Smarter Decisions,
Better Care

UpToDate synthesizes the most recent medical information into evidence-based practical recommendations clinicians trust to make the right point of care decisions.

  • Rigorous editorial process: Evidence-based treatment recommendations
  • World-Renowned physician authors: over 5,100 physician authors around the globe
  • Innovative technology: integrates into the workflow; access from EMRs

For more information, click below.


Subscribers log in here


Related Searches

Flow cytometry for the diagnosis of primary immunodeficiencies

INTRODUCTION

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

TECHNICAL ASPECTS

A basic flow cytometer consists of five main components: a flow cell, 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.

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, current flow cytometers typically have four lasers of different wavelengths, with each laser paired to three different filters to allow for up to 12 different analyses at once.

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

  • The intensity of the forward scatter (FSC) of light, which is a reflection of cell size
  • The intensity of the side scatter of light (SSC), which is a reflection of cell granularity
  • The intensity of light emitted by each fluorochrome, which reflects the level of expression of the antigen of interest

                      

Subscribers log in here

To continue reading this article you must have access through your hospital or your group practice, log in to your personal subscription, or purchase a personal subscription. For more information, click below.
Literature review current through: Apr 2013. | This topic last updated: Apr 9, 2012.
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 ©2013 UpToDate, Inc.
References
Top
  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. 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.
  8. 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.
  9. Conley ME. Early defects in B cell development. Curr Opin Allergy Clin Immunol 2002; 2:517.
  10. 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.
  11. Bleesing JJ, Brown MR, Straus SE, et al. Immunophenotypic profiles in families with autoimmune lymphoproliferative syndrome. Blood 2001; 98:2466.
  12. 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.
  13. 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.
  14. 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.
  15. 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.
  16. 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.
  17. 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.
  18. Ochs HD. Mutations of the Wiskott-Aldrich Syndrome Protein affect protein expression and dictate the clinical phenotypes. Immunol Res 2009; 44:84.
  19. 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.
  20. 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.
  21. Park MA, Li JT, Hagan JB, et al. Common variable immunodeficiency: a new look at an old disease. Lancet 2008; 372:489.
  22. Wehr C, Kivioja T, Schmitt C, et al. The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood 2008; 111:77.
  23. Warnatz K, Schlesier M. Flowcytometric phenotyping of common variable immunodeficiency. Cytometry B Clin Cytom 2008; 74:261.
  24. Filipovich AH. Hemophagocytic lymphohistiocytosis and related disorders. Curr Opin Allergy Clin Immunol 2006; 6:410.
  25. 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.
  26. 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.
  27. Verbsky JW, Grossman WJ. Hemophagocytic lymphohistiocytosis: diagnosis, pathophysiology, treatment, and future perspectives. Ann Med 2006; 38:20.
  28. 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.
  29. Fleisher TA, Dorman SE, Anderson JA, et al. Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin Immunol 1999; 90:425.
  30. Uzel G, Frucht DM, Fleisher TA, Holland SM. Detection of intracellular phosphorylated STAT-4 by flow cytometry. Clin Immunol 2001; 100:270.
  31. Casrouge A, Zhang SY, Eidenschenk C, et al. Herpes simplex virus encephalitis in human UNC-93B deficiency. Science 2006; 314:308.
  32. Picard C, Puel A, Bonnet M, et al. Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science 2003; 299:2076.
  33. 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.
  34. Deering RP, Orange JS. Development of a clinical assay to evaluate toll-like receptor function. Clin Vaccine Immunol 2006; 13:68.