Introduction 53 2 31 10 40 30 21 3 15 12 13 16 50 27 13 50 55 6 ADAMTS13 14 27 52 28 5 Escherichia coli 8 36 47 29 58 51 26 48 15 17 25 38 Materials and methods Subjects n n 1 15 17 Table 1 Clinical and laboratory data regarding thrombotic thrombocytopenic purpura (TTP) patients Patient no. Sex Age at debut  Age at sampling (years) Current age (years) Symptoms during episodes No. of episodes ADAMTS13 activity level ADAMTS13 mutation ADAMTS13 inhibitor  Reference a M 2 d 16 19 Jaundice, hemolytic anemia, thrombocytopenia, macroscopic hematuria, pathological urinalysis, fever, neurological symptoms, elevated serum creatinine >5 <5% bc None  4 20 43 a M 5.3 y 17 18 Jaundice, hemolytic anemia, thrombocytopenia, fever, neurological symptoms, pathological urinalysis >5 <5% bc None 4 20 43 3 F 20 m 15 23 Hemolytic anemia, thrombocytopenia, hematuria, epileptic attacks, slightly elevated serum creatinine >5 <5% d e None 4 4 M 3 y 7 9 Hemolytic anemia, thrombocytopenia, purpura, pathological urinalysis 4 <5% f None 43 5 M 2 d 39 39 Jaundice, hemolytic anemia, petechiae, thrombocytopenia, transitory neurological deficits and aphasia, elevated creatinine >5 <5% b None – 6 F 54 y 70 75 Recurrent hemolytic anemia and thrombocytopenia, reduced consciousness, pathological urinalysis >5 <5% NA 0.5 U/ml – 7 F 25 y 42 44 Thrombocytopenia, hemolytic anemia, elevated creatinine during viral infection and pregnancy 2 <5% NA 0.2 U/ml – a b c d e f NA: not assayed 15 Plasma samples from 20 healthy adult volunteers were used as controls. The study was conducted with the approval of the ethics committee of Lund University and the plasma samples were collected with the informed consent of the patients, their parents, and the controls. Plasma samples 20 Anti-ADAMTS13 antibodies 50 56 Immunoblot analysis for the detection of ADAMTS13 in plasma 19 58 Immunoblotting with the polyclonal antibody revealed, in addition to ADAMTS13, two unspecific bands at 130 kD and 170 kD, which were identified as C3 and alpha-2-macroglobulin. These proteins were removed by incubating the plasma samples with protein A-sepharose coupled rabbit anti-C3 IgG and rabbit anti-alpha-2-macroglobulin IgG. The results obtained using the polyclonal antibody show samples from which these proteins have been removed. n Results Detection of ADAMTS13 in plasma samples from normal individuals 1 1 1 Fig. 1a–g a b c d e f filled circles open circles g  Detection of ADAMTS13 in plasma samples from TTP patients 1 1 1 1 1 Detection of ADAMTS13 in the parents of the TTP patients ADAMTS13 1 Discussion In the present study, we detected ADAMTS13 in plasma using a polyclonal and a monoclonal antibody. This assay was capable of distinguishing TTP patients from normal individuals, as well as differentiating between congenital and acquired TTP. Plasma from the patients with congenital TTP lacked the ADAMTS13 antigen. In contrast, the plasma of patients with acquired TTP expressed a normal ADAMTS13 phenotype. 33 46 11 38 11 38 45 35 42 44 42 44 34 27 11 14 41 45 52 2 48 49 26 Table 2 ADAMTS13 assays ASSAY PRINCIPLE REFERENCE Detection of ADAMTS13 activity Detection of VWF cleavage products  1. VWF multimer structure analysis Detection of the breakdown of high-molecular-weight VWF 15 23  2. Immunoblotting of VWF  Detection of cleavage products of native VWF or recombinant VWF domains 37 50  3. IRMA Detection of VWF cleavage products 32  4. Flow assay Detection of the breakdown of ULVWF-platelet strings attached to endothelial cells 1  5. Various methods using VWF domains or short synthetic VWF  peptides as the substrate, such as the FRETS-VWF73 assay Detection of cleavage products of the VWF domains or VWF peptides 9 18 22 25 59 61 Detection of VWF residual activity    6. Collagen binding Detection of VWF binding to collagen; binding correlates to VWF multimer size 17  7. Ristocetin cofactor activity Detection of platelet aggregates; VWF ability to induce platelet aggregates in the presence of ristocetin correlates to multimer size 7 Detection of ADAMTS13 antigen and auto-antibodies  8. ELISA Detection of ADAMTS13 antigen or anti-ADAMTS13 auto-antibodies 11 38 39 41 54  9. Immunoblotting Detection of anti-ADAMTS13 auto-antibodies 57  10. Present assay (immunoblotting) Detection of ADAMTS13 antigen and size and (indirectly) of auto-antibodies – Mutation analysis  11. PCR ADAMTS13 27 24 2 2 ADAMTS13 2 In the present study, we developed a qualitative, semi-quantitative assay capable of detecting ADAMTS13 antigen and anti-ADAMTS13 auto-antibodies in plasma and, thus, distinguishing between TTP and normal plasma, as well as distinguishing between congenital and acquired TTP. All patients with congenital TTP, regardless of their ADAMTS13 mutations, presented with undetectable levels of ADAMTS13 antigen, whereas patients with acquired TTP presented a normal phenotype. Heterozygous carriers of ADAMTS13 mutations also revealed a normal ADAMTS13 antigen band. Although not a quantitative method like the above-mentioned ELISA assays, the present assay offers the advantage of not only demonstrating the presence or absence of ADAMTS13 antigen, but also the molecular size of ADAMTS13 present in plasma samples. Furthermore, this assay was able to show the presence of anti-ADAMTS13 antibodies indirectly by performing immunoadsorption of plasma immunoglobulins prior to immunoblotting. In conclusion, this assay is able to distinguish between normal and TTP plasma, and also between congenital and acquired TTP. It is easy to perform and can be used at any hospital laboratory routinely using SDS-PAGE electrophoresis and immunoblotting, and offers a complement to existing ADAMTS13 methods in the diagnosis of TTP.