Flow Cytometry Applications in the Study of Immunological Lung Disorders


The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. Flow cytometric analysis provides a rapid qualitative and quantitative description of multiple characteristics of individual cells. For example, it is possible to detect the cell size and granularity, aspects of DNA and RNA content and the presence of cell surface and nuclear markers which are used to characterize the phenotype of single cells. Flow cytometry has been used for the immunophenotyping of a variety of specimens including whole blood, bone marrow, serous cavity fluids, (cerebrospinal fluid) CSF, urine and all types of body fluids. The technique has also been applied to human bronchoalveolar lavage (BAL) fluid, peritoneal fluids and blood. In this review, we describe the current status of the application of flow cytometry as a diagnostic tool in various lung diseases. We focus on the analysis of BAL cell composition in chronic obstructive lung disease (COPD), asthma, lung cancer, sarcoidosis, tuberculosis and idiopathic eosinophilic pneumonia (IEP).

1. Meyer KC. Bronchoalveolar Lavage as a Diagnostic Tool. Semin Respir Crit Care Med 2007; 28(5):546–60.
2. Costabel U, Guzman J. Bronchoalveolar lavage in interstitial lung disease. Curr Opin Pulm Med 2001;7(5):255-61.
3. Welker L, Jorres RA, Costabel U, Magnussen H.Predictive value of BAL cell differentials in the diagnosis of interstitial lung diseases. Eur Respir J 2004;24(6):1000–6.
4. Drent M, Jacobs JA: Bronchoalveolar lavage. In: Baughman RP, Du Bois RM, Lynch III JP, Wells AU, ed. Diffuse Lung Disease: A Practical Approach, New York: Oxford University Press; 2004:56-64.
5. Barnes PJ. Immunology of asthma and chronic obstructive pulmonary disease. Nat Rev Immunol 2008;8(3):183-92.
6. Fromer L, Cooper CB. A review of the GOLD guidelines for the diagnosis and treatment of patients with COPD. Int J Clin Pract 2008; 62(8):1219-36.
7. Givi ME, Peck MJ, Boon L, Mortaz E. The role of dendritic cells in the pathogenesis of cigarette smoke - induced emphysema in mice. Eur J Pharmacol 2013;721(1-3):259-66.
8. O’Shaughnessy TC, Ansari TW, Barnes NC, Jeffery PK.Inflfmation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med 1997;155(3):852-7.
9. Saetta M, Di Stefano A, Turato G, Facchini FM, Corbino L, Mapp CE, et al. CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157(3 Pt1):822-6.
10. Hansel TT, Braunstein JB, Walker C, Blaser K, Bruijnzeel PL, Virchow JC Jr, et al. Sputum eosinophils from asthmatics express ICAM-1 and HLA-DR. Clin Exp Immunol 1991; 86(2):271–7.
11. in 't Veen JC, Grootendorst DC, Bel EH, Smits HH, Van Der Keur M, Sterk PJ, et al. CD11b and L-selectin expression on eosinophils and neutrophils in blood and induced sputum of patients with asthma compared with normal subjects. Clin Exp Allergy 1998; 28(5):606–15.
12. van Rijt LS, Kuipers H, Vos N, Hijdra D, Hoogsteden HC, Lambrecht BN. A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma. J Immunol Methods 2004; 288(1–2):111–21.
13. van Rijt LS, Prins JB, Leenen PJ, Thielemans K, de Vries VC, Hoogsteden HC, et al. Allergen-induced accumulation of airway dendritic cells is supported by an increase in CD31(hi)Ly-6C(neg) bone marrow precursors in a mouse model of asthma. Blood 2002; 100(10):3663-71.
14. Paul WE, Zhu J. How are T(H)2-type immune responses initiated and amplified. Nat Rev Immunol 2010;10(4):225-35.
15. Karp CL. Guilt by intimate association: what makes an allergen an allergen? J Allergy Clin Immunol 2010;125(5):955- 60.
16. Gill MA. The role of dendritic cells in asthma. J Allergy Clin Immunol 2012; 129(4):889–901.
17. Staples KJ, Hinks TS, Ward JA, Gunn V, Smith C,Djukanović R. Phenotypic characterization of lung macrophages in asthmatic patients: overexpression of CCL17. J Allergy Clin Immunol 2012; 130(6):1404-12.
18. Baughman RP, Teirstein AS, Judson MA, Rossman MD, Yeager H Jr, Bresnitz EA, et al. Clinical characteristics of patients in a case control study of sarcoidosis. Am J Respir Crit Care Med 2001; 164(10 Pt 1):1885-9.
19. Statement on sarcoidosis. Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee, February 1999. Am J Respir Crit Care Med 1999; 160(2):736-55.
20. Baughman RP, Teirstein AS, Judson MA, Rossman MD, Yeager H Jr, Bresnitz EA, et al. Clinical characteristics of patients in a case control study of sarcoidosis. Am J Respir Crit Care Med 2001; 164(10 Pt 1):1885-9.
21. Rossman MD, Dauber JH, Daniele RP. Identification of activated T cells in sarcoidosis. Am Rev Respir Dis 1978;117(4):713–20.
22. Muller-Quernheim J, Pfeifer S, Strausz J, Ferlinz R.Correlation of clinical and immunologic parameters of the inflammatory activity of pulmonary sarcoidosis. Am Rev Respir Dis 1991; 144(6):1322–9.
23. Sarcoidosis. Iannuzzi MC, Rybicki BA, Teirstein AS. N Engl J Med 2007; 357(21):2153-65.
24. Baughman RP, Culver DA, Judson MA. A Concise Review of Pulmonary Sarcoidosis. Am J Respir Critical Care Med 2011; 183(5):573-81.
25. Dauber JH, Wagner M, Brunsvold S, Paradis IL, Ernst LA, Waggoner A. Flow cytometric analysis of lymphocyte phenotypes in bronchoalveolar lavage fluid: comparison of a two color technique with a standard immunoperoxidase assay. Am J Respir Cell Mol Biol 1992; 7(5):531-41.
26. Newman LS, Rose CS, Maire LA. Saroidosis. Engl J Med 1997; 336(17):1224-34.
27. Welker L, Jorres RA, Costabel U, Magnussen H.Predictive value of BAL cell differentials in the diagnosis of interstitial lung diseases. Eur Respir J 2004;24(6):1000-6.
28. Drent M, Mulder PG, Wagenaar SJSC, Hoogsteden HC,Van Velzen-Blad H, Van den Bosch JM. Differences in BAL fluid variables in interstitial lung diseases evaluated by discriminant analysis. Eur Respir J 1993;6(6):803-10.
29. Puliyappadamba VT, Cheriyan VT, Thulasidasan AK,Bava SV, Vinod BS, Prabhu PR, et al. Nicotine‐induced survival signaling in lung cancer cells is dependent on their p53 status while its down‐regulation by curcumin is independent. Mol Cancer 2010; 9:220.
30. Pao W, Chmielecki J. Rational, biologically based treatment of EGFR‐mutant non‐small‐cell lung cancer.Nat Rev Cancer 2010; 10(11):760–74.
31. Molecular biology of the lung cancer. Sasho Z. Panov.Radiol Oncol; 2005; 39(3): 197-210.
32. Türecia O, Mackb U, Luxemburgera U, Heinenb H, Krummenauerc F, Sesterd M, et al. Humoral immune responses of lung cancer patients against tumor antigen NYESO- 1. Cancer Lett 2006; 236(1):64-71.
33. Oji Y, Kitamura Y, Kamino E, Kitano A, Sawabata N, Inoue M, et al. WT1 IgG antibody for early detection of nonsmall cell lung cancer and as its prognostic factor. Int J Cancer 2009; 125(2):381-7.
34. Local CD4+, CD8+ and CD56+ T-lymphocyte Reaction on Primary Lung Cancer. Jusufovic E, Prnjavorac B, Iljazovic E, Kosnik M, Keser D, Korosec P, et al. Acta Inform Med 2011; 19(3):132–7.
35. Ruffini E, Asioli S, Filosso PL, Lyberis P, Bruna MC, Macrì L, et al. Clinical significance of tumor-infiltrating lymphocytes in lung neoplasms. Ann Thorac Surg 2009;87(2):365-71.
36. Haridas V, McCloskey TW, Pahwa R, Pahwa S. Discordant expression of perforin and granzyme A in total and HIV-specific CD8 T lymphocytes of HIV infected children and adolescents. AIDS. 2003;17(16):2313-22.
37. Okada R, Kondo T, Matsuki F, Takata H, Takiguchi M.Phenotypic classification of human CD4+ T cell subsets and their differentiation. Int Immunol 2008; 20(9):1189-99.
38. Wyllie AH, Kerr JF, Currie AR. Cell death: significance of apoptosis. Int Rev Cytol 1980; 68:251–306.
39. Thompson CB. Apoptosis in pathogenesis and genes and treatment of disease. Science 1995; 267(5203):1456–62.
40. Nagata S, Golstein P. The Fas death factor 1995;267(5203):1449–56.
41. Cascino I, Fiucci G, Papoff G, Ruberti G. Three functional soluble forms of the human apoptosis-inducing Fas molecule are produced by alternative splicing. J Immunol 1995; 154(6):1157–64.
42. Cheng J, Zhou T, Liu C, Shapiro JP, Brauer MJ, Kiefer MC, et al. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 1994;263(5154):1759–62.
43. Nambu Y1, Hughes SJ, Rehemtulla A, Hamstra D, Orringer MB, Beer DG. Lack of cell surface Fas/Apo-1 expression in pulmonary adenocarcinoma. J Clin Invest 1998; 101(5):1102–10.
44. Hughes SJ, Nambu Y, Soldes OS, Hamstra D, Rehemtulla A, Iannettoni MD, et al. Fas/APO-1 (CD95) is not translocated to the cell membrane in esophageal adenocarcinoma. Cancer Res 1997; 57(24):5571–8.
45. Cheng J, Zhou T, Liu C, Shapiro JP, Brauer MJ, Kiefer MC, et al. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 1994;263(5154):1759–62.
46. Owen-Schaub LB, Angelo LS, Radinsky R, Ware CF, Gesner TG, Bartos DP. Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis? Cancer Lett 1995; 94(1):1–8.
47. Markowitz N, Hansen NI, Hopewell PC, Glassroth J, Kvale PA, Mangura BT, et al. Incidence of tuberculosis in the United States among HIV-infected persons. The Pulmonary Complication of HIV Infection Study Group. Ann Intern Med 1997; 126(2):123-32.
48. Mogues T, Goodrich ME, Ryan L, LaCourse R, North RJ.The relative importance of T cell subsets in immunity and immunopathology of airborne mycobacterium tuberculosis infection in mice. J Exp Med 2001;193(3):271-80.
49. Orme IM, Roberts AD, Griffin JP, Abrams JS. Cytokine secretion by CD4 T lymphocytes acquired in response to Mycobacterium tuberculosis infection. J Immunol 1993;151(1):518-25.
50. Chackerian AA, Perera TV, Behar SM. Gamma Interferon-producing CD4+ T lymphocytes in the lung correlate with resistance to infection with Mycobacterium tuberculosis. Infect Immun 2001; 69(4):2666-74.
51. Jouanguy E, Altare F, Lamhamedi S, Revy P, Emile JF, Newport M, et al. Interferon-Gamma-receptor deficiency in an infant with fatal bacille Calmette-Gue´rin infection. N Engl J Med 1996; 335(26):1956-61.
52. Douvas GS, Looker DL, Vatter AE, Crowle AJ. Gamma Interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria. Infect Immun 1985; 50(1):1-8.
53. Rook GA, Steele J, Ainsworth M, Champion BR.Activation of macrophages to inhibit proliferation of Mycobacterium tuberculosis: comparison of the effects of recombinant - interferon on human monocytes and murine peritoneal macrophages. Immunology 1986;59(3):333-8.
54. Kuo HP, Yu CT. Alveolar Macrophage Subpopulations in Patients With Active Pulmonary Tuberculosis. Chest 1993; 104(6):1773-8.
55. Asemissen AM, Nagorsen D, Keilholz U, Letsch A, Schmittel A, Thiel E, Scheibenbogen C. Flow cytometric determination of intracellular or secreted INF- for the quantification of antigen reactive T cells. Journal of Immunological Methods; 2001; 251:101-8.
56. Tsiavou A, Degiannis D, Hatziagelaki E, Koniavitou K, Raptis S. Flow cytometric detection of intracellular IL-12 release: in vitro effect of widely used immunosuppressants. Int Immunopharmacol 2002;2(12):1713-20.
57. Albera C, Ghio P, Solidoro P, Mabritto I, Marchetti L, Pozzi E. Activated and memory alveolar T-lymphocytes in idiopathic eosinophilic pneumonia. Eur Respir J 1995;8(8):1281–5.
58. Ebisawa M, Bochner BS, Georas SN, Schleimer RP.
Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration. J Immunol 1992; 149(12):4021–8.
59. Resnick MB, Weller PF. Mechanisms of eosinophil recruitment. Am J Respir Cell Mol Biol 1993; 8(4):349–55.
60. Azuma M, Nakamura Y, Sano T, Okano Y, Sone S.Adhesion molecule expression on eosinophils in idiopathic eosinophilic pneumonia. Eur Respir J 1996;9(12):2494–500.

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Asthma Broncho alveolar lavage (BAL) COPD Flow cytometry Lung disease Sarcoidosis

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Mortaz E, Gudarzi H, Tabarsi P, M Adcock I, Masjedi MR, Jamaati HR, Garssen J, Velayati AA, A Redegeld F. Flow Cytometry Applications in the Study of Immunological Lung Disorders. Iran J Allergy Asthma Immunol. 1;14(1):12-18.