Whole Blood Analysis of Leukocyte‐Platelet Aggregates

Anja J. Gerrits1, Andrew L. Frelinger1, Alan D. Michelson1

1 Center for Platelet Research Studies, Division of Hematology/Oncology, Boston Children's Hospital, Dana‐Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.15
DOI:  10.1002/cpcy.8
Online Posting Date:  October, 2016
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In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. This leukocyte‐platelet aggregate formation is initiated primarily through platelet surface expression of P‐selectin (CD62P), following activation‐dependent degranulation of α‐granules, binding to its constitutively expressed counter‐receptor, P‐selectin glycoprotein ligand 1 (PSGL‐1), on leukocytes. Monocyte‐platelet aggregates are a more sensitive marker of platelet activation than platelet surface P‐selectin. Detection of leukocyte‐platelet aggregates is relatively simple by whole‐blood flow cytometry. Light scatter and at least one leukocyte‐specific antibody are used to gate the desired population, and the presence of associated platelets is detected by immunostaining for abundant platelet‐specific markers. © 2016 by John Wiley & Sons, Inc.

Keywords: flow cytometry; leukocytes; platelets; P‐selectin; PSGL‐1; coronary artery disease; activation‐dependent receptors

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Table of Contents

  • Introduction
  • Basic Protocol 1: Whole Blood Analysis of Leukocyte‐Platelet Aggregates
  • Alternate Protocol 1: Whole Blood Analysis of Leukocyte‐Platelet Aggregates in Prefixed Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Whole Blood Analysis of Leukocyte‐Platelet Aggregates

  • Platelet‐specific antibody: monoclonal anti‐CD42a, ‐CD42b, ‐CD41, or ‐CD61 (one or more of these antigens may be missing in rare inherited disorders, e.g., Bernard‐Soulier syndrome and Glanzmann thrombasthenia; see unit 6.10, Krueger et al., )
  • Leukocyte‐specific antibody: monoclonal anti‐CD14, ‐CD64, ‐CD33, or ‐CD45 (this reagent is diluted in an antibody cocktail with the platelet‐specific antibody)
  • Negative control: an isotype‐, fluorophore‐, concentration‐, and F:P ratio‐matched non‐specific antibody for background fluorescence determination (to be used in conjunction with the platelet‐specific antibody)
  • Platelet agonist: such as adenosine diphosphate (ADP; Chrono‐Log, cat. no. 384) or thrombin receptor‐activating peptide (TRAP; Bachem, cat. no. H‐2936; see reciperecipes)
  • Modified HEPES/Tyrode's (HT) buffer (see recipe)
  • Source of human whole blood (patient or donor)
  • Fix/lyse solution: any red‐cell lysis reagent optimized for flow cytometry and containing formaldehyde, e.g., FACS lysing solution, Becton Dickinson, or Optilyse, Beckman Coulter (the performance of these products must be assessed by the individual laboratory)
  • Blood collection tubes containing buffered sodium citrate or other suitable anticoagulant (BD Vacutainers) (see unit 6.10, Krueger et al., )
  • 21‐G (or larger bore) needles
  • Flow cytometer with 488‐nm excitation and suitable filters for collection of fluorophore emissions
NOTE: Individual laboratories must determine optimal assay conditions and sample stability.

Alternate Protocol 1: Whole Blood Analysis of Leukocyte‐Platelet Aggregates in Prefixed Samples

  Additional Materials (also see protocol 1Basic Protocol)
  • 5‐ml tubes
  • Centrifuge
  • Vortex mixer
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Literature Cited

Literature Cited
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