Collection, Storage, and Preparation of Human Blood Cells

Pradeep K. Dagur1, J. Philip McCoy1

1 National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 5.1
DOI:  10.1002/0471142956.cy0501s73
Online Posting Date:  July, 2015
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Abstract

Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non‐flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils, and platelets, prior to flow cytometric analysis. A protocol is also offered for cryopreservation of cells, since clinical research often involves retrospective flow cytometric analysis of samples stored over a period of months or years. © 2015 by John Wiley & Sons, Inc.

Keywords: flow cytometry; human blood; leukocyte preparation; mononuclear blood cells; lymphocyte enrichment; cell population isolation; cell population purification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Storage of Whole Blood Prior to Staining
  • Basic Protocol 2: Preparation of White Cell Suspension by Lysis of Erythrocytes Using Ammonium Chloride Lysis
  • Alternate Protocol 1: Preparation of White Cell Suspension by Lysis of Erythrocytes Using Lysing Reagent Containing a Fixative
  • Basic Protocol 3: Isolation of Mononuclear Cells by Density Gradient Separation
  • Support Protocol 1: Enrichment of Lymphocytes from Mononuclear Cell Preparations by Depletion of Monocytoid Cells Through Adherence to Plastic
  • Basic Protocol 4: Cryopreservation of Mononuclear Cells
  • Basic Protocol 5: Isolation of Peripheral Blood Monocytes by Gradient Centrifugation with Ficoll‐Hypaque Followed by Percoll
  • Basic Protocol 6: Isolation of B Lymphocytes by Positive Magnetic Bead–Antibody Separation
  • Alternate Protocol 2: Isolation of T Cells by Negative Bead Selection
  • Basic Protocol 7: Enrichment of B or NK Cells by Removal of T Lymphocytes Through Complement‐Mediated Lysis
  • Basic Protocol 8: Isolation of Neutrophils by Percoll Gradient Centrifugation
  • Basic Protocol 9: Preparation of Platelet‐Enriched Plasma
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Storage of Whole Blood Prior to Staining

  Materials
  • Anticoagulated whole blood from donor
  • TransFix Cellular Antigen Stabilization Reagent (Cytomark; http://www.cytomark.co.uk)
  • Blood collection tube containing anticoagulant

Basic Protocol 2: Preparation of White Cell Suspension by Lysis of Erythrocytes Using Ammonium Chloride Lysis

  Materials
  • Anticoagulated whole blood from donor
  • Homemade ammonium chloride lysis solution (see appendix 2A), or commercial RBC lysis buffer without fixative, such as ACK lysis buffer (Quality Biological), Optilyse (Beckman Coulter), PharmLyse (BD Bioscience), IOTest3 (Beckman Coulter), Hi‐Yield Lyse (Life Technologies), Easy‐Lyse Erythrocyte Lysis Solution (Dako)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Tissue culture medium (optional; appendix 3B)
  • Rocker
  • Refrigerated centrifuge with swinging‐bucket rotor and adaptors for 15‐ and 50‐ml tubes

Alternate Protocol 1: Preparation of White Cell Suspension by Lysis of Erythrocytes Using Lysing Reagent Containing a Fixative

  Materials
  • Anticoagulated whole blood from donor
  • FACSLyse (Becton Dickinson), ImmunoLyse (Beckman Coulter), or Optilyse (Beckman Coulter)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Refrigerated centrifuge with swinging‐bucket rotor and adaptors for 15‐ and 50‐ml tubes

Basic Protocol 3: Isolation of Mononuclear Cells by Density Gradient Separation

  Materials
  • Cocktail of RosetteSep Tetrameric antibody complexes (Stem Cell Technologies), if necessary.
  • Whole blood anticoagulated with heparin or EDTA
  • 1.077 g/ml Ficoll‐Hypaque (GE Healthcare) or Histopaque‐1077 (Sigma)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Tissue culture medium (optional; appendix 3B)
  • 15‐ and 50‐ml conical centrifuge tubes
  • Refrigerated centrifuge with swinging‐bucket rotor and adaptors for 15‐ and 50‐ml tubes.

Support Protocol 1: Enrichment of Lymphocytes from Mononuclear Cell Preparations by Depletion of Monocytoid Cells Through Adherence to Plastic

  Additional Materials (also see protocol 4)
  • Mononuclear cell preparation (see protocol 4)
  • Tissue culture medium ( appendix 3B) containing ≥10% FBS ( appendix 2A)
  • 75‐cm2 or 150‐cm2 plastic tissue culture flask
  • Hematology analyzer (optional)
  • Additional reagents and equipment for assessing cell viability (unit 9.2; Johnson et al., 2013)

Basic Protocol 4: Cryopreservation of Mononuclear Cells

  Materials
  • Hyclone HI‐FBS (GE Healthcare, cat. no. SH30071.03HI)
  • Dimethylsulfoxide (DMSO; Fisher, cat. no. D128500)
  • Peripheral blood mononuclear cells ( protocol 4 or protocol 5Support Protocol)
  • 0.4% (w/v) trypan blue
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐072)
  • Liquid nitrogen
  • Falcon Blue Max 50‐ml polypropylene conical tube (Becton Dickinson Labware, cat. no. 352098, or equivalent )
  • 0.22‐μm sterile filters
  • 1.8‐ml cryogenic vials (Nalgene, 5000‐0020)
  • Hemacytometer
  • Planer 750Plus Controlled Rate Freezer (Planer PLC)
  • Refrigerated tabletop centrifuge (Kendro Laboratory Products, cat. no. 75004377)
  • Liquid nitrogen storage tank
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A)

Basic Protocol 5: Isolation of Peripheral Blood Monocytes by Gradient Centrifugation with Ficoll‐Hypaque Followed by Percoll

  Materials
  • Anticoagulated whole blood from donor
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1.077 g/ml Ficoll‐Hypaque (GE Healthcare) or Histopaque‐1077 (Sigma)
  • 10× Hanks balanced salt solution (HBSS; appendix 2A)
  • 0.1 N HCl
  • Percoll, specific gravity 1.130 g/ml (Sigma)
  • Tissue culture medium (optional; appendix 3B)
  • 50‐ml conical centrifuge tubes
  • Refrigerated centrifuge with swinging‐bucket rotor and adaptors for 15‐ and 50‐ml tubes
  • 10 × 15‐cm round bottom polypropylene tubes, silanized with Surfasil

Basic Protocol 6: Isolation of B Lymphocytes by Positive Magnetic Bead–Antibody Separation

  Materials
  • Anti‐CD19‐coated magnetic beads (Life Technologies/Stem Cell Technologies/Miltenyi)
  • 1% FBS/PBS: phosphate‐buffered saline (PBS; appendix 2A) with 1% (v/v) FBS (heat inactivated; appendix 2A), 4°C
  • ACK lysed whole blood leukocytes or PBMCs (Basic Protocols protocol 11 and protocol 22)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete tissue culture medium with 10% FBS ( appendix 2A)
  • Detach‐a‐bead (Life Technologies) or other anti‐mouse Fab antibody (optional)
  • 15‐ml and 50‐ml polypropylene round‐bottom or conical bottom test tubes
  • Magnetic separation device (e.g., Life Technologies/Stem Cell Technologies/Miltenyi)
  • Rocker
  • Refrigerated centrifuge with swinging‐bucket rotor and adaptors for 15‐ and 50‐ml tubes

Alternate Protocol 2: Isolation of T Cells by Negative Bead Selection

  Materials
  • ACK lysed whole blood leukocytes or PBMCs (see Basic Protocols protocol 11 and protocol 43)
  • Cocktail of anti‐CD19, anti‐CD14, anti‐CD56, anti‐ CD33, anti‐CD15, anti‐CD16, anti‐CD123, and anti‐CD235a‐coated magnetic beads (Life Technologies/Stem Cell Technologies/Miltenyi)
  • 1% FBS/PBS: phosphate‐buffered saline (PBS; appendix 2A) with 1% (v/v) FBS (heat inactivated; appendix 2A), 4°C
  • Tissue culture medium with 10% FBS ( appendix 2A)
  • 15‐ml polypropylene round‐bottom (or conical bottom) test tube
  • Magnetic column and magnetic separator (Miltenyi) or other magnetic separation device (e.g., Life Technologies/Stem Cell Technologies)
  • 75‐cm2 tissue culture flask
  • Additional reagents and equipment for counting cells ( protocol 6) and flow cytometry (Robinson et al., 2015)

Basic Protocol 7: Enrichment of B or NK Cells by Removal of T Lymphocytes Through Complement‐Mediated Lysis

  Materials
  • Mononuclear cell preparation (see protocol 4)
  • RPMI‐1640 medium supplemented with 1% heat‐inactivated fetal bovine serum (FBS)
  • Anti–T cell antibodies: CD3 (eBioscience, clone OKT3, cat. no. 14‐0037; or BioLegend, clone 17A2, cat. no. 100201)
  • Rabbit complement (Harlan Laboratories)
  • Serum‐free RPMI‐1640 medium
  • 15‐ml conical centrifuge tube (e.g., BD Falcon)
  • Refrigerated centrifuge with swinging‐bucket rotor and adaptors for 15‐ and 50‐ml tubes

Basic Protocol 8: Isolation of Neutrophils by Percoll Gradient Centrifugation

  Materials
  • Blood sample anticoagulated with acid citrate dextrose, formula A
  • 0.6% (w/v) dextran in 0.9% (w/v) NaCl
  • 0.9% NaCl
  • 1.10 g/ml, 1.095 g/ml, and 1.085 g/ml Percoll in phosphate‐buffered saline (PBS; appendix 2A)
  • Refrigerated centrifuge with swinging‐bucket rotor and adaptors for 15‐ and 50‐ml tubes
  • 15‐ml polypropylene tubes

Basic Protocol 9: Preparation of Platelet‐Enriched Plasma

  Materials
  • Peripheral blood in EDTA or appropriate anticoagulant
  • Tyrode's buffer (see recipe)
  • Refrigerated centrifuge with swinging‐bucket rotor and adaptors for 15‐ and 50‐ml tubes
  • 15‐ml conical centrifuge tube
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Literature Cited

Literature Cited
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  Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. Suppl. 21:77‐89.
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Key References
  Clinical and Laboratory Standards Institute. Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline—Second Edition. CLSI document H42‐A2 [ISBN 1‐56238‐000‐0]. Clinical and Laboratory Standards Institute, Wayne, Pa.
  Provides general guidelines for performance of flow cytometric immunophenotyping.
  Clinical and Laboratory Standards Institute. Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition. CLSI document H43‐A2 [ISBN 1‐56238‐000‐0]. Clinical and Laboratory Standards Institute, Wayne, Pa.
  Provides more detailed guidelines for flow cytometric immunophenotyping of leukemias and lymphomas.
  Carey, J.L., McCoy, J.P., and Keren, D.F. 2007. Flow Cytometry in Clinical Diagnosis, 4th ed. ASCP Press, Chicago.
  Provides an overview for the performance of flow cytometric immunophenotyping in the clinical laboratory.
  Stewart, C.C. 1990. Cell preparation for the identification of leukocytes. Methods Cell Biol. 33:411‐426.
  Details sample preparation methods for immunophenotyping studies.
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