Journal Article

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

Kokubo, Tetsuro, Yoshida, Yoko, Fujimura, Yoshihiro, Shirotani-Ikejima, Hiroko, Uchida, Yumiko, Ohyama, Yoshifumi, Matsumoto, Masanori, Miyata, Toshiyuki

2015

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Abnormalities / Animals / Antibodies, Monoclonal - metabolism / Asian Continental Ancestry Group - genetics / Assaying / Atypical Hemolytic Uremic Syndrome - diagnosis / Atypical Hemolytic Uremic Syndrome - genetics / Atypical Hemolytic Uremic Syndrome - immunology / Bioassay / Blood cells / Blood transfusions / Cellular biology / Complement / Complement activation / Complement C3 - genetics / Complement component C3 / Complement factor H / Complement Factor H - genetics / Complement Factor H - metabolism / Complement Hemolytic Activity Assay - methods / Complement system / Diagnosis / E coli / Early Diagnosis / Erythrocytes / Erythrocytes - immunology / Female / Gene mutation / Genetic abnormalities / Genetic aspects / Genetic Predisposition to Disease / Hemolysis / Hemolytic uremic syndrome / Humans / Identification and classification / Infections / Japan / Kinases / Laboratories / Life sciences / Male / Medical diagnosis / Methods / Monoclonal antibodies / Mutation / Ovis aries / Pathogenesis / Patients / Pediatrics / Plasma / Polymorphism / Polymorphism, Restriction Fragment Length / Proteins / Purpura / Restriction fragment length polymorphism / Restriction fragment length polymorphism analysis / Sheep / Sheep - blood / Sheep - immunology / Sheep red blood cells / Shiga toxin / Thrombocytopenic purpura / Thrombotic thrombocytopenic purpura / Toxins / Transplants & implants