Immunophenotypic Analysis of Peripheral Blood Lymphocytes

Alejandro Ruiz‐Argüelles1, Beatriz Pérez‐Romano1

1 Laboratorios Clínicos de Puebla, Puebla, Mexico
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.5
DOI:  10.1002/0471142956.cy0605s11
Online Posting Date:  May, 2001
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Abstract

This unit on basic phenotyping describes two basic and two alternate protocols for the immunophenotypic identification and classification of human peripheral blood lymphocytes. The presented protocols comply with consensus recommendations from professional organizations that regulate the clinical use of such assays. The authors discuss whole blood assay systems as well as enriched systems from several perspectives, including absolute number determination.

Keywords: flow cytometry; peripheral blood; lymphocytes; immunophenotype; lymphocyte subsets

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Immunophenotypic Analysis of the Relative Distribution of Lymphocyte Subsets in Erythrocyte‐Lysed‐and‐then‐Washed Whole Peripheral Blood Samples
  • Alternate Protocol 1: Immunophenotypic Analysis of the Relation Distribution of Lymphocyte Subsets in Erythrocyte‐Lysed‐non‐Washed Whole Peripheral Blood Samples
  • Alternate Protocol 2: Flow Cytometric Immunophenotypic Analysis of Absolute Numbers of Peripheral Blood Lymphocyte Subsets
  • Basic Protocol 2: Lymphocyte Immunophenotypic Analysis of Peripheral Blood Mononuclear Cell (PBMC)–Enriched Suspensions
  • Support Protocol 1: Light‐Scatter Gating Strategy for the Immunophenotypic Analysis of Peripheral Blood Lymphocytes
  • Support Protocol 2: CD45 Immuno‐Gating Strategy for the Phenotypic Analysis of Perpheral Blood Lymphocytes
  • Support Protocol 3: Multiparametric Immuno‐Gating Strategy for the Phneotypic Analysis of Peripheral Blood Lymphocytes
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunophenotypic Analysis of the Relative Distribution of Lymphocyte Subsets in Erythrocyte‐Lysed‐and‐then‐Washed Whole Peripheral Blood Samples

  Materials
  • Blood to be tested, with tripotassium EDTA or heparin as anticoagulant
  • Fluorochrome‐conjugated monoclonal antibodies directed against the cell surface antigens of interest
  • Ammonium chloride lysing solution ( appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A), filtered through a 0.45‐µm filter
  • PBS ( appendix 2A) containing 1% paraformaldehyde (prepare fresh)
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer equipped with four fluorescence detectors

Alternate Protocol 1: Immunophenotypic Analysis of the Relation Distribution of Lymphocyte Subsets in Erythrocyte‐Lysed‐non‐Washed Whole Peripheral Blood Samples

  • Coulter Immunoprep lysing and fixing kit
  • Coulter Q‐Prep immunology workstation

Alternate Protocol 2: Flow Cytometric Immunophenotypic Analysis of Absolute Numbers of Peripheral Blood Lymphocyte Subsets

  • Coulter Immunoprep lysing and fixing kit
  • Coulter Q‐Prep immunology workstation
  • Coulter Flow COUNT beads

Basic Protocol 2: Lymphocyte Immunophenotypic Analysis of Peripheral Blood Mononuclear Cell (PBMC)–Enriched Suspensions

  Materials
  • Blood to be tested, with tripotassium EDTA or heparin as anticoagulant
  • Phosphate‐buffered saline (PBS; appendix 2A), 10°C
  • Lymphocyte separation medium (LSM; e.g., Ficoll‐Paque PLUS from Pharmacia Biotech, Lymphoprep 1077 or Nycoprep 1077 from Life Technologies, LymphoSep from ICN, Histopaque 1077 from Sigma, or LeucoPREP from Becton Dickinson), 10°C
  • Fluorophore‐conjugated monoclonal antibodies directed against the cell surface antigens of interest
  • 15‐ml conical polypropylene centrifuge tubes
  • 12 × 75–mm polypropylene tubes
  • Centrifuge and rotor capable of 2000 × g, refrigerated
  • Flow cytometer
  • Additional reagents and equipment for counting cells ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Bauer, K.D., Duque, R.E., and Shankey, T.V. 1993. Clinical Flow Cytometry: Principles and Application. Williams & Wilkins, Baltimore.
   Keren, D.F., Hanson, C.A., and Hurtubise, P.E. 1994. Flow Cytometry and Clinical Diagnosis. ASCP Press, Chicago, Ill.
   Kishimoto, T., Kikutani, H., von dem Borne, A.E.G.K., Goyert, S.M., Mason, D.Y., Miyasaka, M., Moretta, L., Okumura, K., Shaw, S., Springer, T.A., Sugamura, K., and Zola, H. 1997. Leukocyte Typing VI. White Cell Differentiation Antigens. Garland Publishing, New York.
   Lahita, R.G. 1992. Systemic Lupus Erythematosus. 2nd ed. Churchill Livingston, New York.
   Loken, M.R., Brosnan, J.M., Bach, B.A., and Ault, K.A. 1990. Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry. Cytometry 11:453‐459.
   Macey, M.G. 1999. Flow Cytometry. Clinical Applications. Blackwell Scientific, Oxford.
   National Committee for Clinical Laboratory Standards. 1992. Clinical Applications of Flow Cytometry: Quality Assurance and Immunophenotyping of Peripheral Blood Lymphocytes. NCCLS Publication H42‐T. NCCLS, Wayne, Pa.
   Nicholson, J.K.A., Hubbard, M., and Jones, B.M. 1996. Use of CD45 fluorescence and side scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometry. Cytometry 26:16‐21.
   Rich, R.E. 1996. Clinical Immunology. Principles and Practice. Mosby, Baltimore, Md.
   Roitt, I., Brostoff, J., Male, D. 1998. Immunology. 5th ed. Mosby, London.
   Ruiz‐Argüelles, A., Duque, R.E., and Orfao, A. 1998. Report on the First Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia. Cytometry 34:39‐42.
   U.S. Department of Health and Human Services. Centers for Disease Control and Prevention. 1997. Revised Guidelines for Performing CD4+ T Cell Determinations in Persons Infected With Human Immunodeficiency Virus (HIV). MMWR 46:RR‐2;1‐29.
Internet Resources
  http://www.cyto.purdue.edu
  Purdue University cytometry labs.
  http://www.nih.gov
   National Institutes of Health home page.
  http://www.cdc.gov
   U.S. Centers for Disease Control home page.
  http://www.cyto.purdue.edu/step/stephome.htm
   ISAC Specialty Training and Education Program home page.
  http://www.journals.wiley.com/cytometry/
   John Wiley & Sons cytometry Web site.
  http://www.cytometry.org/
   Clinical Cytometry Society Web site.
  http://dpalm2.med.uth.tmc.edu/edprog/00000014.htm
   Diagnostic flow cytometry tutorial.
  http://www.club.ch/scs/cytometr.htm
   Cytometry HotLinks; links to many flow cytometry–related sites.
  http://flosun.salk.edu
   Salk Institute flow cytometry laboratory home page.
  http://www.ncrr.nih.gov/ncrrprog/btceltec.htm#flow
   National Flow Cytometry Resource.
  http://www.coulter.com
   Beckman Coulter home page.
  http://www.enterprise.net/appcysys/
   Applied Cytometry Systems page.
  http://www.phnxflow.com
   Phoenix Flow Systems home page.
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