Immunophenotyping of Plasma Cells

Andy C. Rawstron1

1 Leeds Teaching Hospitals
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.23
DOI:  10.1002/0471142956.cy0623s36
Online Posting Date:  May, 2006
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Abstract

Plasma cell enumeration and immunophenotyping has been shown to be of value in predicting the outcome for patients with myeloma. Detection of abnormal plasma cells at a leukocyte level >0.01% at the end of therapy predicts early relapse. Although clonally related populations at varying stages of differentiation are reported to be present in myeloma, only the CD138+ fraction has been shown to directly correlate with the outcome. In addition, the plasma cell immunophenotype in monoclonal gammopathy of undetermined significance (MGUS) is reported to correlate with the risk of progression to myeloma. This protocol provides a four‐color flow cytometry method for enumeration and classification of plasma cells in both diagnostic and post‐treatment samples. Plasma cells are identified by strong expression of CD38 and CD138. Neoplastic plasma cells are separated from their normal counterparts by aberrant expression of CD19 and other relevant markers in combination with intracellular immunoglobulin light chain restriction.

Keywords: plasma cells; myeloma; monoclonal gammopathy of undetermined significance (MGUS); minimal residual disease (MRD); CD138

     
 
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Table of Contents

  • Basic Protocol 1: Plasma Cell Enumeration and Cell Surface Immunophenotyping
  • Basic Protocol 2: Assessment of Intracellular Immunoglobulin Expression
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Plasma Cell Enumeration and Cell Surface Immunophenotyping

  Materials
  • Erythrocytes: bone marrow sample in EDTA (<24 hr old)
  • Ammonium chloride–based lysing solution (8.6 g/liter in distilled water or commercially available equivalent; also see appendix 2A)
  • Phosphate buffered saline (PBS) solution with protein support (e.g., FACSFlow with 0.1% bovine serum albumin)
  • Antibodies:
    • Tube 1: CD38‐FITC, CD3‐PE, CD45‐PE‐CY5.5, CD138‐APC
    • Tube 2: CD38‐FITC, CD19‐PE, CD45‐PE‐CY5.5, CD138 APC
    • Tube 3: CD38‐FITC, antigen x‐PE, CD45‐PE‐CY5.5, CD138 APC
    • where x = antigen of interest
  • CELLFix (or 1% formalin in PBS) (BD Biosciences)
  • 96‐well round‐bottom microtiter plate
  • Centrifuge with a microtiter plate holder
  • Microtiter plate mixer
  • Flow cytometer with 488‐nm and 635‐nm excitation and filter sets to detect green (FITC), orange (PE), red (PE‐Cy5.5), and deep red (APC)

Basic Protocol 2: Assessment of Intracellular Immunoglobulin Expression

  Materials
  • Bone marrow sample in EDTA (<24 hr old)
  • PBS solution with protein support (e.g., FACSFlow with 0.1% bovine serum albumin), 37°C
  • Reagent kit for intracellular analysis (e.g., Intrastain, DAKO) containing:
    • Fixative reagent A
    • Permeabilization reagent B
  • Gating antibodies:
  • Intracellular antibodies:
  • CELLFix (or 1% formalin in PBS) (BD Biosciences)
  • 12 × 75–mm tubes
  • Flow cytometer with 488‐nm and 635‐nm excitation, at least four fluorescence detectors, and appropriate filter sets for detection of FITC, PE, PE‐Cy5.5, and APC fluorescence
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Figures

Videos

Literature Cited

Literature Cited
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