title: A novel quantitative hemolytic assay coupled with restriction fragment length polymorphisms analysis enabled early diagnosis of atypical hemolytic uremic syndrome and identified unique predisposing mutations in Japan.
abstract: For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients.
authors.affiliation: Department of Blood Transfusion Medicine, Nara Medical University, Kashihara, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Miyata
authors.affiliation: Department of Blood Transfusion Medicine, Nara Medical University, Kashihara, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Shirotani-Ikejima
authors.affiliation: Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: Molecular and Cellular Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Ohyama
authors.affiliation: Molecular and Cellular Biology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Kokubo
authors.affiliation: Department of Blood Transfusion Medicine, Nara Medical University, Kashihara, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Fujimura
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D044466:Asian People / Q000235:genetics*; D065766:Atypical Hemolytic Uremic Syndrome / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D003176:Complement C3 / Q000235:genetics*; D017242:Complement Factor H / Q000235:genetics / Q000378:metabolism; D015941:Complement Hemolytic Activity Assay / Q000379:methods*; D042241:Early Diagnosis; D004912:Erythrocytes / Q000276:immunology; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D007564:Japan; D008297:Male; D009154:Mutation; D012150:Polymorphism, Restriction Fragment Length; D012756:Sheep / Q000097:blood / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; C493495:CFH protein, human; D003176:Complement C3; D017242:Complement Factor H