ADAMTS‐13 assays in thrombotic thrombocytopenic purpura
2010; Elsevier BV; Volume: 8; Issue: 4 Linguagem: Inglês
10.1111/j.1538-7836.2010.03761.x
ISSN1538-7933
AutoresFlora Peyvandi, Roberta Palla, Luca A. Lotta, Ian Mackie, Marie Scully, Samuel J. Machin,
Tópico(s)Platelet Disorders and Treatments
ResumoSummaryADAMTS‐13, the thirteenth member of the ADAMTS (A Disintegrin And Metalloprotease with Thrombo‐Spondin 1 repeats) family, is the plasma metalloprotease responsible for regulating the multimeric structure of VWF. In congenital or acquired deficiency it is actively involved in the pathophysiology of thrombotic thrombocytopenic purpura (TTP), a rare but life threatening disease characterized by microangiopathic haemolytic anaemia and consumptive thrombocytopenia leading to disseminated microvascular thrombosis and variable signs and symptoms of organ ischemia and damage. In the last few years, a number of in house and commercial laboratory assays for ADAMTS‐13 and its autoantibodies have been developed. The features and clinical utility of ADAMTS‐13 assays are summarized in this review. ADAMTS‐13, the thirteenth member of the ADAMTS (A Disintegrin And Metalloprotease with Thrombo‐Spondin 1 repeats) family, is the plasma metalloprotease responsible for regulating the multimeric structure of VWF. In congenital or acquired deficiency it is actively involved in the pathophysiology of thrombotic thrombocytopenic purpura (TTP), a rare but life threatening disease characterized by microangiopathic haemolytic anaemia and consumptive thrombocytopenia leading to disseminated microvascular thrombosis and variable signs and symptoms of organ ischemia and damage. In the last few years, a number of in house and commercial laboratory assays for ADAMTS‐13 and its autoantibodies have been developed. The features and clinical utility of ADAMTS‐13 assays are summarized in this review. ADAMTS‐13 is a metalloprotease that limits platelet‐rich thrombus formation in arterioles by cleaving von Willebrand factor (VWF) at its Tyr 1605–Met 1606 bond. Deficiency of ADAMTS‐13 activity, either because of an inherited or an acquired etiology, may lead to a potentially lethal syndrome, thrombotic thrombocytopenic purpura (TTP) [1Sadler J.E. Von Willebrand factor, ADAMTS‐13, and thrombotic thrombocytopenic purpura.Blood. 2008; 112: 11-8Crossref PubMed Scopus (0) Google Scholar]. Evaluation of ADAMTS‐13 in plasma is now being used more frequently to support the acute diagnosis of TTP, although this is usually primarily a clinical diagnosis. The possibility of using ADAMTS‐13 data to manage TTP patients depends on ADAMTS‐13 assays becoming widely available, rapid, reliable and feasible for most clinical laboratories. However, only a few laboratories have been able to accumulate sufficient experience in such a rare disease, to enable them to give useful advice to less specialized laboratories. ADAMTS‐13 testing includes assays of ADAMTS‐13 activity, antigen, neutralizing or non‐neutralizing anti‐ADAMTS‐13 autoantibodies and genetic characterization of the ADAMTS‐13 gene. Since 1998, several assays for measuring ADAMTS‐13 activity have been developed [2Tsai H.M. Lian E.C. Antibodies to von Willebrand factor cleaving protease in acute thrombotic thrombocytopenic purpura.N Engl J Med. 1998; 339: 1585-9Crossref PubMed Scopus (1474) Google Scholar, 3Furlan M. Robles R. Galbusera M. Remuzzi G. Kyrle P.A. Brenner B. Krause M. Scharrer I. Aumann V. Mittler U. Solenthaler M. Lämmle B. von Willebrand factor‐cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic‐uremic syndrome.N Engl J Med. 1998; 339: 1578-84Crossref PubMed Scopus (0) Google Scholar, 4Gerritsen H.E. Turecek P.L. Schwarz H.P. Lämmle B. Furlan M. Assay of von Willebrand factor (vWF)‐cleaving protease based on decreased collagen binding affinity of degraded vWF: a tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP).Thromb Haemost. 1999; 82: 1386-9Crossref PubMed Scopus (0) Google Scholar, 5Rick M.E. Moll S. Taylor M.A. Krizek D.M. White 2nd, G.C. Aronson D.L. Clinical use of a rapid collagen binding assay for von Willebrand factor cleaving protease in patients with thrombotic thrombocytopenic purpura.Thromb Haemost. 2002; 88: 598-604Crossref PubMed Scopus (44) Google Scholar, 6Knovich M.A. Lawson H.L. Burke M.H. McCoy T.P. Owen J. Rapid quantitative assay of ADAMTS13 activity on an automated coagulation analyzer: clinical applications and comparison with immunoblot method.Am J Hematol. 2008; 83: 654-6Crossref PubMed Scopus (10) Google Scholar, 7Bohm M. Vigh T. Scharrer I. Evaluation and clinical application of a new method for measuring activity of von Willebrand factor‐cleaving metalloprotease (ADAMTS13).Ann Hematol. 2002; 81: 430-5Crossref PubMed Scopus (0) Google Scholar, 8Kostousov V. Fehr J. Bombeli T. Novel, semi‐automated, 60‐min‐assay to determine von Willebrand factor cleaving activity of ADAMTS‐13.Thromb Res. 2006; 118: 723-31Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 9Obert B. Tout H. Veyradier A. Fressinaud E. Meyer D. Girma J.P. Estimation of the von Willebrand factor‐cleaving protease in plasma using monoclonal antibodies to vWF.Thromb Haemost. 1999; 82: 1382-5Crossref PubMed Scopus (0) Google Scholar, 10Knovich M.A. Craver K. Matulis M.D. Lawson H. Owen J. Simplified assay for VWF cleaving protease (ADAMTS13) activity and inhibitor in plasma.Am J Hematol. 2004; 76: 286-90Crossref PubMed Scopus (12) Google Scholar, 11Whitelock J.L. Nolasco L. Bernardo A. Moake J. Dong J.F. Cruz M.A. ADAMTS‐13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF‐A2 domain as substrate.J Thromb Haemost. 2004; 2: 485-91Crossref PubMed Scopus (0) Google Scholar, 12Cruz M.A. Whitelock J. Dong J.F. Evaluation of ADAMTS‐13 activity in plasma using recombinant von Willebrand Factor A2 domain polypeptide as substrate.Thromb Haemost. 2003; 90: 1204-9Crossref PubMed Google Scholar, 13Wu J.J. Fujikawa K. Lian E.C. McMullen B.A. Kulman J.D. Chung D.W. A rapid enzyme‐linked assay for ADAMTS‐13.J Thromb Haemost. 2006; 4: 129-36Crossref PubMed Scopus (0) Google Scholar, 14Zhou W. Tsai H.M. An enzyme immunoassay of ADAMTS13 distinguishes patients with thrombotic thrombocytopenic purpura from normal individuals and carriers of ADAMTS13 mutations.Thromb Haemost. 2004; 91: 806-11Crossref PubMed Google Scholar, 15Kokame K. Nobe Y. Kokubo Y. Okayama A. Miyata T. FRETS‐VWF73, a first fluorogenic substrate for ADAMTS13 assay.Br J Haematol. 2005; 129: 93-100Crossref PubMed Scopus (0) Google Scholar, 16Kremer Hovinga J.A. Mottini M. Lämmle B. Measurement of ADAMTS‐13 activity in plasma by the FRETS‐VWF73 assay: comparison with other assay methods.J Thromb Haemost. 2006; 4: 1146-8Crossref PubMed Scopus (0) Google Scholar, 17Kokame K. Matsumoto M. Fujimura Y. Miyata T. VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS‐13.Blood. 2003; 103: 607-12Crossref PubMed Scopus (0) Google Scholar, 18Ai J. Smith P. Wang S. Zhang P. Zheng X.L. The proximal carboxyl terminal domains of ADAMTS13 determine substrate specificity and are all required for cleavage of von Willebrand factor.J Biol Chem. 2005; 280: 29428-34Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 19Kato S. Matsumoto M. Matsuyama T. Isonishi A. Hiura H. Fujimura Y. Novel monoclonal antibody‐based enzyme immunoassay for determining plasma levels of ADAMTS13 activity.Transfusion. 2006; 46: 1444-52Crossref PubMed Scopus (0) Google Scholar, 20Jin M. Casper T.C. Cataland S.R. Kennedy M.S. Lin S. Li Y.J. Wu H.M. Relationship between ADAMTS13 activity in clinical remission and the risk of TTP relapse.Br J Haematol. 2008; 141: 651-8Crossref PubMed Scopus (98) Google Scholar]. They are based on the degradation of purified, plasma‐derived or recombinant VWF multimers or of synthetic VWF peptides, by ADAMTS‐13 in patient plasma and the direct or indirect detection of the VWF cleavage products (Table 1). The latter are usually measured by electrophoresis, platelet aggregation techniques, fluorescence resonance energy transfer (FRET) technique or immunoassay. The assays based on multimeric VWF are sensitive (3%–6% of ADAMTS‐13 activity) and reproducible, but cumbersome, time‐consuming (2–3 days) and performed in non‐physiological conditions, using denaturing agents. On the other hand, the assays based on VWF peptides are very sensitive (1%–3% of ADAMTS‐13 activity), reproducible, easy and rapid (1–4 h), performed in the absence of denaturing agents, but they use non‐physiologic VWF substrates. A recent multicenter study showed that assays based on multimeric VWF displayed good performance characteristics, whereas assays based on VWF peptides were excellent [21Tripodi A. Peyvandi F. Chantarangkul V. Palla R. Afrasiabi A. Canciani M.T. Chung D.W. Ferrari S. Fujimura Y. Karimi M. Kokame K. Kremer Hovinga J.A. Lämmle B. De Meyer S.F. Plaimauer B. Vanhoorelbeke K. Varadi K. Mannucci P.M. Second international collaborative study evaluating performance characteristics of methods measuring the von Willebrand factor cleaving protease (ADAMTS‐13).J Thromb Haemost. 2008; 6: 1534-41Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. However, this study analyzed test plasmas made by dilutions of one ADAMTS‐13‐deficient plasma (with no autoantibodies to ADAMTS‐13) into one pooled normal plasma, so that the estimate of some characteristics may be biased by the absence of variable ADAMTS‐13 deficiencies and/or the presence/absence of anti‐ADAMTS‐13 autoantibodies that would have occurred in different patients with TTP.Table 1Different available assays reported in the literature for measuring ADAMTS‐13 activity, together with main characteristics of each assay. The white shaded area shows assays using the direct detection of von Willebrand factor (VWF) cleavage products and the gray shaded area those using indirect detection Open table in a new tab In all the reported activity assays, a number of variables may interfere with assay results. First, all assays developed to date measure ADAMTS‐13 activity in static conditions which do not reflect the in vivo physiologic flow conditions, necessary for conformational changes in VWF that allow optimal ADAMTS‐13 enzymatic activity. Another important variable is introduced by the use of denaturing agents (i.e. guanidine HCl or 1.5 m urea) that are required to promote the susceptibility of VWF multimers to cleavage by ADAMTS‐13, but these agents could also affect the enzymatic activity of ADAMTS‐13 and inhibit antibody–ADAMTS‐13 interactions, which may artifactually reduce ADAMTS‐13 activity measurement. The use of the short VWF peptides, instead of full‐length VWF in enzyme immunoassay‐based methods improved, but did not resolve completely the issue of intra‐ or inter‐laboratory variability. It has been previously shown that the results of some of the ADAMTS‐13 activity assays could be influenced by high levels of endogenous VWF, hyperlipemia, elevated plasma hemoglobin level (acting as an inhibitor of ADAMTS‐13) [22Studt J.D. Hovinga J.A. Antoine G. Hermann M. Rieger M. Scheiflinger F. Lämmle B. Fatal congenital thrombotic thrombocytopenic purpura with apparent ADAMTS13 inhibitor: in vitro inhibition of ADAMTS13 activity by hemoglobin.Blood. 2005; 105: 542-4Crossref PubMed Scopus (0) Google Scholar] or hyperbilrubinemia (bilirubin concentrations higher than 100 μmol L−1 may interfere with the fluorescence assays) [23Meyer S.C. Sulzer I. Lämmle B. Kremer Hovinga J.A. Hyperbilirubinemia interferes with ADAMTS‐13 activity measurement by FRETS‐VWF73 assay: diagnostic relevance in patients suffering from acute thrombotic microangiopathies.J Thromb Haemost. 2007; 5: 866-7Crossref PubMed Scopus (0) Google Scholar, 24Eckmann C.M. De Laaf R.T. Van Keulen J.M. Van Mourik J.A. De Laat B. Bilirubin oxidase as a solution for the interference of hyperbilirubinemia with ADAMTS‐13 activity measurement by FRETS‐VWF73 assay.J Thromb Haemost. 2007; 5: 1330-1Crossref PubMed Scopus (20) Google Scholar]. In addition, the presence of other proteases in plasma samples might interfere with VWF cleavage [25Raife T.J. Cao W. Atkinson B.S. Bedell B. Montgomery R.R. Lentz S.R. Johnson G.F. Zheng X.L. Leukocyte proteases cleave von Willebrand factor at or near the ADAMTS13 cleavage site.Blood. 2009; 114: 1666-74Crossref PubMed Scopus (91) Google Scholar], or degrade ADAMTS‐13 itself [26Lam J.K. Chion C.K. Zanardelli S. Lane D.A. Crawley J.T. Further characterization of ADAMTS‐13 inactivation by thrombin.J Thromb Haemost. 2007; 5: 1010-8Crossref PubMed Scopus (0) Google Scholar]. Therefore, all these reported factors could influence the clinical utility of each assay. The sensitivity, specificity, linearity, precision, ease of use, optimal dilution and sensitivity to ADAMTS‐13 mutations and specific types of autoantibodies may also differ between the assays. However, none of these assays employ flow conditions and some of them utilize denaturing agents which could potentially alter the equilibrium between free ADAMTS‐13 metalloprotease and that portion bound to autoantibodies. Only two test systems, capable of observing plasma ADAMTS‐13 in vitro under flowing conditions have been developed [27Dong J.F. Moake J.L. Nolasco L. Bernardo A. Arceneaux W. Shrimpton C.N. Schade A.J. McIntire L.V. Fujikawa K. Lopez J.A. ADAMTS‐13 rapidly cleaves newly secreted ultralarge vonWillebrand factor multimers on the endothelial surface under flowing conditions.Blood. 2002; 100: 4033-9Crossref PubMed Scopus (0) Google Scholar, 28Shenkman B. Inbal A. Tamarin I. Lubetsky A. Savion N. Varon D. Diagnosis of thrombotic thrombocytopenic purpura based on modulation by patient plasma of normal platelet adhesion under flow condition.Br J Haematol. 2003; 120: 597-604Crossref PubMed Scopus (24) Google Scholar]. One of these assays [27Dong J.F. Moake J.L. Nolasco L. Bernardo A. Arceneaux W. Shrimpton C.N. Schade A.J. McIntire L.V. Fujikawa K. Lopez J.A. ADAMTS‐13 rapidly cleaves newly secreted ultralarge vonWillebrand factor multimers on the endothelial surface under flowing conditions.Blood. 2002; 100: 4033-9Crossref PubMed Scopus (0) Google Scholar] was evaluated in a multicenter study [29Tripodi A. Chantarangkul V. Böhm M. Budde U. Dong J.F. Friedman K.D. Galbusera M. Girma J.P. Moake J. Rick M.E. Studt J.D. Turecek P.L. Mannucci P.M. Measurement of von Willebrand factor cleaving protease (ADAMTS‐13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas.J Thromb Haemost. 2004; 2: 1601-9Crossref PubMed Scopus (0) Google Scholar] showing that this assay may only be reliable in discriminating ADAMTS‐13 levels higher or lower than 20% without measuring a precise value of ADAMTS‐13 activity. However, these two assays are cumbersome, not applicable in non‐specialized laboratories and not suitable for routine laboratory tests. Several immunoassays employing different monoclonal and polyclonal antibodies are available to quantify plasma ADAMTS‐13 antigen levels (Table 2) [30Feys H.B. Liu F. Dong N. Pareyn I. Vauterin S. Vandeputte N. Noppe W. Ruan C. Deckmyn H. Vanhoorelbeke K. ADAMTS‐13 plasma level determination uncovers antigen absence in acquired thrombotic thrombocytopenic purpura and ethnic differences.J Thromb Haemost. 2006; 4: 955-62Crossref PubMed Scopus (0) Google Scholar, 31Rieger M. Ferrari S. Kremer Hovinga J.A. Konetschny C. Herzog A. Koller L. Weber A. Remuzzi G. Dockal M. Plaimauer B. Scheiflinger F. Relation between ADAMTS13 activity and ADAMTS13 antigen levels in healthy donors and patients with thrombotic microangiopathies (TMA).Thromb Haemost. 2006; 95: 212-20Crossref PubMed Scopus (0) Google Scholar, 32Ishizashi H. Yagi H. Matsumoto M. Soejima K. Nakagaki T. Fujimura Y. Quantitative Western blot analysis of plasma ADAMTS13 antigen in patients with Upshaw‐Schulman syndrome.Thromb Res. 2007; 120: 381-6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. These vary in their ability to discriminate between full‐length, mutant and truncated forms of ADAMTS‐13 and it is unclear how they may be affected by the presence of autoantibodies and immune complexes. In acquired TTP, antigen levels may be normal (owing to immune complexes) or reduced (as a result of removal) and they are not clinically helpful in the absence of ADAMTS‐13 activity assays.Table 2Available assays for the measurement of ADAMTS‐13 antigen levelsMethodAntibodyStandard CurveNormal rangePerformance characteristicsReferenceELISAThree monoclonalsNHP (1 mg mL−1) in PBS, 0.3% milk951 ± 261 ng mL−1DL = 16 ng mL−1 (1.6%) Intra‐assay CV = 4.4% Inter‐assay CV = 8.3%[30Feys H.B. Liu F. Dong N. Pareyn I. Vauterin S. Vandeputte N. Noppe W. Ruan C. Deckmyn H. Vanhoorelbeke K. ADAMTS‐13 plasma level determination uncovers antigen absence in acquired thrombotic thrombocytopenic purpura and ethnic differences.J Thromb Haemost. 2006; 4: 955-62Crossref PubMed Scopus (0) Google Scholar]ELISATwo polyclonalsrADAMTS‐13 in depleted plasma740–1420 ng mL−1DL = 62.5 ng mL−1 (6.25%) Intra‐assay CV = 2.5% Inter‐assay CV = 11.1%[31Rieger M. Ferrari S. Kremer Hovinga J.A. Konetschny C. Herzog A. Koller L. Weber A. Remuzzi G. Dockal M. Plaimauer B. Scheiflinger F. Relation between ADAMTS13 activity and ADAMTS13 antigen levels in healthy donors and patients with thrombotic microangiopathies (TMA).Thromb Haemost. 2006; 95: 212-20Crossref PubMed Scopus (0) Google Scholar]WBOne monoclonalNormal plasma101.6% ± 49.4%DL = 3% Intra‐assay CV = nr Inter‐assay CV = nr[32Ishizashi H. Yagi H. Matsumoto M. Soejima K. Nakagaki T. Fujimura Y. Quantitative Western blot analysis of plasma ADAMTS13 antigen in patients with Upshaw‐Schulman syndrome.Thromb Res. 2007; 120: 381-6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]NHP, normal human plasma; DL, detection limit; CV, coefficient of variation; WB, western blotting. Open table in a new tab NHP, normal human plasma; DL, detection limit; CV, coefficient of variation; WB, western blotting. Two types of anti‐ADAMTS‐13 antibodies have been described, one inhibiting (neutralizing) ADAMTS‐13 proteolytic activity [2Tsai H.M. Lian E.C. Antibodies to von Willebrand factor cleaving protease in acute thrombotic thrombocytopenic purpura.N Engl J Med. 1998; 339: 1585-9Crossref PubMed Scopus (1474) Google Scholar, 3Furlan M. Robles R. Galbusera M. Remuzzi G. Kyrle P.A. Brenner B. Krause M. Scharrer I. Aumann V. Mittler U. Solenthaler M. Lämmle B. von Willebrand factor‐cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic‐uremic syndrome.N Engl J Med. 1998; 339: 1578-84Crossref PubMed Scopus (0) Google Scholar] and the other binding to the protease and accelerating its clearance from plasma through opsonization and/or other yet unresolved mechanisms (non‐neutralizing) [33Scheiflinger F. Knobl P. Trattner B. Plaimauer B. Mohr G. Dockal M. Dorner F. Rieger M. Nonneutralizing IgM and IgG antibodies to vonWillebrand factor–cleaving protease (ADAMTS‐13) in a patient with thrombotic thrombocytopenic purpura.Blood. 2003; 102: 3241-3Crossref PubMed Scopus (0) Google Scholar]; both of these may be simultaneously present in many TTP patients. Neutralizing ADAMTS‐13 autoantibodies (inhibitor) can be titrated in vitro using classic mixing studies of heat‐inactivated patient and normal plasmas at a 1:1 dilution or several dilutions. However, although useful, Bethesda assays are far from being optimized and generally lack sensitivity. Less frequently (about 30%), autoantibodies are non‐neutralizing and probably promote the clearance of ADAMTS‐13 from blood without inhibiting its activity [33Scheiflinger F. Knobl P. Trattner B. Plaimauer B. Mohr G. Dockal M. Dorner F. Rieger M. Nonneutralizing IgM and IgG antibodies to vonWillebrand factor–cleaving protease (ADAMTS‐13) in a patient with thrombotic thrombocytopenic purpura.Blood. 2003; 102: 3241-3Crossref PubMed Scopus (0) Google Scholar]. These non‐neutralizing antibodies can be detected using western blotting or ELISA assays [34Peyvandi F. Lavoretano S. Palla R. Feys H.B. Vanhoorelbeke K. Battaglioli T. Valsecchi C. Canciani M.T. Fabris F. Zver S. Réti M. Mikovic D. Karimi M. Giuffrida G. Laurenti L. Mannucci P.M. ADAMTS13 and anti‐ADAMTS13 antibodies as markers for recurrence of acquired thrombotic thrombocytopenic purpura during remission.Haematologica. 2008; 93: 232-9Crossref PubMed Scopus (0) Google Scholar, 35Rieger M. Mannucci P.M. Kremer Hovinga J.A. Herzog A. Gerstenbauer G. Konetschny C. Zimmermann K. Scharrer I. Peyvandi F. Galbusera M. Remuzzi G. Böhm M. Plaimauer B. Lämmle B. Scheiflinger F. ADAMTS13 autoantibodies in patients with thrombotic microangiopathies and other immunomediated diseases.Blood. 2005; 106: 1262-7Crossref PubMed Scopus (235) Google Scholar]. Western blotting, although apparently more sensitive than ELISA [36Palla R. Valsecchi C. Bajetta M.T. Canciani M.T. Lavoretano S. Mannucci P.M. Peyvandi F. Determination of anti‐ADAMTS13 autoantibodies in thrombotic trombocitopenic purpura (TTP) patients: comparison of two different methods.J Thromb Haemost. 2007; 5: PT‐303Crossref Google Scholar], is not a quantitative assay. The ELISA methods may vary in relation to the dilution of plasma employed, the nature of the ADAMTS‐13 antigen coated onto the plates and the method of detection. All of these variables could affect the sensitivity and cut‐off value for positive results and it is not clear whether clinically irrelevant antibodies may sometimes be detected as a result of alterations in antigen presentation (similar to those observed in anti‐phospholipid syndrome with anti‐beta2‐glycoprotein‐I assays [37Sanmarco M. Bardin N. Blank M. Pascal V. Christine Alessi M. Dignat‐George F. Shoenfeld Y. Harlé J.R. Heterogeneity of anti‐beta2‐glycoprotein I antibodies. A factor of variability in test results.Thromb Haemost. 2005; 93: 80-7Crossref PubMed Scopus (15) Google Scholar]). In the past 2 years a number of commercial kits have become available (Table 3). They are based on previously reported methods and show different normal ranges and cut‐off values. Manufacturers generally advise that each laboratory assesses its local normal range and cut‐off value for each type of kit. However, in daily practice, it is difficult to obtain a statistically relevant number of healthy normal volunteers (> 100 required) and to match them to the local patient population. Therefore, it is important that manufacturers provide sufficient information about the geographic distribution of their study sample as well as the demographic data of the control group used to determine their normal range and cut‐off values, as these parameters could affect the status (positive/negative) of the samples. Unfortunately there are no validated evaluation exercises for direct comparison and standardization of the performance characteristics of these kits.Table 3Commercial kits presently available on the market (October 2009)Name (company)Intended useDetection methodSample dilutionNormal range of activity and performance characteristics (as reported in the information sheet)Standard and controlsTECHNOZYM ADAMTS‐13 Activity ELISA (Technoclone GmbH, Wien, Austria)ADAMTS13 activityChromogenic; based on [19Kato S. Matsumoto M. Matsuyama T. Isonishi A. Hiura H. Fujimura Y. Novel monoclonal antibody‐based enzyme immunoassay for determining plasma levels of ADAMTS13 activity.Transfusion. 2006; 46: 1444-52Crossref PubMed Scopus (0) Google Scholar]1:31 in reaction buffer40%–130%Five calibrators 1 high control 1 low control calibrated against an internal reference standard (defined activity of 100%) produced from a pool of 300 normal donorsATS‐13 activity assay (GTI Diagnostics Inc., Waukesha, WI, USA)ADAMTS13 activityFluorogenic; based on [16Kremer Hovinga J.A. Mottini M. Lämmle B. Measurement of ADAMTS‐13 activity in plasma by the FRETS‐VWF73 assay: comparison with other assay methods.J Thromb Haemost. 2006; 4: 1146-8Crossref PubMed Scopus (0) Google Scholar]1:8.3–Five calibrators 1 high‐range positive control 1 low‐range positive controlACTIFLUOR ADAMTS13 Activity Assay (American Diagnostica Inc, Stamford, CT, USA)ADAMTS13 activityFluorogenic; based on the cleavage of rVWF86‐ALEXA FRET substrate1:2 in inactivated plasma, then 1:5 in assay buffer633 ± 76 ng mL−1 Intra‐assay CV: 4.1% Inter‐assay CV: 4.4%One standard (750 ng mL−1) to be serially diluited 1 positive Control The standard is prepared from normal plasma calibrated against a recombinant ADAMTS13TECHNOZYM ADAMTS‐13 (Technoclone GmbH, Wien, Austria)ADAMTS13 activity and antigenActivity: fluorogenic, based on [16Kremer Hovinga J.A. Mottini M. Lämmle B. Measurement of ADAMTS‐13 activity in plasma by the FRETS‐VWF73 assay: comparison with other assay methods.J Thromb Haemost. 2006; 4: 1146-8Crossref PubMed Scopus (0) Google Scholar]. Antigen: chromogenicIndiluted samplesActivity: 50%–110% Antigen: 75%–110%Five calibrators 1 high control 1 low control calibrated against an internal reference standard (defined activity or antigen of 100%) produced from a pool of 100 normal donorsTECHNOZYM ADAMTS‐13 Antigen (Technoclone GmbH, Wien, Austria)ADAMTS13 antigenChromogenic; Capture antibody: anti ADAMTS13 MAb, directed against the CUB domain; Detection antibody: unspecified anti‐ADAMTS13Indiluted samples70%–160%Five calibrators 1 high control 1 low control calibrated against an internal reference standard (defined activity of 100%) produced from a pool of 300 normal donorsIMUBIND ADAMTS13 ELISA (American Diagnostica Inc, Stamford, CT, USA)ADAMTS13 antigenChromogenic; Capture antibody: anti‐ ADAMTS13 MAb; Detection antibody: biotinylated rabbit anti‐ADAMTS13 polyclonal Ab1:20 in Assay buffer740 ± 110 ng mL−1 Intra‐assay CV: 4% Inter‐assay CV: 7.3%One standard (100 ng mL−1) to be serially diluited 1 positive Control The standard is prepared from normal plasma calibrated against a recombinant ADAMTS13HUMAN ADAMTS13 ELISA KIT (BETHIL Laboratorie Inc, Montgomery, TX, USA)ADAMTS13 antigenChromogenic; 2 polyclonal Abs produced against distinct and disparate regions of human ADAMTS131:10 in ELISA diluent–One calibrator (100%) to be serially diluited 1 positive Control 1 negative ControlTECHNOZYM ADAMTS‐13 INH (Technoclone GmbH,Wien, Austria)Anti‐ ADAMTS13 IgGChromogenic; Antigen used: full‐length recombinant ADAMTS13; Detection antibody: anti‐human IgG antibody POX, diluted 1:50 in Incubation buffer1:100 in Incubation bufferNegative samples: < 12 U mL−1 borderline: 12–15 U mL−1 positive samples: > 15 U mL−1Five calibrators 1 positive control 1 negative control calibrated against a plasma with a very high titre of ADAMTS13 IgG (1:200 dilution defined to contain 100 U mL−1)IMUBIND ADAMTS13 Autoantibody ELISA (American Diagnostica Inc, Stamford, CT, USA)Anti‐ ADAMTS13 IgGChromogenic; Antigen used: full‐length recombinant ADAMTS13; Detection antibody: goat antihuman IgG antibody HRPconjugated, diluted 1:100 in assay buffer1:20 in Assay bufferPositivity cut‐off = 9.6 AU mL−1One standard (60 AU mL−1) to be serially diluited 1 positive Control Open table in a new tab The human ADAMTS13 gene is located at chromosome 9q34, spans approximately 37 kb and contains 29 exons [38Levy G.G. Nichols W.C. Lian E.C. Foroud T. McClintick J.N. McGee B.M. Yang A.Y. Siemieniak D.R. Stark K.R. Gruppo R. Sarode R. Shurin S.B. Chandrasekaran V. Stabler S.P. Sabio H. Bouhassira E.E. Upshaw Jr, J.D. Ginsburg D. Tsai H.M. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.Nature. 2001; 413: 488-94Crossref PubMed Scopus (1430) Google Scholar]. In suspected congenital TTP cases, genetic analysis, carried out by polymerase chain reaction (PCR) amplification and sequencing of the exon areas and the intron–exon boundaries of ADAMTS13, is performed to identify the presence of genetic variants responsible for the disease. Up to now, at least 76 ADAMTS‐13 mutations have been reported [see review 39Lotta L.A. Garagiola I. Palla R. Cairo A. Peyvandi F. ADAMTS13 mutations and polymorphisms in congenital thrombotic thrombocytopenic purpura.Hum Mutat. 2009; 10.1002/humu.21143Google Scholar], including: 45 missense mutations (59%), 10 nonsense mutations (13%), 10 deletions (13%), 4 insertions (6%) and 7 splice site mutations (9%). Nineteen non‐synonymous single nucleotide polymorphisms (SNPs) of ADAMTS13 are recorded in dbSNP(http://www.ncbi.nlm.nih.gov/projects/SNP/); eight of them have also been reported in patients with congenital TTP [see review 39Lotta L.A. Garagiola I. Palla R. Cairo A. Peyvandi F. ADAMTS13 mutations and polymorphisms in congenital thrombotic thrombocytopenic purpura.Hum Mutat. 2009; 10.1002/humu.21143Google Scholar]. Notably, two polymorphisms of ADAMTS13 (c.1423C>T leading to p.P475S, and c.1852C>G leading to p.P618A) have been demonstrated to result in a significant reduction of mutant recombinant ADAMTS‐13 activity in vitro [40Kokame K. Matsumoto M. Soejima K. Yagi H. Ishizashi H. Funato M. Tamai H. Konno M. Kamide K. Kawano Y. Miyata T. Fujimura Y. Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factorcleaving protease activity.Proc Natl Acad Sci USA. 2002; 99: 11902-7Crossref PubMed Scopus (0) Google Scholar, 41Plaimauer B. Fuhrmann J. Mohr G. Wernhart W. Bruno K. Ferrari S. Konetschny C. Antoine G. Rieger M. Scheiflinger F. Modulation of ADAMTS13 secretion and specific activity by a combination of common amino acid polymorphisms and a missense mutation.Blood. 2006; 107: 118-25Crossref PubMed Scopus (90) Google Scholar], indicating that genetic variants of ADAMTS13 may profoundly affect in vivo protease activity. The congenital form of the disease should be suspected in patients who present with TTP and severe ADAMTS‐13 deficiency in the absence of anti‐ADAMTS‐13 autoantibodies. Plasma therapy (plasma exchange or plasma infusion) is usually carried out before the diagnosis of congenital TTP is confirmed by the id
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