Immunophenotyping of Paucicellular Samples

Alessandra Stacchini1, Anna Demurtas1, Sabrina Aliberti1

1 Flow Cytometry Unit, Anatomical Pathology, Diagnostic Laboratory Department, Città della Salute e della Scienza, Turin
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.46
DOI:  10.1002/0471142956.cy0946s68
Online Posting Date:  April, 2014
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Abstract

ABSTRACT

Immunophenotyping of paucicellular samples may represent a diagnostic challenge in the flow cytometry (FC) laboratory routine, as the scarcity of cells limits the number of tests that can be performed. Specimens such as fine needle aspirates (FNA), human body fluids (BF), cerebrospinal fluid (CSF), or ocular fluid (OF) sent for FC investigations in the case of suspicion of lymphoma, or for lymphoma monitoring, may contain very low numbers of cells. In these cases, it is mandatory to obtain the largest amount possible of useful information from a single tube. The basic protocol described in this unit provides a method that combines the use of multiple monoclonal antibodies (MAbs) with a Boolean gating strategy to identify and quantify the main lymphocyte populations, as well as to detect lymphomatous B cells or any aberrant T cell expression, if present, in paucicellular samples. Curr. Protoc. Cytom. 68:9.46.1‐9.46.14. © 2014 by John Wiley & Sons, Inc.

Keywords: multiparameter flow cytometry; paucicellular samples; non‐Hodgkin lymphomas

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Single Tube Assay (STA)
  • Basic Protocol 2: Analysis of Paucicellular Fine Needle Aspirates
  • Basic Protocol 3: Analysis of Paucicellular Effusions (Pleural, Pericardial, Peritoneal)
  • Basic Protocol 4: Analysis of Paucicellular Cerebrospinal Fluid (CSF) or Ocular Fluid (OF)
  • Support Protocol 1: Morphological Control
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Single Tube Assay (STA)

  Materials
  • Paucicellular sample
  • Phosphate‐buffered saline (PBS; see recipe)
  • STA combination (Table 9.46.1)
  • Cleaning buffer (commercial flow cytometer cleaning solution, e.g., BD Biosciences)
  • Rinse buffer (commercial flow cytometer rinse solution, e.g., BD Biosciences)
  • Flow buffer (commercial flow cytometer buffer, e.g., BD Biosciences)
  • 12 × 75 mm polystyrene tubes
  • Benchtop centrifuge
  • Flow cytometer
  • Additional reagents and equipment for flow cytometer setup and calibration (units 1.3 & 1.4)

Basic Protocol 2: Analysis of Paucicellular Fine Needle Aspirates

  Materials
  • Fine‐needle aspirate (FNA), ultrasound–fine needle aspirate (US‐FNA), or endoscopic‐ultrasound–fine needle aspirate (EUS‐FNA) sample, freshly collected
  • Hold medium (HM; see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Ammonium chloride lysis solution (optional; see recipe)
  • Sterile syringe
  • 15‐ml conical centrifuge tubes
  • Benchtop centrifuge
  • Counting chamber ( appendix 3A)
  • Additional reagents and equipment for counting cells ( appendix 3A), staining, flow cytometry, and data analysis ( protocol 1), and morphological control ( protocol 5Support Protocol)

Basic Protocol 3: Analysis of Paucicellular Effusions (Pleural, Pericardial, Peritoneal)

  Materials
  • Effusion sample
  • Phosphate‐buffered saline (PBS; see recipe)
  • Ammonium chloride lysis solution (optional; see recipe)
  • 15‐ or 50‐ml centrifuge tubes
  • Benchtop centrifuge
  • Counting chamber ( appendix 3A)
  • Additional reagents and equipment for counting cells ( appendix 3A), staining, flow cytometry, and data analysis ( protocol 1), and morphological control ( protocol 5Support Protocol)

Basic Protocol 4: Analysis of Paucicellular Cerebrospinal Fluid (CSF) or Ocular Fluid (OF)

  Materials
  • Cerebrospinal fluid or ocular fluid
  • Hold medium (HM; see recipe)
  • PBS‐FBS (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 10‐ml tubes, sterile
  • Benchtop centrifuge
  • Counting chamber ( appendix 3A)
  • Additional reagents and equipment for counting cells ( appendix 3A), staining, flow cytometry, and data analysis ( protocol 1), and morphological control ( protocol 5Support Protocol)

Support Protocol 1: Morphological Control

  Materials
  • Cell suspension
  • May‐Grünwald‐Giemsa stain
  • Glass microscope slides
  • Cytocentrifuge (Thermo Scientific Shandon) with slide chambers and filter cards
  • Optical microscope
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Figures

Videos

Literature Cited

Literature Cited
  Barnett, D., Louzao, R., Gambell, P., De, J., Oldaker, T., Hanson, C.A., and ICSH/ICCS Working group. 2013. Validation of cell‐based fluorescence assay: Practice guidelines for the ICHS and ICCS‐part IV‐Postanalytic considerations. Cytometry B 84:309‐314.
  Barrena, S., Almeida, J., Del Carmen García‐Macias, M., López, A., Rasillo, A., Sayagués, J.M., Rivas, R.A., Gutiérrez, M.L., Ciudad, J., Flores, T., Balanzategui, A., Caballero, M.D., and Orfao, A. 2011. Flow cytometry immunophenotyping of fine‐needle aspiration specimens: Utility in the diagnosis and classification of non‐Hodgkin lymphomas. Histopathology 58:906‐918.
  Bertram, H.C., Check, I.J., and Milano, M.A. 2001. Immunophenotyping large B‐cell lymphomas. Flow cytometric pitfalls and pathologic correlation. Am. J. Clin. Pathol. 116:191‐203.
  Cesana, C., Klersy, C., Scarpati, B., Brando, B., Volpato, E., Bertani, G., Faleri, M., Nosari, A., Cantoni, S., Ferri, U., Scampini, L., Barba, C., Lando, G., Morra, E., and Cairoli, R. 2010. Flow cytometry vs. cytomorphology for the detection of hematologic malignancy in body cavity fluids. Leuk. Res. 34:1027‐1034.
  Craig, F.E. and Foon, K.A. 2008. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111:3941‐3967.
  Craig, F.E., Ohori, N.P., Gorrill, T.S., and Swerdlow, S.H. 2011. Flow cytometric immunophenotyping of cerebrospinal fluid specimens. Am. J. Clin. Pathol. 135:22‐34.
  Davis, J.L., Ruiz, P., Shah, M., and Mandelcorn, E.D. 2012. Evaluation of the reactive T‐cell infiltrate in uveitis and intraocular lymphoma with flow cytometry of vitreous fluid (an American Ophthalmological Society thesis). Trans. Am. Ophthalmol. Soc. 110:117‐129.
  Demurtas, A., Accinelli, G., Pacchioni, D., Godio, L., Novero, D., Bussolati, G., Palestro, G., Papotti, M., and Stacchini, A. 2010. 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. 18:311‐322.
  Hegde, U., Filie, A., Little, R.F., Janik, J.E., Grant, N., Steinberg, S.M., Dunleavy, K., Jaffe, E.S., Abati, A., Stetler‐Stevenson, M., and Wilson, W.H. 2005. High incidence of occult leptomeningeal disease detected by flow cytometry in newly diagnosed aggressive B‐cell lymphomas at risk for central nervous system involvement: The role of flow cytometry versus cytology. Blood 105:496‐502.
  Iqbal, J., Liu, T., Mapow, B., Swami, V.K., and Hou, J.S. 2010. Importance of flow cytometric analysis of serous effusions in the diagnosis of hematopoietic neoplasms in patients with prior hematopoietic malignancies. Anal. Quant. Cytol. Histol. 32:161‐165.
  Mehra, M., Tamhane, A., and Eloubeidi, M. 2005. EUS‐guided FNA combined with flow cytometry in the diagnoses of suspected or recurrent intrathoracic or retroperitoneal lymphoma. Gastrointest. Endosc. 62:508‐513.
  Missotten, T., Tielemans, D., Bromberg, J.E., van Hagen, P.M., van Lochem, E.G., van Dongen, J.J., Baarsma, G.S., and Langerak, A.W. 2013. Multicolor flowcytometric immunophenotyping is a valuable tool for detection intraocular lymphoma. Ophthalmology 120:991‐996.
  Quijano, S., López, A., Manuel Sancho, J., Panizo, C., Debén, G., Castilla, C., Antonio García‐Vela, J., Salar, A., Alonso‐Vence, N., González‐Barca, E., Peñalver, F.J., Plaza‐Villa, J., Morado, M., García‐Marco, J., Arias, J., Briones, J., Ferrer, S., Capote, J., Nicolás, C., and Orfao A. 2009. Identification of leptomeningeal disease in aggressive B‐cell non‐Hodgkin's lymphoma: Improved sensitivity of flow cytometry. J. Clin. Oncol. 27:1462‐1469.
  Ribeiro, A., Pereira, D., Escalon, M.P., Goodman, M., and Byrne, G.E. 2010. EUS‐guided biopsy for the diagnosis and classification of lymphoma. Gastrointest. Endosc. 71:851‐855.
  Schmid, S., Tinguely, M., Cione, P., Moch, H., and Bode, B. 2011. Flow cytometry as an accurate tool to complement fine needle aspiration cytology in the diagnosis of low grade malignant lymphomas. Cytopathology 22:397‐406.
  Schwock, J. and Geddie, W.R. 2012. Diagnosis of B‐cell non‐Hodgkin lymphomas with small‐/intermediate‐sized cells in cytopathology. Pathol. Res. Int. doi: 10.1155/2012/164934.
  Sen, N.H., Bodaghi, B., and Nussemblat, R. 2009. Primary intraocular lymphoma: Diagnosis and differential diagnosis. Ocul. Immunol. Inflamm. 17:133‐141.
  Sodikoff, J.B., Johnson, H.L., Lewis, M.M., Garud, S.S., Bharmal, S.J., Keilin, S.A., Siddiqui, M.T., Cai, Q., and Willingham, F.F. 2013. Increased diagnostic yield of endoscopic ultrasound‐guided fine needle aspirates with flow cytometry and immunohistochemistry. Diagn. Cytopathol. 41:1043‐1051.
  Stacchini, A., Demurtas, A., Aliberti, S., Francia di Celle, P., Godio, L., and Novero, D. 2007. The usefulness of flow cytometric CD10 detection in the differential diagnosis of peripheral T‐cell lymphomas. Am. J. Clin. Pathol. 128:854‐864.
  Stacchini, A., Aliberti, S., Demurtas, A., Benevolo, G., and Godio, L. 2012a. Ten antibodies, six colors, twelve parameters: A multiparameter flow cytometric approach to evaluate leptomeningeal disease in B‐cell non Hodgkin's lymphomas. Cytometry B 82:139‐144.
  Stacchini, A., Carucci, P., Pacchioni, D., Accinelli, G., Demurtas, A., Aliberti, S., Bosco, M., Bruno, M., Balbo Mussetto, A., Rizzetto, M., Bussolati, G., and De Angelis, C. 2012b. Diagnosis of deep‐seated lymphomas by endoscopic ultrasound‐guided fine needle aspiration combined with flow cytometry. Cytopathology 23:50‐56.
  Stelzer, G.T., Shults, K.E., and Loken, M.R. 1993. CD45 gating for routine flow cytometric analysis of human bone marrow specimens. Ann. N.Y. Acad. Sci. 677:265‐280.
  Sun, T., Sangaline, R., Ryder, J., Gibbens, K., Rollo, C., Stewart, S., and Rajagopalan, C. 1997. Gating strategy for immunophenotyping of leukemia and lymphoma. Am. J. Clin. Pathol. 108:152‐157.
  Sutherland, D.R., Keeney, M., and Illingworth, A. 2012. A practical guideline for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry B 82:195‐208.
  Wood, B. 2006. 9‐and 10‐color flow cytometry in the clinical laboratory. Arch. Pathol. Lab. Med. 130:680‐690.
  Zembruski, N.C., Stache, V., Haefeli, W.E., and Weiss, J. 2012. 7‐Aminoactinomycin D for apoptosis staining in flow cytometry. Anal. Biochem. 429:79‐81.
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