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

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.