Flow Cytometric Immunophenotyping of Cerebrospinal Fluid

Jaco Kraan1, Jan W. Gratama1, Corinne Haioun2, Alberto Orfao3, Anne Plonquet4, Anna Porwit5, Sandra Quijano3, Maryalice Stetler‐Stevenson6, Dolores Subira7, Wyndham Wilson8

1 Department of Medical Oncology, Erasmus Medical Center, Rotterdam, The Netherlands, 2 Service d'Hématologie Clinique, Hospital Henri Mondor, Créteil, France, 3 CSIC, University of Salamanca, Salamanca, Spain, 4 Immunologie Biologique, Hospital Henri Mondor, Créteil, France, 5 Department of Pathology, Karolinska University Hospital, Stockholm, Sweden, 6 Flow Cytometry Unit, National Institutes of Health, Bethesda, Maryland, 7 Department of Hematology, Jiménez Díaz Foundation, Madrid, Spain, 8 Center for Cancer Research, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.25
DOI:  10.1002/0471142956.cy0625s45
Online Posting Date:  July, 2008
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Leptomeningeal disease is an important adverse complication occurring in patients with B and T cell lymphomas and acute leukemias of lymphoid and myeloid origin. Recent reports suggest that multiparameter flow cytometry immunophenotypic assessment of spinal fluid samples could improve the efficiency of detection of CNS involvement, due to its high specificity and greater sensitivity. However, spinal fluid samples are frequently paucicellular with a rapidly decreasing cell viability. Staining of spinal fluid therefore requires dedicated sample storage/transport, staining, and preparation protocols. The Basic Protocol in this unit outlines a consensus multiparameter (3‐ to 8‐color) flow cytometry immunophenotypic protocol for the evaluation of CNS involvement of cerebrospinal fluid (CSF) samples by neoplastic cells. A Support Protocol describing the simultaneous assessment of surface and cytoplasmic antigens is also provided. Finally, in the Alternate Protocol, we describe a method to calculate absolute numbers of both normal and pathological cell subpopulations by adding counting beads to the assay. Curr. Protocol. Cytom. 45:6.25.1‐6.25.16. © 2008 by John Wiley & Sons, Inc.

Keywords: CSF; cerebrospinal fluid; diagnosis; flow cytometry; immunophenotyping; leukemia; lymphoma

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunophenotypic Analysis of White Blood Cells with Multiple Monoclonal Antibodies to Surface Antigens
  • Support Protocol 1: Combined Staining for Surface and Intracellular Antigens
  • Alternate Protocol 1: Enumeration of Absolute Leukocyte Subset Counts
  • Support Protocol 2: Sample Collection
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Immunophenotypic Analysis of White Blood Cells with Multiple Monoclonal Antibodies to Surface Antigens

  • Phosphate‐buffered saline (PBS), pH 7.6 ( appendix 2A)
  • Cerebrospinal fluid (CSF) sample ( protocol 4)
  • Antibody cocktail (see and Tables 6.25.1 6.25.5)
  • Phosphate‐buffered saline (PBS), pH 7.6) ( appendix 2A) containing 1% (w/v) paraformaldehyde (PFA)
  • Rinse buffer (commercial flow cytometer rinse buffer, e.g., Beckman Coulter or BD Biosciences)
  • Flow buffer (commercial flow cytometer buffer, e.g., Beckman Coulter or BD Biosciences)
  • Centrifuge
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer

Support Protocol 1: Combined Staining for Surface and Intracellular Antigens

  • Surface antibody cocktail, titrated (see )
  • Cytoplasmic antibody (cocktail), titrated (see )
  • Fix & Perm kit from Invitrogen, Intrastain kit from Dako, or Intraprep from Beckman Coulter

Alternate Protocol 1: Enumeration of Absolute Leukocyte Subset Counts

  • Aqueous suspension of microspheres (Perfect Count from Invitrogen; alternatives include FlowCount from Beckman Coulter, CytoCount from Dako, and Cyto‐Cal Count Control from Duke Scientific)

Support Protocol 2: Sample Collection

  • Patient/CSF donor
  • Stabilization medium 1—complete RPMI ( appendix 3B) containing 5% FBS—or stabilization medium 2—Transfix (Cytomark, http://www.cytomark.co.uk)
  • Equipment for lumbar puncture
  • 15‐ml tubes
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Literature Cited

Literature Cited
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