Summary. Background: Thrombotic thrombocytopenic purpura (TTP) is a rare life‐threatening disease. Of surviving patients, 45% develops an exacerbation or a late recurrence. Severe ADAMTS‐13 deficiency, both during the acute episode and remission, is a well‐established predictor of recurrence. The predictive value of anti‐ADAMTS‐13 antibodies, their inhibitory activity and Ig class subtype for disease recurrence is still to be established. Objectives: To analyze ADAMTS‐13‐related biomarkers (ADAMTS‐13 and anti‐ADAMTS‐13 immunoglobulins, classes and subclasses) and their potential relationship with prognosis. Patients/Methods: In 115 patients with TTP, we assessed the association between levels of these biomarkers and the severity of acute episodes; we analysed also the hazard ratio (HR) and 95% confidence interval (CI) of recurrence in association with biomarkers levels retrieved at the previous acute episode or during remission, using Cox regression models. Results: During the acute phase, higher IgA, IgG1 and IgG3 titers showed the strongest association with acute episode severity. In the survival analyzes, the only biomarker significantly associated with a high hazard of recurrence after an acute episode was the presence of IgG. Conversly, low ADAMTS‐13 activity or antigen levels (< 10%), the presence of ADAMTS‐13 inhibitor or IgG during remission were all significantly associated with a higher hazard of recurrence. Conclusions: Both the Ig class and subclass are of predictive value for acute episode severity in patients with TTP. Although markers that could predict the risk of recurrence in the acute phase are limited, a thorough assessment of ADAMTS‐13‐related parameters during remission is warranted.

Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) characterized by the severe deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity (< 10%). Rapid ADAMTS13 testing is crucial for an early diagnosis and optimal management of acute TTP. We evaluated the performance of the HemosIL AcuStar ADAMTS13 activity assay (Instrumentation Laboratory, Bedford, Massachusetts, United States), a fully automated chemiluminescent immunoassay with an analytical time of 33 minutes. A method comparison study was performed on 176 samples from 49 healthy donors and 127 TMA patients (109 TTP, 7 atypical hemolytic uremic syndrome, 11 other TMAs), comparing this new assay with an in-house FRETS-VWF73 assay and a commercial enzyme-linked immunosorbent assay (ELISA) (TECHNOZYM ADAMTS-13 Activity, Technoclone GmbH, Vienna, Austria). Agreement between methods was assessed with focus on ADAMTS13 activity less than 10%, the medical decision level relevant for TTP diagnosis. The HemosIL AcuStar ADAMTS13 Activity showed good correlation with both the FRETS-VWF73 (r = 0.96) and ELISA (r = 0.96) methods. Slope of the Passing–Bablok regression was 1.05 for FRETS-VWF73 and 1.02 for ELISA, and absolute bias at the medical decision level was +0.1 and +0.3%, respectively. The study also revealed high agreement with FRETS-VWF73 (kappa 0.97) and ELISA (kappa 0.98) methods in classifying TTP patients with a severe deficiency of ADAMTS13 activity. Because of its short turnaround time and full automation, the HemosIL AcuStar ADAMTS13 activity assay might become the assay of choice to rapidly test ADAMTS13 activity in plasma and thus establish the diagnosis of acute TTP in emergency settings.

Recently, we showed that during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP), ADAMTS13 circulates in an open conformation. Although the cause of this conformational change in acute iTTP remains elusive, ADAMTS13 is mainly closed in iTTP patients (i) in remission with an ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, and (ii) after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, IgGs from 18 acute iTTP patients were purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14/18 (78%) samples, proving that indeed anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n=197) that also included plasma samples of iTTP patients in remission where ADAMTS13 activity was <50%. The open ADAMTS13 conformation was not only found during acute iTTP but also in patients in remission with an ADAMTS13 activity 50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus open ADAMTS13 is not only a hallmark of acute iTTP, but also a novel biomarker to detect subclinical iTTP in patients in remission. Finally, a long term follow-up study in one iTTP patient showed that the open conformation precedes a severe drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management.

Acquired thrombotic thrombocytopaenic purpura (TTP) is a rare thrombotic microangiopathy caused by the immune-mediated severe deficiency of ADAMTS13. We hereby report the demographic and disease-related data of acquired TTP patients recorded in the Milan TTP Registry (www.ttpdatabase.org). We performed a cross-sectional study of 302 individuals enrolled in our registry for an acute episode of acquired TTP occurred between 2002 and 2015 (female 77%; median age at onset 40 years, interquartile range: 30–50). Twenty per cent of patients had concomitant autoimmune disorders. Among potential triggers of acute episodes, infections were the most prevalent (27%), followed by estroprogestinics use and pregnancy (5 and 4% of women, respectively). At presentation, systemic (72%), bleeding (68%) and neurological (43%) symptoms were the most frequent, whereas a lower prevalence of renal (18%) and cardiovascular (10%) signs and symptoms was observed. Almost all acute events were treated by plasma exchange and steroids, and 15% by rituximab. Exacerbation of acute TTP occurred in 15% of events. The TTP-related mortality was 5%. In survivors, the median number of plasma exchange procedures to remission was 9 (interquartile range: 6–14), longer for first events than relapses (median difference 3, 95% confidence interval: 2–4). Of 251 survivors of the first TTP episode with at least a 6-month follow-up, 55% had a relapse. In conclusion, acquired TTP is a severe disease with highly variable clinical presentation, usually requiring a long hospitalization. The Milan TTP Registry represents a powerful tool to improve our knowledge and management of acquired TTP. I. Mancini designed the study, performed experiments, performed quality control and statistical analysis, interpreted the results and wrote the manuscript; S. Pontiggia performed experiments, collected clinical and/or experimental data, performed quality control and statistical analysis and contributed to write the manuscript; R. Palla interpreted the results, contributed to write and critically reviewed the manuscript; A. Artoni collected clinical data, interpreted the results and critically reviewed the manuscript; C. Valsecchi performed experiments, interpreted the results and critically reviewed the manuscript; B. Ferrari collected clinical data, interpreted the results and critically reviewed the manuscript; D. Mikovic, S.M. Trisolini, S. Capria, L. Facchini, K. Codeluppi, E. Rinaldi, D. Pastore, S. Campus, R.A. Podda, C. Caria, A. Caddori, D. Nicolosi, G. Giuffrida, V. Agostini, U. Roncarati, C. Mannarella, A. Fragasso, G.M. Podda, S. Birocchi, A.M. Cerbone, A. Tufano, G. Loffredo, G. Menna, M. Pizzuti, G. Re, M. Ronchi, A. De Fanti, S. Amarri, M. Defina, M. Bocchia and S. Cerù collected clinical data; F. Peyvandi designed the study, interpreted the results and critically reviewed the manuscript. The Italian Group of TTP Investigators includes: Silvia Maria Trisolini and Saveria Capria (Hematology, Department of Cellular Biotechnologies and Hematology, Policlinico Umberto 1, Sapienza University, Rome, Italy), Luca Facchini and Katia Codeluppi (Hematology Unit, Oncology Department, Azienda Unità Sanitaria Locale – IRCCS di Reggio Emilia, Reggio Emilia, Italy), Erminia Rinaldi and Domenico Pastore (Hematology Unit, A. Perrino Hospital, Brindisi, Italy), Simona Campus and Rosa Anna Podda (Pediatric Unit, Ospedale Microcitemico, Cagliari, Italy), Cinzia Caria and Aldo Caddori (Internal Medicine Unit, S.S. Trinità Hospital, Cagliari, Italy), Daniela Nicolosi and Gaetano Giuffrida (Hematology Division, Department of Clinical and Molecular Biomedicine, University of Catania, Catania, Italy), Vanessa Agostini and Umberto Roncarati (U.O. Immunoematologia e Medicina Trasfusionale/Officina Trasfusionale, Cesena e Forlì, Cesena, Italy), Clara Mannarella and Alberto Fragasso (Hematology Unit, Madonna delle Grazie Hospital, Matera, Italy), Gian Marco Podda and Simone Birocchi (Unità di Medicina III, ASST Santi Paolo e Carlo, Milano, Italy), Anna Maria Cerbone and Antonella Tufano (Department of Clinical Medicine and Surgery, AOU Federico II, Napoli, Italy), Giuseppe Loffredo and Giuseppe Menna (Department of Oncology, AORN Santobono-Pausilipon, Naples, Italy), Michele Pizzuti (Hematology Unit, AO San Carlo, Potenza, Italy), Giuseppe Re and Michela Ronchi (Internal Medicine Unit, Department of Medicine, Lugo Hospital, Ravenna, Italy), Alessandro De Fanti (Departmental Simple Unit of Pediatric Rheumatology, AUSL-IRCSS Reggio Emilia, Reggio Emilia, Italy), Sergio Amarri (Pediatrics Unit, AUSL-IRCSS Reggio Emilia, Reggio Emilia, Italy), Marzia Defina and Monica Bocchia (Università degli studi di Siena, Department of Medical, Surgery and Neuroscience, Hematology Unit, Azienda Ospedaliera Universitaria Senese, Siena, Italy) and Silvia Cerù (Hematology Unit, Santa Chiara Hospital, Trento, Italy). ^* A complete list of the members of the Italian Group of TTP Investigators appears in section ”Study Group Members.”

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), by the ADAMTS13 Assay Working Group, which comprises an international group of both clinical and laboratory experts. The document provides recommendations on best practice for the performance of ADAMTS13 assays in clinical laboratories. ADAMTS13 assays support the differential diagnosis of thrombotic microangiopathies and have utility in the management of thrombotic thrombocytopenic purpura (TTP). There are three types of assay: activity, antigen and autoantibody/inhibitor assays. Methods for activity assays differ in terms of sensitivity, specificity, precision and turnaround time. The most widely used assays involve VWF peptide substrates and either chromogenic ELISA or FRET techniques, although chemiluminescence assays and rapid screening tests have recently become available. Tests for autoantibodies and inhibitors allow confirmation of acquired, immune‐mediated TTP, while antigen assays may be useful in congenital TTP and as prognostic markers. In this document, we have attempted to describe ADAMTS13 assays and the conditions that affect them, as well as: blood collection, sample processing, quality control, standardization and clinical utility; recognizing that laboratories in different parts of the world have varying levels of sophistication. The recommendations are based on expert opinion, published literature and good clinical laboratory practice.