A rapid test for the diagnosis of thrombotic thrombocytopenic purpura using surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF)-mass spectrometry

Authors


Haifeng M. Wu, Clinical Coagulation Laboratory, Department of Pathology, Ohio State University College of Medicine and Public Health, 288 Medical Research Facility, 420 West 12th Avenue, Columbus, OH 43210, USA.
Tel.: +1 614 292 1798; fax: +1 614 292 3144; e-mail: wu-6@medctr.osu.edu

Abstract

Summary. Background: Thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy, requires immediate diagnosis and plasma exchange therapy. Development of TTP is related to functional deficiency of ADAMTS-13 protease that leads to the accumulation of ultra large von Willebrand factor (VWF) and subsequent platelet thrombosis. Currently no clinical test is available for the rapid detection of ADAMTS-13 activity. Objectives: The goal is to devise a novel method to rapidly detect functional activity of ADAMTS-13 and improve clinical outcome. Methods and results: A recombinant VWF substrate containing the ADAMTS-13 cleavage site and a 6X Histidine tag was cleaved by ADAMTS-13 in a dose-dependent manner, generating approximately 7739 Da peptide containing a 6X Histidine tag. This cleaved peptide, bound to an IMAC/Nickel ProteinChip, was quantified using Surface Enhanced Laser Desorption/Ionization Time-of-flight Mass Spectrometry (SELDI-TOF-MS). The assay is capable of quantifying ADAMTS-13 activity as low as 2.5% in plasma within 4 h. When the cleaved peptide was quantified as a ratio of an internal control peptide, the test displayed good reproducibility, with an average inter-assay coefficient of variation (CV) of < 33%. Further validation revealed a mean ADAMTS-13 activity of 92.5% ± 16.6% in 39 healthy donors. Sixteen patients with idiopathic TTP displayed mean ADAMTS-13 activity of 1.73% ± 3.62%. Further utility of this novel method includes determining the inhibitory titer of ADAMTS-13 antibody in cases of acquired TTP. Conclusions: We have devised a novel SELDI-TOF-MS assay that offers a rapid, cost-effective, and functionally relevant test for timely diagnosis and management of TTP.

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