A New Automatic Method to Measure Adamts 13

Abstract number: P0100

Lattuada A, Preda L, Sacchi E, Candolfi R, Rossi E

Adamts 13 is a metalloprotease that cleaves von Willebrand Factor (VWF) physiologically. The absence of this protease results in the circulation of ultralarge VWF multimers that cause intravascular thrombi under high shear stress in patients with thrombotic microangiopathies (TMA). Since the value of ADAMTS13 is important for the diagnosis in patients with TTP, we developed a fast and automatic method for its measurement.

Materials:   Ristocetin 50 mg/mL (Mascia Brunelli) was reconstituted with buffer supplied with the kit. Lyophilized platelets (Dade–Behringer) were diluted to obtain 340 ÷ 360 10³/microLiter of platelets. VWF-substrate for ADAMTS13 was a commercial FVIII concentrate.

Method:   Serial dilutions in Urea and BaCl₂ of the plasma samples were incubated for 30 minutes at 37°C. Digested samples were incubated over night at room temperature with VWF-substrate. Protease value were read from a dose-response curve obtained by testing a serial dilutions of reference plasma. An automated VWF:RCo test (ref.: Lattuada, BCF 2004) was used to determine Adamts13. We analysed: 60 normal controls, 10 pregnant women, 2 TTP patients. To verify the accuracy of the new test different dilutions of normal plasma (100% of Adamts13) and TTP plasma (<6% of Adamts13) to obtain 0, 10, 20, 40, 80 and 100% of the Adamts13 were tested.

Results:   The coefficient of variation within- and between- assay was 5% and 12% respectively. Normal range of Adamts13 is 60–130%. The values of ADAMTS13 in TTP patients were 3%, 4%; the means during normal pregnancy were 99% in the first, 87% in the second, 83% in the third trimester and in the different dilutions of normal / TTP plasma, were 4%, 11%, 21%, 37%, 83%, 95%.

Conclusion:   This method is fully automated, reproducible, rapid and does not require any special laboratory equipment and specialized technical staff.