title: 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: Division of Hematology, Department of Medicine.
authors: Eleni
authors: http://orcid.org/0000-0002-8883-8208
authors: E
authors: Gavriilaki
authors: Division of Hematology, Department of Medicine.
authors: Xuan
authors: X
authors: Yuan
authors: Division of Hematology, Department of Medicine.
authors: Zhaohui
authors: Z
authors: Ye
authors: Division of Hematology, Department of Medicine.
authors: Alexander J
authors: AJ
authors: Ambinder
authors: Division of Hematology, Department of Medicine.
authors: Satish P
authors: SP
authors: Shanbhag
authors: Division of Hematology, Department of Medicine.
authors: Michael B
authors: MB
authors: Streiff
authors: Department of Pathology, and.
authors: Thomas S
authors: TS
authors: Kickler
authors: Division of Hematology, Department of Medicine.
authors: Alison R
authors: AR
authors: Moliterno
authors: Division of Nephrology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD.
authors: C John
authors: CJ
authors: Sperati
authors: Division of Hematology, Department of Medicine.
authors: Robert A
authors: RA
authors: Brodsky
mesh_terms: D000328:Adult; D000368:Aged; D065766:Atypical Hemolytic Uremic Syndrome / Q000097:blood* / Q000175:diagnosis* / Q000235:genetics; D002470:Cell Survival; D005260:Female; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000097:blood / Q000235:genetics; D008875:Middle Aged; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D044967:Serum / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein