A Rapid Test for the Diagnosis of Thrombotic Thrombocytopenic Purpura Using SELDI-TOF-Mass Spectrometry.

Thrombotic Thrombocytopenic Purpura (TTP), a life-threatening microangiopathic thrombotic disorder, requires immediate diagnosis and plasma exchange therapy. Development of TTP is related to functional deficiency of a metalloprotease, ADAMTS13, that leads to accumulation of ultra large von Willebrand factor (vWF) and subsequent platelet thrombosis. Currently no clinical test is available for a rapid detection of functional ADAMTS13 activity. In this study, a recombinant vWF peptide containing the ADAMTS13 cleavage site and a 6 X Histidine tag was used as a substrate for ADAMTS13. ADAMTS13 cleaved the substrate in a dose-dependent manner, generating a ~7739 Dalton peptide containing a 6 X 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 ADAMTS13 activity as low as 2.5 % in plasma within 3–4 hours. When the cleaved peptide was quantified as a ratio of an internal control peptide, the test displayed excellent reproducibility, with an average inter-assay CV of < 33 %. Further validation of the test in healthy donors (n=39) revealed normal ADAMTS13 activity with a mean of 92.5% + 16.6. Sixteen patients with idiopathic TTP displayed mean ADAMTS13 activity of 1.73% + 3.62 at presentation, prior to any therapy. Further utility of this novel method includes functional determination of ADAMTS13 antibody titers in cases of acquired TTP. In summary, 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 patients.