title: Novel, semi-automated, 60-min-assay to determine von Willebrand factor cleaving activity of ADAMTS-13.
abstract: BACKGROUND
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Coagulation Laboratory, Division of Haematology, University Hospital of Zürich, Raemistrasse 100, 8091 Zürich, Switzerland. kostousow@yahoo.com
authors.forename: Vadim
authors.initials: V
authors.lastname: Kostousov
authors.forename: Jörg
authors.initials: J
authors.lastname: Fehr
authors.forename: Thomas
authors.initials: T
authors.lastname: Bombeli
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D001331:Automation; D001681:Biological Assay / Q000379:methods; D019411:Clinical Laboratory Techniques; D006801:Humans; D008403:Mass Screening; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human