Determination of anti-ADAMTS13 autoantibodies in thrombotic thrombocytopenic purpura (TTP) patients : comparison of two different methods

Introduction : about 70 to 80% of TTP patients could develop autoantibodies that bind and/or inhibit ADAMTS13 by blocking its proteolytic activity or increasing its clearance in vivo, leading to a more or less severe deficiency of ADAMTS13. Therefore, determination of these autoantibodies could be very important for TTP diagnosis and for therapeutic strategies Methods : we selected 38 TTP patients with no ADAMTS13 inhibitor; the presence of anti-ADAMTS13 autoantibodies was determined by two methods: Western Blotting (WB) and ELISA. The proteolytic activity of ADAMTS13 and the presence of the ADAMTS13 inhibitor were determined by the method previously reported by Gerritsen (Thromb Haemost, 1999). The WB determination of anti-ADAMTS13 autoantibodies was performed by diluting the patients' plasma 1/100 and using conditioned media of cells stably transfected with ADAMTS13 wild type as a source of ADAMTS13 antigen. The ELISA method was performed as previously reported (Zhou, JBC 2005) with some modifications Results : of the 38 patients with no inhibitor, 42% (16/38) had ADAMTS13 activity <10%; 50% (19/38) had values ranging from 10 to 46%, and 8% (3/38) had normal values. The WB method determined the presence of anti-ADAMTS13 autoantibodies in 90% (34/38) of our patients, while only 61% (23/38) resulted positive according to the ELISA assay. All 16 patients with severe ADAMTS13 deficiency had anti-ADAMTS13 autoantibodies by WB, and only 11 out of 16 patients were positive by ELISA Conclusions : this study confirms that a negative result using the inhibitor assay does not exclude a diagnosis of autoimmune TTP, but this diagnosis also requires to use a second assay, either WB or ELISA. Our results found WB to be more sensitive method than ELISA