Modified Ham test for atypical hemolytic uremic syndrome.

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.

• Authors: Eleni Gavriilaki, Xuan Yuan, Zhaohui Ye, Alexander J Ambinder, Satish P Shanbhag, Michael B Streiff, Thomas S Kickler, Alison R Moliterno, C John Sperati, Robert A Brodsky
• Journal: Blood (Blood) Vol. 125 Issue 23 Pg. 3637-46 (Jun 04 2015) ISSN: 1528-0020 [Electronic] United States
• PMID: 25862562 (Publication Type: Clinical Trial, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 by The American Society of Hematology.

Chemical References

• Membrane Proteins
• phosphatidylinositol glycan-class A protein

Topics

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