Mouse Models for Immune‐Mediated Platelet Destruction or Immune Thrombocytopenia (ITP)

Anton Neschadim1, Donald R. Branch2

1 Centre for Innovation, Canadian Blood Services, Toronto, Ontario, 2 Division of Advanced Diagnostics, Infection and Immunity Group, Toronto General Research Institute, Toronto, Ontario
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.30
DOI:  10.1002/0471142735.im1530s113
Online Posting Date:  April, 2016
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Abstract

Immune thrombocytopenia (ITP) is a debilitating, life‐threatening autoimmune disorder affecting more than 4 in every 100,000 adults annually, stemming from the production of antiplatelet antibody resulting in accelerated platelet destruction and thrombocytopenia. Numerous animal models of ITP have been developed that contributed to the basic understanding of the underlying mechanisms of ITP onset, progression, and maintenance. Rodent models that develop ITP spontaneously, or by passive transfer of an antiplatelet sera or antibody, play an instrumental role in the investigation of ITP mechanisms responsible for the breakdown of tolerance in human ITP, in studies of the immunopathology underlying the progression of platelet destruction, and in elucidation of the mechanisms of therapeutic amelioration of ITP by existing and new therapeutic modalities. This unit captures the protocols for the implementation and readout of passive antibody transfer mouse models of ITP, established by the infusion of a commercially‐available monoclonal rat anti‐mouse CD41 platelet antibody. © 2016 by John Wiley & Sons, Inc.

Keywords: immune thrombocytopenia; ITP; intravenous immunoglobulin; IVIg; platelets; autoimmune disease: mouse model

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Escalating‐Dose Anti‐Mouse CD41 (Anti‐Glycoprotein IIb) Passive Transfer ITP Model
  • Alternate Protocol 1: Constant‐Dose Anti‐Mouse CD41 (Anti‐Glycoprotein IIb) Passive Transfer ITP Model
  • Reagents and Solutions
  • COMMENTARY
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Escalating‐Dose Anti‐Mouse CD41 (Anti‐Glycoprotein IIb) Passive Transfer ITP Model

  Materials
  • Female inbred Balb/c or C57BL/6 mice, 6 to 8 weeks of age (e.g., Charles River Laboratories, Jackson Laboratories)
  • Rat monoclonal anti‐mouse glycoprotein IIb antibody, unconjugated and fluorescein isothiocyanate (FITC)‐conjugated (CD41; clone MWReg30, rat IgG1, k; e.g., BD Biosciences)
  • PBS, pH 7.2 (with calcium and magnesium; see appendix 2A)
  • Treatment solutions (optional)
  • 10% citrate‐phosphate‐dextrose‐adenine (CPDA) in PBS (see recipe)
  • Vaseline or glycerin
  • 10% or 100 mg/ml human IVIg (Gammagard S/D; e.g., Baxter Healthcare)
  • 1.5‐ml microcentrifuge tubes
  • 50‐ml conical polypropylene tubes with sawed‐off tip (for use as conical tube animal restrainer)
  • 25‐G (5/8 in.) and 27‐G (1/2 in.) needles with syringes
  • P20 (0.5 to 20 μl) pipet tips
  • Gauze
  • 5‐ml round‐bottom tubes
  • Flow cytometer (e.g., BD Biosciences, FACSCalibur)
  • Hematology analyzer (e.g., Beckman Coulter, cat. no. LH750)

Alternate Protocol 1: Constant‐Dose Anti‐Mouse CD41 (Anti‐Glycoprotein IIb) Passive Transfer ITP Model

  Materials
  • Female inbred Balb/c or C57BL/6 mice, 6 to 8 weeks of age (e.g., Charles River Laboratories, Jackson Laboratories)
  • Rat monoclonal anti‐mouse glycoprotein IIb antibody, unconjugated and fluorescein isothiocyanate (FITC)‐conjugated (CD41; clone MWReg30, rat IgG1, k; e.g., BD Biosciences)
  • PBS, pH 7.2 (with calcium and magnesium, see appendix 2A)
  • Treatment solutions (optional)
  • 10% citrate‐phosphate‐dextrose‐adenine (CPDA) in PBS (see recipe)
  • Vaseline or glycerin
  • 10% or 100 mg/ml human IVIg (Gammagard S/D; e.g., Baxter Healthcare)
  • 1.5‐ml microcentrifuge tubes
  • 50‐ml conical polypropylene tubes with sawed‐off tip (for use as conical tube animal restrainer)
  • 25‐G (5/8 in.) and 27‐G (1/2 in.) needles with syringes
  • P20 (0.5 to 20 μl) pipet tips
  • Gauze
  • 5‐ml round‐bottom tubes
  • Flow cytometer (e.g., BD Biosciences, FACSCalibur)
  • Hematology analyzer (e.g., Beckman Coulter, cat. no. LH750)
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Figures

Videos

Literature Cited

Literature Cited
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