In the last decades, lymph node fine-needle cytology (FNC), coupled with flow cytometry (FC), has gained a role in the diagnosis and classification of non-Hodgkin lymphoma (NHL). The combination of FNC/FC allows the diagnosis and classification of NHL in lymph node samples with a high sensitivity and specificity by combining cytological features and specific phenotypic profiles. The present review provides a brief technical description of FC and a detailed analysis of the current markers and their combinations (diagnostic algorithm) for the diagnosis and classification of NHL. The basic principles of clonality assessment, as well as the diagnostic strengths and weaknesses of the procedure, are reported. The current diagnostic algorithms for NHL classification are critically reviewed with a focus on specific problems related to single entities. Moreover, this review provides a detailed analysis of the different clinical contexts in which FNC/FC is performed and related implications. Future and further applications of FNC/FC for NHL are also discussed.

Keywords:
Lymph node, Fine-needle cytology, Flow cytometry, Lymphoma
1.
Jaffe ES, Harris NL, Stein H, et al: World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, IARC Press, 2008.
2.
Saboorian MH, Ashfaq R: The use of fine needle aspiration biopsy in the evaluation of lymphadenopathy. Semin Diagn Pathol 2001;18:110-123.
3.
Craig FE, Foon KA: Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008;111:3941-3967.
4.
Dey P, Amir T, Al Jassar A, Al Shemmari S, Jogai S, Bhat MG, Al Quallaf A, Al Shammari Z: Combined applications of fine needle aspiration cytology and flow cytometric immunphenotyping for diagnosis and classification of non Hodgkin lymphoma. Cytojournal 2006;27;3-24.
5.
Zardawi IM, Jain S, Bennett G: Flow-cytometric algorithm on fine-needle aspirates for the clinical workup of patients with lymphadenopathy. Diagn Cytopathol 1998;19:274-278.
6.
Swart GJ, Wright C, Brundyn K, Mansvelt E, du Plessis M, ten Oever D, Jacobs P: Fine needle aspiration biopsy and flow cytometry in the diagnosis of lymphoma. Transfus Apher Sci 2007;37:71-79.
7.
Dunphy CH, Ramos R: Combining fine-needle aspiration and flow cytometric immunophenotyping in evaluation of nodal and extranodal sites for possible lymphoma: a retrospective review. Diagn Cytopathol 1997;16:200-206.
8.
Young NA, Al-Saleem TI, Ehya H, Smith MR: Utilization of fine-needle aspiration cytology and flow cytometry in the diagnosis and sub-classification of primary and recurrent lymphoma. Cancer 1998;84:252-261.
9.
Jeffers MD, Milton J, Herriot R, McKean M: Fine needle aspiration cytology in the investigation of non-Hodgkin's lymphoma. J Clin Pathol 1998;51:189-196.
10.
Meda BA, Buss DH, Woodruff RD, et al: Diagnosis and sub-classification of primary and recurrent lymphoma: the usefulness and limitations of combined fine-needle aspiration cytomorphology and flow cytometry. Am J Clin Pathol 2000;113:688-699.
11.
Liu K, Stern RC, Rogers RT, Dodd LG, Mann KP: Diagnosis of hematopoietic processes by fine-needle aspiration in conjunction with flow cytometry: a review of 127 cases. Diagn Cytopathol 2001;24:1-10.
12.
Mourad WA, Tulbah A, Shoukri M, Al Dayel F, Akhtar M, Ali MA, Hainau B, Martin J: Primary diagnosis and REAL/WHO classification of non-Hodgkin's lymphoma by fine-needle aspiration: cytomorphologic and immunophenotypic approach. Diagn Cytopathol 2003;28:191-195.
13.
Zeppa P, Marino G, Troncone G, Fulciniti F, De Renzo A, Picardi M, Benincasa G, Rotoli B, Vetrani A, Palombini L: Fine-needle cytology and flow cytometry immunophenotyping and subclassification of non-Hodgkin lymphoma: a critical review of 307 cases with technical suggestions. Cancer 2004;102:55-65.
14.
Bangerter M, Brudler O, Heinrich B, Griesshamnuer M: Fine needle aspiration cytology and flow cytometry in the diagnosis and subclassification of non-Hodgkin's lymphoma based on the World Health Organization classification. Acta Cytol 2007;51:390-398.
15.
Barrena S, Almeida J, Del Carmen García-Macias M, López A, Rasillo A, Sayagués JM, Rivas RA, Gutiérrez ML, Ciudad J, Flores T, Balanzategui A, Caballero MD, Orfao A: Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas. Histopathology 2011;58:906-918.
16.
Stacchini A, Carucci P, Pacchioni D, Accinelli G, Demurtas A, Aliberti S, Bosco M, Bruno M, Balbo Mussetto A, Rizzetto M, Bussolati G, De Angelis C: Diagnosis of deep-seated lymphomas by endoscopic ultrasound-guided fine needle aspiration combined with flow cytometry. Cytopathology 2012;23:50-56.
17.
Dong HY, Harris NL, Preffer FI, Pitman MB: Fine-needle aspiration biopsy in the diagnosis and classification of primary and recurrent lymphoma: a retrospective analysis of the utility of cytomorphology and flow cytometry. Mod Pathol 2001;14:472-481.
18.
Zeppa P, Vigliar E, Cozzolino I, Troncone G, Picardi M, De Renzo A, Grimaldi F, Pane F, Vetrani A, Palombini L: Fine needle aspiration cytology and flow cytometry immunophenotyping of non-Hodgkin lymphoma: can we do better? Cytopathology 2010;21:300-310.
19.
Demurtas A, Accinelli G, Pacchioni D, Godio L, Novero D, Bussolati G, Palestro G, Papotti M, Stacchini A: Utility of flow cytometry immunophenotyping in fine-needle aspirate cytologic diagnosis of non-Hodgkin lymphoma: a series of 252 cases and review of the literature. Appl Immunohistochem Mol Morphol 2010;18:311-322.
20.
Kalina T, Flores-Montero J, van der Velden VH, Martin-Ayuso M, Böttcher S, Ritgen M, Almeida J, Lhermitte L, Asnafi V, Mendonça A, de Tute R, Cullen M, Sedek L, Vidriales MB, Pérez JJ, te Marvelde JG, Mejstrikova E, Hrusak O, Szczepański T, van Dongen JJ, Orfao A; EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708): EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia 2012;26:1986-2010.
21.
Laane E, Tani E, Björklund E, Elmberger G, Everaus H, Skoog L, Porwit-MacDonald A: Flow cytometric immunophenotyping including Bcl-2 detection on fine needle aspirates in the diagnosis of reactive lymphadenopathy and non-Hodgkin's lymphoma. Cytometry B Clin Cytom 2005;64:34-42.
22.
Dey P: Role of ancillary techniques in diagnosing and subclassifying non-Hodgkin's lymphomas on fine needle aspiration cytology. Cytopathology 2006;17:275-287.
23.
Ohmoto A, Maeshima AM, Taniguchi H, Tanioka K, Makita S, Kitahara H, Fukuhara S, Munakata W, Suzuki T, Maruyama D, Kobayashi Y, Tobinai K: Histopathological analysis of B-cell non-Hodgkin lymphomas without light chain restriction by using flow cytometry. Leuk Lymphoma 2015;56:3301-3305.
24.
Nicol TL, Silberman M, Rosenthal DL, Borowitz MJ: The accuracy of combined cytopathologic and flow cytometric analysis of fine-needle aspirates of lymph nodes. Am J Clin Pathol 2000;114:18-28.
25.
Ravoet C, Demartin S, Gerard R, Dehon M, Peny MO, Petit B, Delannoy A, Husson B: Contribution of flow cytometry to the diagnosis of malignant and non malignant conditions in lymph node biopsies. Leuk Lymphoma 2004;45:1587-1593.
26.
Liu J, Song B, Fan T, Huang C, Xie C, Li J, Zhong W, Li S, Yu J: Pathological and clinical characteristics of 1,248 non-Hodgkin's lymphomas from a regional cancer hospital in Shandong, China. Asian Pac J Cancer Prev 2011;12:3055-3061.
27.
Bertram HC, Check IJ, Milano MA: Immunophenotyping large B-cell lymphomas: flow cytometric pitfalls and pathologic correlation. Am J Clin Pathol 2001;116:191-203.
28.
Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, Müller-Hermelink HK, Campo E, Braziel RM, Jaffe ES, Pan Z, Farinha P, Smith LM, Falini B, Banham AH, Rosenwald A, Staudt LM, Connors JM, Armitage JO, Chan WC: Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004;103:275-282.
29.
Visco C, Li Y, Xu-Monette ZY, Miranda RN, Green TM, Li Y, Tzankov A, Wen W, Liu WM, Kahl BS, d'Amore ES, Montes-Moreno S, Dybkær K, Chiu A, Tam W, Orazi A, Zu Y, Bhagat G, Winter JN, Wang HY, O'Neill S, Dunphy CH, Hsi ED, Zhao XF, Go RS, Choi WW, Zhou F, Czader M, Tong J, Zhao X, van Krieken JH, Huang Q, Ai W, Etzell J, Ponzoni M, Ferreri AJ, Piris MA, Møller MB, Bueso-Ramos CE, Medeiros LJ, Wu L, Young KH: Comprehensive gene expression profiling and immunohistochemical studies support application of immunophenotypic algorithm for molecular subtype classification in diffuse large B-cell lymphoma: a report from the International DLBCL Rituximab-CHOP Consortium Program Study. Leukemia 2012;26:2103-2113; erratum in Leukemia 2014;28:980.
30.
Cozzolino I, Varone V, Picardi M, Baldi C, Memoli D, Ciancia G, Selleri C, De Rosa G, Vetrani A, Zeppa P: CD10, BCL6, and MUM1 expression in diffuse large B-cell lymphoma on FNA samples. Cancer Cytopathol 2016;124:135-143.
31.
Pai RK, Mullins FM, Kim YH, Kong CS: Cytologic evaluation of lymphadenopathy associated with mycosis fungoides and Sézary syndrome: role of immunophenotypic and molecular ancillary studies. Cancer 2008;114:323-332.
32.
Rahemtullah A, Reichard KK, Preffer FI, Harris NL, Hasserjian RP: A double-positive CD4+CD8+ T-cell population is commonly found in nodular lymphocyte predominant Hodgkin lymphoma. Am J Clin Pathol 2006;126:805-814.
33.
Rahemtullah A, Harris NL, Dorn ME, Preffer FI, Hasserjian RP: Beyond the lymphocyte predominant cell: CD4+CD8+ T-cells in nodular lymphocyte predominant Hodgkin lymphoma. Leuk Lymphoma 2008;49:1870-1878.
34.
David JA, Huang JZ: Diagnostic utility of flow cytometry analysis of reactive T cells in nodular lymphocyte-predominant Hodgkin lymphoma. Am J Clin Pathol 2016;145:107-115.
35.
Kim YH, Liu HL, Mraz-Gernhard S: Long-term outcome of 525 patients with mycosis fungoides and Sézary syndrome: clinical prognostic factors and risk for disease progression. Arch Dermatol 2003, 139:857-866.
36.
Olsen E, Vonderheid E, Pimpinelli N, Willemze R, Kim Y, Knobler R, Zackheim H, Duvic M, Estrach T, Lamberg S, Wood G, Dummer R, Ranki A, Burg G, Heald P, Pittelkow M, Bernengo MG, Sterry W, Laroche L, Trautinger F, Whittaker S; ISCL/EORTC: Revisions to the staging and classification of mycosis fungoides and Sézary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the Cutaneous Lymphoma Task Force of the European Organization of Research and Treatment of Cancer (EORTC). Blood 2007;110:1713-1722.
37.
Vigliar E, Cozzolino I, Picardi M, Peluso AL, Fernandez LV, Vetrani A, Botti G, Pane F, Selleri C, Zeppa P: Lymph node fine needle cytology in the staging and follow-up of cutaneous lymphomas. BMC Cancer 2014;14:18.
38.
Galindo LM, Garcia FU, Hanau CA, Lessin SR, Jhala N, Bigler RD, Vonderheid EC: Fine-needle aspiration biopsy in the evaluation of lymphadenopathy associated with cutaneous T-cell. Am J Clin Pathol 2000;113:865-871.
39.
Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, Marcé S, López-Guillermo A, Campo E, Montserrat E: ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348:1764-1765.
40.
Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG, Neuberg DS, Flinn IW, Rai KR, Byrd JC, Kay NE, Greaves A, Weiss A, Kipps TJ: ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 2004;351:893-901.
41.
Orchard JA, Ibbotson RE, Davis Z, Wiestner A, Rosenwald A, Thomas PW, Hamblin TJ, Staudt LM, Oscier DG: ZAP-70 expression and prognosis in chronic lymphocytic leukaemia. Lancet 2004;363:105-111.
42.
Dürig J, Naschar M, Schmücker U, Renzing-Köhler K, Hölter T, Hüttmann A, Dührsen U: CD38 expression is an important prognostic marker in chronic lymphocytic leukaemia. Leukemia 2002;16:30-35.
43.
Del Poeta G, Maurillo L, Venditti A, Buccisano F, Epiceno AM, Capelli G, Tamburini A, Suppo G, Battaglia A, Del Principe MI, Del Moro B, Masi M, Amadori S: Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood 2001;98:2633-2639.
44.
Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW, Stevenson FK, Oscier DG: CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002;99:1023-1029.
45.
Ibrahim S, Keating M, Do KA, O'Brien S, Huh YO, Jilani I, Lerner S, Kantarjian HM, Albitar M: CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 2001;98:181-186.
46.
Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH: A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 2000;100:655-669.
47.
Bob R, Falini B, Marafioti T, Paterson JC, Pileri S, Stein H: Nodal reactive and neoplastic proliferation of monocytoid and marginal zone B cells: an immunoarchitectural and molecular study highlighting the relevance of IRTA1 and T-bet as positive markers. Histopathology 2013;63:482-498.
48.
Jöhrens K, Shimizu Y, Anagnostopoulos I, Schiffmann S, Tiacci E, Falini B, Stein H: T-bet-positive and IRTA1-positive monocytoid B cells differ from marginal zone B cells and epithelial-associated B cells in their antigen profile and topographical distribution. Haematologica 2005;90:1070-1077.
49.
Went P, Ascani S, Strøm E, Brorson SH, Musso M, Zinzani PL, Falini B, Dirnhofer S, Pileri S: Nodal marginal-zone lymphoma associated with monoclonal light-chain and heavy-chain deposition disease. Lancet Oncol 2004;5:381-383.
50.
Ondrejka SL, Hsi ED: Pathology of B-cell lymphomas: diagnosis and biomarker discovery. Cancer Treat Res 2015;165:27-50.
51.
Amador-Ortiz C, Goolsby CL, Peterson LC, Wolniak KL, McLaughlin JL, Gao J, Chen YH: Flow cytometric analysis of lymphoid enhancer-binding factor 1 in diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma. Am J Clin Pathol 2015;143:214-222.
52.
Wasik AM, Priebe V, Lord M, Jeppsson-Ahlberg Å, Christensson B, Sander B: Flow cytometric analysis of SOX11: a new diagnostic method for distinguishing B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma from Mantle cell lymphoma. Leuk Lymphoma 2015;56:1425-1431.
53.
Dagan LN, Jiang X, Bhatt S, Cubedo E, Rajewsky K, Lossos IS: miR-155 regulates HGAL expression and increases lymphoma cell motility. Blood 2012;119:513-520.
© 2016 S. Karger AG, Basel
2016
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.