Modified Ham test for atypical hemolytic uremic syndrome

Blood. 2015 Jun 4;125(23):3637-46. doi: 10.1182/blood-2015-02-629683. Epub 2015 Apr 10.

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by excessive activation of the alternative pathway of complement (APC). Atypical HUS is frequently a diagnosis of exclusion. Differentiating aHUS from other TMAs, especially thrombotic thrombocytopenic purpura (TTP), is difficult due to overlapping clinical manifestations. We sought to develop a novel assay to distinguish aHUS from other TMAs based on the hypothesis that paroxysmal nocturnal hemoglobinuria cells are more sensitive to APC-activated serum due to deficiency of glycosylphosphatidylinositol- anchored complement regulatory proteins (GPI-AP). Here, we demonstrate that phosphatidylinositol-specific phospholipase C-treated EA.hy926 cells and PIGA-mutant TF-1 cells are more susceptible to serum from aHUS patients than parental EA.hy926 and TF-1 cells. We next studied 31 samples from 25 patients with TMAs, including 9 with aHUS and 12 with TTP. Increased C5b-9 deposition was evident by confocal microscopy and flow cytometry on GPI-AP-deficient cells incubated with aHUS serum compared with heat-inactivated control, TTP, and normal serum. Differences in cell viability were observed in biochemically GPI-AP-deficient cells and were further increased in PIGA-deficient cells. Serum from patients with aHUS resulted in a significant increase of nonviable PIGA-deficient TF-1 cells compared with serum from healthy controls (P < .001) and other TMAs (P < .001). The cell viability assay showed high reproducibility, sensitivity, and specificity in detecting aHUS. In conclusion, we developed a simple, rapid, and serum-based assay that helps to differentiate aHUS from other TMAs.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Atypical Hemolytic Uremic Syndrome / blood*
  • Atypical Hemolytic Uremic Syndrome / diagnosis*
  • Atypical Hemolytic Uremic Syndrome / genetics
  • Cell Survival
  • Female
  • Humans
  • Male
  • Membrane Proteins / blood
  • Membrane Proteins / genetics
  • Middle Aged
  • Purpura, Thrombotic Thrombocytopenic / blood
  • Purpura, Thrombotic Thrombocytopenic / diagnosis
  • Purpura, Thrombotic Thrombocytopenic / genetics
  • Serum / metabolism

Substances

  • Membrane Proteins
  • phosphatidylinositol glycan-class A protein