Enumeration of Absolute Cell Counts Using Immunophenotypic Techniques

Frank Mandy1, Bruno Brando2

1 Health Canada, Ottawa, Canada, 2 Niguarda‐Ca' Granda Hospital, Milano, Italy
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.8
DOI:  10.1002/0471142956.cy0608s13
Online Posting Date:  May, 2001
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Abstract

Absolute counting of cells or cell subsets has a number of significant clinical applications: monitoring the disease status of HIV‐infected patients, enumerating residual white blood cells in leukoreduced blood products, and assessing immunodeficiency in a variety of situations. The single‐platform method (flow cytometry alone) has emerged as the method of choice for absolute cell enumeration. This technology counts only the cells of interest in a precisely determined blood volume. Exact cell identification is accomplished by a logical electronic gating algorithm capable of identifying lineage‐specific immunofluorescent markers. Exclusion of unwanted cells is automatic. This extensive and detailed unit presents protocols for both volumetric and flow‐rate determination of residual white blood cells and of leukocyte subsets. Keywords: absolute count; volumetric counting; flow rate cytometry; counting microsphere standards; single‐platform absolute count; dual‐platform absolute count; logical gate; lineage‐specific markers; immunophenotyping; absolute residual WBC

     
 
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Table of Contents

  • Basic Protocol 1: Single‐Platform Enumeration of Absolute Numbers of Residual WBCs in Leukoreduced Blood Products Using Flow‐Count Fluorospheres
  • Alternate Protocol 1: Single‐Platform Enumeration of Absolute Numbers of Residual WBCs in Leukoreduced Blood Products Using Fluorosphere‐Containing TruCount Tubes
  • Alternate Protocol 2: Single‐Platform Enumeration of Absolute Numbers of Residual WBCs in Leukoreduced Blood Products Using the Partec‐PAS/DAKO Galaxy Volumetric Flow Cytometer
  • Basic Protocol 2: Universal Single‐Platform Method for Absolute Leukocyte Subset Counting
  • Support Protocol 1: Setting Up the Universal Template
  • Alternate Protocol 3: Single‐Platform Method for Absolute Leukocyte Subset Counting Using Flow‐Count Fluorospheres with Epics XL Instruments
  • Alternate Protocol 4: Single‐Platform Method for Absolute Leukocyte Subset Counting with TruCount Tubes Using FACScan/FACScalibur Instruments
  • Alternate Protocol 5: Single‐Platform Method for Absolute Leukocyte Subset Counting Using a Partec‐PAS/DAKO Galaxy Volumetric Flow Cytometer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Single‐Platform Enumeration of Absolute Numbers of Residual WBCs in Leukoreduced Blood Products Using Flow‐Count Fluorospheres

  Materials
  • Sample of interest: leukoreduced blood sample or leukoreduced platelet sample
  • Staining buffer (see recipe)
  • Flow‐Count beads (aqueous suspension of microspheres; Beckman‐Coulter)
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer with at least two fluorescence detectors and appropriate filter set to detect red flurorescence from propidium iodide (>650 nm) and green fluorescence from beads (530 ± 15 nm).

Alternate Protocol 1: Single‐Platform Enumeration of Absolute Numbers of Residual WBCs in Leukoreduced Blood Products Using Fluorosphere‐Containing TruCount Tubes

  • TruCount absolute count tubes (Becton Dickinson Immunocytometry)

Alternate Protocol 2: Single‐Platform Enumeration of Absolute Numbers of Residual WBCs in Leukoreduced Blood Products Using the Partec‐PAS/DAKO Galaxy Volumetric Flow Cytometer

  • Partec‐PAS/DAKO Galaxy flow cytometer with at least two fluorescence detectors and appropriate filter set to detect red fluorescence from propidium iodide (>650 nm) and green fluorescence from beads (530 ± 15 nm)

Basic Protocol 2: Universal Single‐Platform Method for Absolute Leukocyte Subset Counting

  Materials
  • TruCount absolute count tubes (lyophylized pellet of beads in a test tube; Becton Dickinson Immunocytometry) or Flow‐Count beads (aqueous suspension of microspheres; Beckman‐Coulter)
  • Sample of interest: whole blood anticoagulated with EDTA
  • Commercial “lyse‐no‐wash” erythrocyte lysing solution (e.g., Beckman‐Coulter or Becton Dickinson)
  • 1% to 2% (v/v) formaldehyde or paraformaldehyde in PBS (see appendix 2A for PBS)
  • Premixed monoclonal antibody combinations (available from a number of suppliers; also see unit 6.2)
  •  4 color panel: CD45/CD3/CD4/CD8
  •  3 color panel: CD45/CD3/CD4 and CD45/CD3/CD8
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer
  • Additional reagents and equipment for immunophenotyping (unit 6.2) and setting up the universal template (see protocol 5)

Support Protocol 1: Setting Up the Universal Template

  Materials
  • Premixed monoclonal antibody: Cyto‐Stat reagents (Beckman‐Coulter); 2‐color, 3‐color, or 4‐color:
  •  CD3‐FITC/T4‐RD1 and CD3‐FITC/T8‐RD1
  •  CD45‐FITC/CD4‐RD1/CD3‐PC5 and CD45‐FITC/CD8‐RD1/CD3‐PC5
  •  CD45‐FITC/CD4‐RD1/CD8‐ECD /CD3‐PC5
  • Sample of interest: whole blood anticoagulated with EDTA
  • ImmunoPrep Reagent System for whole blood lysis (Beckman‐Coulter)
  •  ImmunoPrep solution A: formic acid
  •  ImmunoPrep solution B: sodium carbonate/sodium chloride/sodium sulfate
  •  ImmunoPrep solution C: paraformaldehyde
  • Flow‐Count beads (aqueous suspension of microspheres; Beckman‐Coulter)
  • 1% to 2% (v/v) formaldehyde or paraformaldehyde in PBS (see appendix 2A for PBS)
  • 12 × 75–mm polystyrene test tubes
  • Q‐Prep, Multi‐Q‐Prep, or TQ‐Prep immunocytometry workstation (Beckman‐Coulter)
  • Flow cytometer with at least 3 (or 4) fluorescence detectors.

Alternate Protocol 3: Single‐Platform Method for Absolute Leukocyte Subset Counting Using Flow‐Count Fluorospheres with Epics XL Instruments

  Materials
  • TruCount absolute count tubes (lyophilized pellet of beads in a test tube; Becton Dickinson Biosciences)
  • TriTest premixed monoclonal antibody combinations (Becton Dickinson):
  •  CD45‐PerCP/CD3‐FITC/CD4‐PE
  •  CD45‐PerCP/CD3‐FITC/CD8‐PE
  • Sample of interest: whole blood anticoagulated with EDTA
  • 1× FACS lysing solution (Becton Dickinson)
  • 1% to 2% (v/v) formaldehyde or paraformaldehyde in PBS (see appendix 2A for PBS)
  • FACScan or FACScalibur flow cytometer (Becton Dickinson)

Alternate Protocol 4: Single‐Platform Method for Absolute Leukocyte Subset Counting with TruCount Tubes Using FACScan/FACScalibur Instruments

  Materials
  • Sample of interest: whole blood anticoagulated with EDTA
  • 3‐color antibody mixture including CD4‐FITC/CD8‐PE/CD3‐PE‐Cy5 (or PerCP) or 4‐color antibody mixture including CD4‐FITC/CD8‐PE/CD3‐PE‐Cy5 (or ‐PerCP)/CD19‐APC
  • Staining buffer (see recipe)
  • Ammonium chloride lysing buffer, pH 7.2 ( appendix 2A)
  • 12 × 75–mm polystyrene test tubes
  • Partec‐PAS/DAKO Galaxy flow cytometer with at least two fluorescence detectors and appropriate filter sets
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Figures

Videos

Literature Cited

Literature Cited
   Bene, M.C., Kolopp Sarda, M.N., Kaissouni, J.E., De March Kennel, A., Mole, C. Kohler, C., and Faure, G. 1998. Automated cell counting in flow cytometry. Am J. Clin. Path. 110:321‐326.
   Bland, J.M. and Altman, D.G. 1986. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet I:307‐310.
   Connely, M.C.M., Knight, M., Giorgy, J.V., Kagan, J., Landay, A.L., Parker, J.W., Page, E., Spino, C., Wilkening, C., and Mercolino, T.J. 1995. Standardization of Absolute CD4+ lymphocyte count across laboratories: An evaluation of the Ortho CytoronAbsolute flow cytometry system on normal donors. Cytometry 22:200‐210.
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   Schlenke, P., Frohn, C., Klueter, H., Saballus, M., Hammers, H.J., Zajac, S.R., and Kirchner, H. 1998. Evaluation of a flow cytometric method for simultaneous leukocyte phenotyping and quantification by fluorescent microspheres. Cytometry 33:310‐317.
   Schnizlein‐Bick, C.T., Spritzler, J., Wilkening, C., Nicholson, J.K.A., O'Gorman, R.G. 2000. Evaluation of the TruCount absolute‐counting tubes for detrminating CD4 and CD8 cell numbers in human immunodeficiency virus‐positive adults. Clin. Diag. Lab. Immunol. 7:336‐343.
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