Ex vivo assays to detect complement activation in complementopathies

Xuan Yuan, Jia Yu, Gloria Gerber, Shruti Chaturvedi, Michael Cole, Hang Chen, Ara Metjian, C. John Sperati, Evan M. Braunstein, Robert A. Brodsky

Research output: Contribution to journalArticlepeer-review

Abstract

In complement-driven thrombotic microangiopathies, failure to regulate complement activation leads to end-organ damage. The modified Ham (mHam) test measures complement-mediated killing of a nucleated cell in vitro but lacks a confirmatory assay and reliable positive controls. We demonstrate that C5b-9 accumulation on the surface of TF1 PIGAnull cells correlates with cell killing in the mHam. We also show that Sialidase treatment of cells or addition of Shiga toxin 1 to human serum serve as a more reliable positive control for the mHam than cobra venom factor or lipopolysaccharide. Simultaneously performing the mHam and measuring C5b-9 accumulation either in GVB++ or GVB0 MgEGTA buffer with the addition of complement pathway specific inhibitors (anti-C5 antibody or a factor D inhibitor, ACH-145951) can be used to localize defects in complement regulation. As more targeted complement inhibitors become available, these assays may aid in the selection of personalized treatments for patients with complement-mediated diseases.

Original languageEnglish (US)
Article number108616
JournalClinical Immunology
Volume221
DOIs
StatePublished - Dec 2020

Keywords

  • Atypical hemolytic uremic syndrome
  • Complement
  • Complement inhibitors
  • Modified HAM assay
  • Shiga toxin 1
  • Sialidase

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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