Introduction: The clinical entity of idiopathic thrombotic thrombocytopenic purpura (TTP) has been closely associated with a severe deficiency of the von Willebrand factor (vWF)-cleaving protease (ADAMTS-13). Levels below 5% are highly specific for TTP basically excluding hemolytic uremic syndrome (HUS). Deficiency of ADAMTS-13 can either be inherited or caused by inhibitory IgG autoantibodies to ADAMTS-13. Diagnosis of TTP is often difficult since it often manifests oligosymptomatic exhibiting only laboratory signs of thrombocytopenia and hemolysis or involving atypical organs. In such cases rapid knowledge of the ADAMTS-13 activity is very useful in helping to establish a correct diagnosis rapidly and to initiate the appropriate therapy.
Methods and results: We have therefore designed a rapid, sensitive and simple assay for the determination of ADAMTS-13 activity and ADAMTS-13 inhibitors. The test is based on the measurement of the ristocetin-cofactor activity of purified, urea-denaturated vWF concentrate after incubation with diluted patient plasma that has been preactivated with barium chloride. While this assay has already previously been established, we have further modified and simplified the test thereby improving markedly the sensitivity, accuracy and turn-around time. A major improvement proved to be the change from buffer to heat-treated and vWF-replenished plasma as diluent for making the serial dilutions of normal pool plasma (NPP) for the calibration curve. Set up as a semi-automated assay the test is performed in only 50 to 60 min. Tested in 15 patients with ADAMTS-13 deficiency this new assay compared favorably and even more accurately with the classic electrophoretic ADAMTS-13 assay, especially in the low range below 10%. Mixing studies with patient plasma and NPP (ratio 1:1) revealed that a relatively short period of only 30 min at room temperature is sufficient for most samples to detect an inhibitor. With a single mixing experiment it is possible to discriminate inhibitor levels in the range of 0.5-4 Bethesda units.
Conclusion: Using this novel assay, determination of ADAMTS-13 activity and ADAMTS-13 inhibitors can be performed easily, rapidly (less than 2 h), and accurately, providing rapid information about the severity of ADAMTS-13 deficiency and the presence of an inhibitor. In addition, the test is suitable for full automation enabling to screen large populations and, thus, help to get further insights into the clinical significance of ADAMTS-13 deficiency in other diseases.