015; 125:591.
  48. Renner ED, Rylaarsdam S, Anover-Sombke S, et al. Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome. J Allergy Clin Immunol 2008; 122:181.
Topic Outline