Abstract
Multiparametric flow cytometry is a powerful diagnostic tool that permits rapid assessment of cellular antigen expression to quickly provide immunophenotypic information suitable for disease classification. This chapter describes a general approach for the identification of abnormal lymphoid populations by flow cytometry, including B, T, NK, and Hodgkin lymphoma cells suitable for the clinical and research environment. Knowledge of the common patterns of antigen expression of normal lymphoid cells is critical to permit identification of abnormal populations at disease presentation and for minimal residual disease assessment. We highlight an overview of procedures for processing and immunophenotyping non-Hodgkin B- and T-cell lymphomas and also describe our strategy for the sensitive and specific diagnosis of classical Hodgkin lymphoma and nodular lymphocyte predominant Hodgkin lymphoma.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jaffe ES, Campo E, Harris NL, Pileri SA, Stein H, Swerdlow SH (2017) Introduction and overview of the classification of lymphoid neoplasms. In: Swerdlow SH, Campos E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J (eds) WHO classification of tumours of haematopoietic and lymphoid tissues. World Health Organization classification of tumors. IARC Press, Lyon, France, pp 190–198
Craig FE, Foon KA (2008) Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111(8):3941–3967. https://doi.org/10.1182/blood-2007-11-120535
Wood BL, Borowitz MJ (2017) The flow cytometric evaluation of hematopoietic neoplasia. In: RA MP, Pincus MR (eds) Henry’s clinical diagnosis and management by laboratory methods. Elsevier, St. Louis, Missouri, pp 659–679
Givan AL (2011) Flow cytometry: an introduction. Methods Mol Biol 699:1–29. https://doi.org/10.1007/978-1-61737-950-5_1
Redelman D (2000) Flow cytometric analyses of cell phenotypes. In: Stewart CC, Nicholson, J.K.A. (ed) Immunophenotyping. Wiley-Liss, New York,
Keeney M, Hedley BD, Chin-Yee IH (2017) Flow cytometry-recognizing unusual populations in leukemia and lymphoma diagnosis. Int J Lab Hematol 39 Suppl 1:86–92. https://doi.org/10.1111/ijlh.12666
Gorczyca W, Weisberger J, Liu Z, Tsang P, Hossein M, Wu CD, Dong H, Wong JY, Tugulea S, Dee S, Melamed MR, Darzynkiewicz Z (2002) An approach to diagnosis of T-cell lymphoproliferative disorders by flow cytometry. Cytometry 50(3):177–190. https://doi.org/10.1002/cyto.10003
Jamal S, Picker LJ, Aquino DB, McKenna RW, Dawson DB, Kroft SH (2001) Immunophenotypic analysis of peripheral T-cell neoplasms. A multiparameter flow cytometric approach. Am J Clin Pathol 116(4):512–526. https://doi.org/10.1309/QF6N-VAQW-N74H-4JE2
Beck RC, Stahl S, O’Keefe CL, Maciejewski JP, Theil KS, Hsi ED (2003) Detection of mature T-cell leukemias by flow cytometry using anti-T-cell receptor V beta antibodies. Am J Clin Pathol 120(5):785–794. https://doi.org/10.1309/835B-04QX-GNNF-NRJU
Langerak AW, van Den Beemd R, Wolvers-Tettero IL, Boor PP, van Lochem EG, Hooijkaas H, van Dongen JJ (2001) Molecular and flow cytometric analysis of the Vbeta repertoire for clonality assessment in mature TCRalphabeta T-cell proliferations. Blood 98(1):165–173
Morice WG, Kimlinger T, Katzmann JA, Lust JA, Heimgartner PJ, Halling KC, Hanson CA (2004) Flow cytometric assessment of TCR-Vbeta expression in the evaluation of peripheral blood involvement by T-cell lymphoproliferative disorders: a comparison with conventional T-cell immunophenotyping and molecular genetic techniques. Am J Clin Pathol 121(3):373–383. https://doi.org/10.1309/3A32-DTVM-H640-M2QA
Tembhare P, Yuan CM, Xi L, Morris JC, Liewehr D, Venzon D, Janik JE, Raffeld M, Stetler-Stevenson M (2011) Flow cytometric immunophenotypic assessment of T-cell clonality by Vbeta repertoire analysis: detection of T-cell clonality at diagnosis and monitoring of minimal residual disease following therapy. Am J Clin Pathol 135(6):890–900. https://doi.org/10.1309/AJCPV2D1DDSGJDBW
Gibson JF, Huang J, Liu KJ, Carlson KR, Foss F, Choi J, Edelson R, Hussong JW, Mohl R, Hill S, Girardi M (2016) Cutaneous T-cell lymphoma (CTCL): current practices in blood assessment and the utility of T-cell receptor (TCR)-Vbeta chain restriction. J Am Acad Dermatol 74(5):870–877. https://doi.org/10.1016/j.jaad.2015.12.018
Wu D, Anderson MM, Othus M, Wood BL (2016) Clinical experience with modified, single-tube T-cell receptor Vbeta flow cytometry analysis for T-cell clonality. Am J Clin Pathol 145(4):467–485. https://doi.org/10.1093/ajcp/aqw015
Karube K, Aoki R, Nomura Y, Yamamoto K, Shimizu K, Yoshida S, Komatani H, Sugita Y, Ohshima K (2008) Usefulness of flow cytometry for differential diagnosis of precursor and peripheral T-cell and NK-cell lymphomas: analysis of 490 cases. Pathol Int 58(2):89–97. https://doi.org/10.1111/j.1440-1827.2007.02195.x
de Mel S, Li JB, Abid MB, Tang T, Tay HM, Ting WC, Poon LM, Chung TH, Mow B, Tso A, Ong KH, Chng WJ, Liu TC (2018) The utility of flow cytometry in differentiating NK/T cell lymphoma from indolent and reactive NK cell proliferations. Cytometry B Clin Cytom 94(1):159–168. https://doi.org/10.1002/cyto.b.21529
Chan WC (2001) The Reed-Sternberg cell in classical Hodgkin’s disease. Hematol Oncol 19(1):1–17
Stein H, Hummel M (1999) Cellular origin and clonality of classic Hodgkin’s lymphoma: immunophenotypic and molecular studies. Semin Hematol 36(3):233–241
Marafioti T, Hummel M, Foss HD, Laumen H, Korbjuhn P, Anagnostopoulos I, Lammert H, Demel G, Theil J, Wirth T, Stein H (2000) Hodgkin and Reed-Sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription. Blood 95(4):1443–1450
Stein H (1999) Diagnosis of Hodgkin’s disease, Hodgkin’s like anaplastic large cell lymphoma, and T cell/histiocyte-rich B cell lymphoma. In: Mason DY, Harris NL (eds) Human lymphoma: clinical implications of the REAL classification, vol 52. Springer, London, pp 0–4
Stein H, Pileri SA, Weiss LM, Poppema S, Gascoyne RD, Jaffe ES (2017) Hodgkin lymphomas: introduction. In: Swerdlow SH, Campos E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J (eds) WHO classification of tumours of haematopoietic and lymphoid tissues. World Health Organization classification of tumors. IARC Press, Lyon, France, pp 424–430
Dorreen MS, Habeshaw JA, Stansfeld AG, Wrigley PF, Lister TA (1984) Characteristics of Sternberg-Reed, and related cells in Hodgkin’s disease: an immunohistological study. Br J Cancer 49(4):465–476
Kadin ME, Newcom SR, Gold SB, Stites DP (1974) Letter: origin of Hodgkin’s cell. Lancet 2(7873):167–168
Payne SV, Jones DB, Wright DH (1977) Reed-Sternberg-cell/lymphocyte interaction. Lancet 2(8041):768–769
Payne SV, Newell DG, Jones DB, Wright DH (1980) The Reed-Sternberg cell/lymphocyte interaction: ultrastructure and characteristics of binding. Am J Pathol 100(1):7–24
Sanders ME, Makgoba MW, Sussman EH, Luce GE, Cossman J, Shaw S (1988) Molecular pathways of adhesion in spontaneous rosetting of T-lymphocytes to the Hodgkin’s cell line L428. Cancer Res 48(1):37–40
Stuart AE, Williams AR, Habeshaw JA (1977) Rosetting and other reactions of the Reed-Sternberg cell. J Pathol 122(2):81–90
Fromm JR, Kussick SJ, Wood BL (2006) Identification and purification of classical Hodgkin cells from lymph nodes by flow cytometry and flow cytometric cell sorting. Am J Clin Pathol 126(5):764–780. https://doi.org/10.1309/7371-XK6F-6P74-74XX
Harris NL (1999) Hodgkin’s disease: classification and differential diagnosis. Mod Pathol 12(2):159–175
Kuppers R, Engert A, Hansmann ML (2012) Hodgkin lymphoma. J Clin Invest 122(10):3439–3447. https://doi.org/10.1172/JCI61245
Mathas S, Hartmann S, Kuppers R (2016) Hodgkin lymphoma: pathology and biology. Semin Hematol 53(3):139–147. https://doi.org/10.1053/j.seminhematol.2016.05.007
Fromm JR, Thomas A, Wood BL (2009) Flow cytometry can diagnose classical Hodgkin lymphoma in lymph nodes with high sensitivity and specificity. Am J Clin Pathol 131(3):322–332. https://doi.org/10.1309/AJCPW3UN9DYLDSPB
Fromm JR, Wood BL (2012) Strategies for immunophenotyping and purifying classical Hodgkin lymphoma cells from lymph nodes by flow cytometry and flow cytometric cell sorting. Methods 57(3):368–375. https://doi.org/10.1016/j.ymeth.2012.03.028
Fromm JR, Wood BL (2014) A six-color flow cytometry assay for immunophenotyping classical Hodgkin lymphoma in lymph nodes. Am J Clin Pathol 141(3):388–396. https://doi.org/10.1309/AJCP0Q1SVOXBHMAM
Cherian S, Fromm JR (2018) Evaluation of primary mediastinal large B cell lymphoma by flow cytometry. Cytometry B Clin Cytom 94(3):459–467. https://doi.org/10.1002/cyto.b.21544
Schmitz R, Stanelle J, Hansmann ML, Kuppers R (2009) Pathogenesis of classical and lymphocyte-predominant Hodgkin lymphoma. Annu Rev Pathol 4:151–174. https://doi.org/10.1146/annurev.pathol.4.110807.092209
Stein H, Swerdlow SH, Gascoyne RD, Poppema S, Jaffe ES, Pileri SA (2017) Nodular lymphocyte predominant Hodgkin lymphoma. In: Swerdlow SH, Campos E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J (eds) WHO classification of tumours of haematopoietic and lymphoid tissues. IARC Press, Lyon, France, pp 431–434
Uherova P, Valdez R, Ross CW, Schnitzer B, Finn WG (2003) Nodular lymphocyte predominant Hodgkin lymphoma. An immunophenotypic reappraisal based on a single-institution experience. Am J Clin Pathol 119(2):192–198. https://doi.org/10.1309/38RK-238F-CDCH-5R22
Harris NL (1999) Hodgkin’s lymphomas: classification, diagnosis, and grading. Semin Hematol 36(3):220–232
Fromm JR, Thomas A, Wood BL (2017) Characterization and purification of neoplastic cells of nodular lymphocyte predominant Hodgkin lymphoma from lymph nodes by flow cytometry and flow cytometric cell sorting. Am J Pathol 187(2):304–317. https://doi.org/10.1016/j.ajpath.2016.10.007
Carbone A, Gloghini A, Pinto A (1996) CD40: a sensitive marker of Reed-Sternberg cells. Blood 87(11):4918–4919
Carbone A, Gloghini A, Gattei V, Aldinucci D, Degan M, De Paoli P, Zagonel V, Pinto A (1995) Expression of functional CD40 antigen on Reed-Sternberg cells and Hodgkin’s disease cell lines. Blood 85(3):780–789
Schmid C, Sargent C, Isaacson PG (1991) L and H cells of nodular lymphocyte predominant Hodgkin’s disease show immunoglobulin light-chain restriction. Am J Pathol 139(6):1281–1289
Stoler MH, Nichols GE, Symbula M, Weiss LM (1995) Lymphocyte predominance Hodgkin’s disease. Evidence for a kappa light chain-restricted monotypic B-cell neoplasm. Am J Pathol 146(4):812–818
Torlakovic E, Torlakovic G (2002) B-cell markers in lymphocyte predominance Hodgkin disease. Arch Pathol Lab Med 126(7):862–863. https://doi.org/10.1043/0003-9985(2002)126<0862:BCMILP>2.0.CO;2
Gruss HJ, Kadin ME (1996) Pathophysiology of Hodgkin’s disease: functional and molecular aspects. Baillieres Clin Haematol 9(3):417–446
Lim SH, Vaughan AT, Ashton-Key M, Williams EL, Dixon SV, Chan HT, Beers SA, French RR, Cox KL, Davies AJ, Potter KN, Mockridge CI, Oscier DG, Johnson PW, Cragg MS, Glennie MJ (2011) Fc gamma receptor IIb on target B cells promotes rituximab internalization and reduces clinical efficacy. Blood 118(9):2530–2540. https://doi.org/10.1182/blood-2011-01-330357
Seegmiller AC, Karandikar NJ, Kroft SH, McKenna RW, Xu Y (2009) Overexpression of CD7 in classical Hodgkin lymphoma-infiltrating T lymphocytes. Cytometry B Clin Cytom 76(3):169–174. https://doi.org/10.1002/cyto.b.20459
Fromm JR, Thomas A, Wood BL (2010) Increased expression of T cell antigens on T cells in classical Hodgkin lymphoma. Cytometry B Clin Cytom 78(6):387–388. https://doi.org/10.1002/cyto.b.20535
Wu D, Thomas A, Fromm JR (2016) Reactive T cells by flow cytometry distinguish Hodgkin lymphomas from T cell/histiocyte-rich large B cell lymphoma. Cytometry B Clin Cytom 90(5):424–432. https://doi.org/10.1002/cyto.b.21261
Rahemtullah A, Reichard KK, Preffer FI, Harris NL, Hasserjian RP (2006) A double-positive CD4+CD8+ T-cell population is commonly found in nodular lymphocyte predominant Hodgkin lymphoma. Am J Clin Pathol 126(5):805–814. https://doi.org/10.1309/Y8KD-32QG-RYFN-1XQX
Rahemtullah A, Harris NL, Dorn ME, Preffer FI, Hasserjian RP (2008) Beyond the lymphocyte predominant cell: CD4+CD8+ T-cells in nodular lymphocyte predominant Hodgkin lymphoma. Leuk Lymphoma 49(10):1870–1878. https://doi.org/10.1080/10428190802308728
Li S, Eshleman JR, Borowitz MJ (2002) Lack of surface immunoglobulin light chain expression by flow cytometric immunophenotyping can help diagnose peripheral B-cell lymphoma. Am J Clin Pathol 118(2):229–234. https://doi.org/10.1309/57G0-1BNF-KB9R-L4HN
Jaffe ES, Harris NL, Stein H, Campo E, Pileri SA, Swerdlow SH (2008) Introduction and overview of the classification of lymphoid neoplasms. In: Swerdlow SH, Campos E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds) WHO classification of tumours of haematopoietic and lymphoid tissues. World Health Organization classification of Tumors. IARC Press, Lyon, pp 158–166
Mantei K, Wood BL (2009) Flow cytometric evaluation of CD38 expression assists in distinguishing follicular hyperplasia from follicular lymphoma. Cytometry B Clin Cytom 76(5):315–320. https://doi.org/10.1002/cyto.b.20477
Yang W, Agrawal N, Patel J, Edinger A, Osei E, Thut D, Powers J, Meyerson H (2005) Diminished expression of CD19 in B-cell lymphomas. Cytometry B Clin Cytom 63(1):28–35. https://doi.org/10.1002/cyto.b.20030
Ray S, Craig FE, Swerdlow SH (2005) Abnormal patterns of antigenic expression in follicular lymphoma: a flow cytometric study. Am J Clin Pathol 124(4):576–583. https://doi.org/10.1309/2GFKU23XA1DH38L7
Kussick SJ, Kalnoski M, Braziel RM, Wood BL (2004) Prominent clonal B-cell populations identified by flow cytometry in histologically reactive lymphoid proliferations. Am J Clin Pathol 121(4):464–472. https://doi.org/10.1309/4EJ8-T3R2-ERKQ-61WH
Chen HI, Akpolat I, Mody DR, Lopez-Terrada D, De Leon AP, Luo Y, Jorgensen J, Schwartz MR, Chang CC (2006) Restricted kappa/lambda light chain ratio by flow cytometry in germinal center B cells in Hashimoto thyroiditis. Am J Clin Pathol 125(1):42–48
Hurwitz CA, Raimondi SC, Head D, Krance R, Mirro J Jr, Kalwinsky DK, Ayers GD, Behm FG (1992) Distinctive immunophenotypic features of t(8;21)(q22;q22) acute myeloblastic leukemia in children. Blood 80(12):3182–3188
Rawstron AC, Green MJ, Kuzmicki A, Kennedy B, Fenton JA, Evans PA, O’Connor SJ, Richards SJ, Morgan GJ, Jack AS, Hillmen P (2002) Monoclonal B lymphocytes with the characteristics of “indolent” chronic lymphocytic leukemia are present in 3.5% of adults with normal blood counts. Blood 100(2):635–639
Rawstron AC, Shanafelt T, Lanasa MC, Landgren O, Hanson C, Orfao A, Hillmen P, Ghia P (2010) Different biology and clinical outcome according to the absolute numbers of clonal B-cells in monoclonal B-cell lymphocytosis (MBL). Cytometry B Clin Cytom 78(Suppl 1):S19–S23. https://doi.org/10.1002/cyto.b.20533
Strati P, Shanafelt TD (2015) Monoclonal B-cell lymphocytosis and early-stage chronic lymphocytic leukemia: diagnosis, natural history, and risk stratification. Blood 126(4):454–462. https://doi.org/10.1182/blood-2015-02-585059
Rodriguez-Caballero A, Garcia-Montero AC, Barcena P, Almeida J, Ruiz-Cabello F, Tabernero MD, Garrido P, Munoz-Criado S, Sandberg Y, Langerak AW, Gonzalez M, Balanzategui A, Orfao A (2008) Expanded cells in monoclonal TCR-alphabeta+/CD4+/NKa+/CD8-/+dim T-LGL lymphocytosis recognize hCMV antigens. Blood 112(12):4609–4616. https://doi.org/10.1182/blood-2008-03-146241
Rahemtullah A, Longtine JA, Harris NL, Dorn M, Zembowicz A, Quintanilla-Fend L, Preffer FI, Ferry JA (2008) CD20+ T-cell lymphoma: clinicopathologic analysis of 9 cases and a review of the literature. Am J Surg Pathol 32(11):1593–1607. https://doi.org/10.1097/PAS.0b013e31817d7452
Rizzo K, Stetler-Stevenson M, Wilson W, Yuan CM (2009) Novel CD19 expression in a peripheral T cell lymphoma: a flow cytometry case report with morphologic correlation. Cytometry B Clin Cytom 76(2):142–149. https://doi.org/10.1002/cyto.b.20442
Hsu SM, Jaffe ES (1984) Leu M1 and peanut agglutinin stain the neoplastic cells of Hodgkin’s disease. Am J Clin Pathol 82(1):29–32
Stein H, Mason DY, Gerdes J, O’Connor N, Wainscoat J, Pallesen G, Gatter K, Falini B, Delsol G, Lemke H et al (1985) The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells. Blood 66(4):848–858
Stein H, Uchanska-Ziegler B, Gerdes J, Ziegler A, Wernet P (1982) Hodgkin and Sternberg-Reed cells contain antigens specific to late cells of granulopoiesis. Int J Cancer 29(3):283–290
Quintanilla-Martinez L, Fend F, Moguel LR, Spilove L, Beaty MW, Kingma DW, Raffeld M, Jaffe ES (1999) Peripheral T-cell lymphoma with Reed-Sternberg-like cells of B-cell phenotype and genotype associated with Epstein-Barr virus infection. Am J Surg Pathol 23(10):1233–1240
Mao Z, Quintanilla-Martinez L, Raffeld M, Richter M, Krugmann J, Burek C, Hartmann E, Rudiger T, Jaffe ES, Muller-Hermelink HK, Ott G, Fend F, Rosenwald A (2007) IgVH mutational status and clonality analysis of Richter’s transformation: diffuse large B-cell lymphoma and Hodgkin lymphoma in association with B-cell chronic lymphocytic leukemia (B-CLL) represent 2 different pathways of disease evolution. Am J Surg Pathol 31(10):1605–1614. https://doi.org/10.1097/PAS.0b013e31804bdaf8
Momose H, Jaffe ES, Shin SS, Chen YY, Weiss LM (1992) Chronic lymphocytic leukemia/small lymphocytic lymphoma with Reed-Sternberg-like cells and possible transformation to Hodgkin’s disease. Mediation by Epstein-Barr virus. Am J Surg Pathol 16(9):859–867
Ohno T, Smir BN, Weisenburger DD, Gascoyne RD, Hinrichs SD, Chan WC (1998) Origin of the Hodgkin/Reed-Sternberg cells in chronic lymphocytic leukemia with “Hodgkin’s transformation”. Blood 91(5):1757–1761
Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, Marce S, Lopez-Guillermo A, Campo E, Montserrat E (2003) ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 348(18):1764–1775. https://doi.org/10.1056/NEJMoa023143
Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N (1999) Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 94(6):1840–1847
Acknowledgments
The authors thank the medical technologists in the hematopathology laboratory at the University of Washington for their expert technical assistance.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Glynn, E., Soma, L., Wu, D., Wood, B.L., Fromm, J.R. (2019). Flow Cytometry for Non-Hodgkin and Hodgkin Lymphomas. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 1956. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9151-8_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9151-8_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9150-1
Online ISBN: 978-1-4939-9151-8
eBook Packages: Springer Protocols