Modified Ham test for atypical hemolytic uremic syndrome

Area of Science:

• Complement System Biology
• Hematology
• Diagnostic Assay Development

Background:

• Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) driven by alternative complement pathway overactivation.
• Distinguishing aHUS from other TMAs like thrombotic thrombocytopenic purpura (TTP) is challenging due to similar clinical presentations.
• Diagnosis of aHUS is often a diagnosis of exclusion, highlighting the need for specific diagnostic tools.

Purpose of the Study:

• To develop a novel, serum-based assay for differentiating aHUS from other TMAs.
• To leverage the heightened sensitivity of glycosylphosphatidylinositol-anchored complement regulatory protein (GPI-AP)-deficient cells to excessive complement activation in aHUS.

Main Methods:

• Utilized phosphatidylinositol-specific phospholipase C-treated EA.hy926 cells and PIGA-mutant TF-1 cells, which are deficient in GPI-APs.
• Assessed susceptibility of these deficient cells to serum from aHUS patients compared to healthy controls and other TMAs using confocal microscopy and flow cytometry.
• Measured C5b-9 deposition and cell viability as indicators of complement activation and cell damage.

Main Results:

• GPI-AP-deficient cells showed increased susceptibility and C5b-9 deposition when incubated with aHUS serum compared to control and TTP serum.
• Significant differences in cell viability were observed, with PIGA-deficient cells exhibiting greater non-viability with aHUS serum.
• The assay demonstrated high reproducibility, sensitivity, and specificity in identifying aHUS.

Conclusions:

• A novel, simple, and rapid serum-based assay was successfully developed to differentiate aHUS from other TMAs.
• The assay's efficacy relies on the increased sensitivity of GPI-AP-deficient cells to the overactivated complement system in aHUS.
• This assay provides a valuable tool for the accurate diagnosis of aHUS, aiding in clinical management.