Quantitative Flow Cytometric Analysis of Membrane Antigen Expression

Jean‐Luc D'hautcourt1

1 Centre Hospitalier Régional Mons, Warquignies, Boussu
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.12
DOI:  10.1002/0471142956.cy0612s22
Online Posting Date:  November, 2002
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Abstract

Immunological analysis for cell antigens has been performed by flow cytometry in a qualitative fashion for over thirty years. During that time it has become increasingly apparent that quantitative measurements such as number of antigens per cell provide unique and useful information. This unit on quantitative flow cytometry (QFCM) describes the most commonly used protocols, both direct and indirect, and the major methods of analysis for the number of antibody binding sites on a cell or particle. Practical applications include detection of antigen underÔÇÉ or overexpression in hematological malignancies, distinguishing between B cell lymphoproliferative disorders, and precise diagnosis of certain rare diseases.

Keywords: flow cytometry; quantitative flow cytometry (QFCM); calibration; fluorescence; antibody binding capacity (ABC); antibodies bound per cell (AB/C)

     
 
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Table of Contents

  • Basic Protocol 1: Quantitative Determination of Cell Surface Antigens by Indirect Immunofluorescence Assay Using Qifikit Calibrator Beads
  • Alternate Protocol 1: Quantitative Determination of Cell Surface Antigens by Direct or Indirect Immunofluorescence Assay Using Quantum Simply Cellular Calibrator Beads
  • Support Protocol 1: Titration of Monoclonal Antibodies to Use with Quantum Simply Cellular Calibrator Beads
  • Alternate Protocol 2: Flow Cytometric Quantitative Determination of Cell Surface Antigens by Direct Immunofluorescence Assay Using 1:1 PE‐Labeled Antibodies and QuantiBRITE PE Calibrator Beads
  • Alternate Protocol 3: Quantitative Evaluation of Cell Surface Antigen Expression on Leukocyte Subsets by Indirect Immunofluorescence Assay Using Counter‐Staining Reagents and Cellquant Calibrator Beads
  • Support Protocol 2: Selection and Titration of Monoclonal Antibody Reagent for Indirect Immunofluorescence Staining of Cell Surface Antigens
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitative Determination of Cell Surface Antigens by Indirect Immunofluorescence Assay Using Qifikit Calibrator Beads

  Materials
  • Cell suspension: whole blood, cell lines, or isolated cells
  • Unconjugated primary antibody: mouse monoclonal IgG antibody specific for the cell surface antigen under evaluation
  • Unconjugated negative control antibody: irrelevant mouse monoclonal antibody of the same isotype as the unconjugated primary antibody
  • PBS/BSA/azide: PBS ( appendix 2A) with 0.5% (w/v) BSA and 0.1% (w/v) NaN 3
  • QIFIKIT (Dako):
  •  Setup beads
  •  Calibration beads
  •  F(ab′) 2 fragment of FITC‐conjugated goat anti‐mouse (GAM) IgG
  • PBS/BSA: 1× PBS with 0.5% BSA
  • 1× ammonium chloride lysing solution ( appendix 2A) or equivalent (for whole blood only)
  • 1% paraformaldehyde in 1× PBS
  • PBS/azide: 1× PBS with 0.1% (w/v) NaN 3
  • Computer with spreadsheet software (e.g., MS Excel)
  • TallyCAL software for Windows: dedicated software for automatic calculation of antigen density (optional)

Alternate Protocol 1: Quantitative Determination of Cell Surface Antigens by Direct or Indirect Immunofluorescence Assay Using Quantum Simply Cellular Calibrator Beads

  • Quantum Simply Cellular calibration beads (Bangs Laboratories)
  • 1× PBS ( appendix 2A)
  • Fluorochrome‐conjugated mouse IgG monoclonal antibody or antibodies (see protocol 3)
  • Computer with spreadsheet software (e.g., MS Excel)
  • QuickCal data diskette to be used with QuickCal v2.1 (optional)
  • QuickCal v2.1 for Windows and Macintosh platforms (optional)
  • Additional reagents and solutions for determining optimal labeling conditions of cells with conjugated mouse IgG (see protocol 3)

Support Protocol 1: Titration of Monoclonal Antibodies to Use with Quantum Simply Cellular Calibrator Beads

  • Phycoerythrin (PE) Fluorescence Quantitation kit (BDB):
  •  QuantiBRITE PE tubes; store up to the expiration date at 2° to 4°C in manufacturer's foil pouch
  • PE‐conjugated monoclonal antibody with 1:1 PE/protein ratio
  • QuantiQuest v1.0 for Macintosh platforms (optional)
  • Additional reagents and equipment for direct immunostaining (e.g., unit 6.2)

Alternate Protocol 2: Flow Cytometric Quantitative Determination of Cell Surface Antigens by Direct Immunofluorescence Assay Using 1:1 PE‐Labeled Antibodies and QuantiBRITE PE Calibrator Beads

  • CELLQUANT Calibrator (BioCytex):
  •  FITC‐conjugated polyclonal anti‐mouse IgG
  •  1× dilution buffer: prepare appropriate volume (∼7 ml/tube) by diluting 10× concentrate 1:10 with distilled water; store up to 15 days at 2°C to 8°C
  •  Neutralizing solution: purified mouse IgG
  •  Calibration beads: Four different populations of beads bearing increasing and precise amounts of Mouse IgG MAb molecules. Lot‐specific information on the exact number of MAb is provided with each kit.
  • Unconjugated primary antibody (see protocol 6): mouse monoclonal antibody directed to a cell surface antigen of interest of the IgG 1 or IgG 2a isotype
  • Isotypic control antibody (see protocol 6): irrelevant mouse monoclonal antibody of the same isotype as the unconjugated primary mouse antibody
  • Monoclonal antibody conjugated to a different fluorochrome (e.g., PE) for counterstaining
  • Additional reagents and equipment for compensation (unit 1.14)
NOTE: Use all reagents according to manufacturer's instructions.NOTE: Any fluorochrome compatible with FITC can be employed instead of PE.

Alternate Protocol 3: Quantitative Evaluation of Cell Surface Antigen Expression on Leukocyte Subsets by Indirect Immunofluorescence Assay Using Counter‐Staining Reagents and Cellquant Calibrator Beads

  • Unconjugated primary antibody
  • Cellular sample containing both negative and positive cells for the antigen detected by the unconjugated primary antibody
  • FITC‐conjugated GAM antibody
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Figures

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Literature Cited

Literature Cited
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   Lenkei, R., Gratama, J.W., Rothe, G., Schmitz, G., D'hautcourt, J‐L., Arekrans, A., Mandy, F., and Marti, G. 1998. Performance of calibration standards for antigen quantitation with flow cytometry. Cytometry 33:188‐196.
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Key References
   Cytometry Volume 33, Number 2, October 1, 1998. A special issue of Cytometry: Quantitative Fluorescence Cytometry: An Emerging Consensus.
  This issue contains summaries of three international conferences held in Europe and the United States in late 1997, review papers that provide a general background on QFCM, original reports on clinical relevance of CD38 expression, use of QFCM in multi‐center multi‐platform situations, as well as papers on more fundamental aspects of QFCM.
   Poncelet et al., 2000. See above.
  A recent and exhaustive review, illustrating the potential of antigen quantitation with emphasis on clinical applications of QFCM. With more than one hundred references.
Internet Resources
   http://cyto.mednet.ucla.edu
  Provides a protocol written by L.E. Hultin and J.V. Giorgi for estimating the number of CD38 molecules on the CD8+ T lymphocytes of HIV‐infected individuals. Protocol for general distribution developed with support from U.S. Public Health Service Award U01‐A1‐37613:
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