AbstractDespite clinical remission and normal platelet counts, congenital TTP (cTTP) is associated with non‐overt symptoms. Prophylactic ADAMTS13 replacement therapy such as plasma infusion (PI) prevents acute episodes and improves symptomatology. There is no current method to investigate disease severity or monitor the impact of treatment. We utilize a dynamic high shear flow assay to further understand disease pathophysiology and determine the impact of cTTP on symptomatology and therapy, despite normal platelet counts. Whole blood, under high shear, was run over collagen‐coated channels, causing platelet adhesion to von Willebrand factor (VWF) multimers. The resulting surface coverage by platelet‐VWF thrombus was assessed. The normal range was 6–39% in 50 controls. Twenty‐two cTTP patients with normal platelet counts were evaluated. Median pre‐treatment surface coverage was 89%, and PI reduced coverage to a median of 44% (p = 0.0005). Patients taking antiplatelets had further reduced coverage when combined with PI and improved non‐overt symptoms such as headache, lethargy, and abdominal pain in 100% of patients compared to 74% with PI alone (p = 0.046). We use a dynamic assay to report increased in vitro platelet adhesion and aggregation and additionally demonstrate significantly decreased thrombi following PI, with levels in the normal range levels achieved in patients taking additional antiplatelet therapy.

BACKGROUND:Thrombotic thrombocytopenic purpura (TTP), caused by a genetic or autoimmune-driven lack of ADAMTS-13 activity, leads to high levels of the ultra-large von Willebrand factor (VWF) multimers produced by endothelial cells, causing excess platelet recruitment into forming thrombi, often with mortal consequences. Treatments include plasma infusion or replacement to restore ADAMTS-13 activity, or prevention of platelet recruitment to VWF.OBJECTIVES:We tested a different approach, exploiting the unique cell biology of the endothelium. Upon activation, the VWF released by exocytosis of Weibel-Palade bodies (WPBs), transiently anchored to the cell surface, unfurls as strings into flowing plasma, recruiting platelets. Using plasma from patients with TTP increases platelet recruitment to the surface of cultured endothelial cells under flow. WPBs are uniquely plastic, and shortening WPBs dramatically reduces VWF string lengths and the recruitment of platelets. We wished to test whether the TTP plasma-driven increase in platelet recruitment would be countered by reducing formation of the longest WPBs that release longer strings.METHODS:Endothelial cells grown in flow chambers were treated with fluvastatin, one of 37 drugs shown to shorten WPBs, then activated under flow in the presence of platelets and plasma of either controls or patients with TTP.RESULT:We found that the dramatic increase in platelet recruitment caused by TTP plasma is entirely countered by treatment with fluvastatin, shortening the WPBs.CONCLUSIONS:This potential approach of ameliorating the endothelial contribution to thrombotic risk by intervening far upstream of hemostasis might prove a useful adjunct to more conventional and direct therapies.

Acute ischemic stroke (IS) and transient ischemic attack (TIA) are associated with raised von Willebrand factor (VWF) and decreased ADAMTS13 activity (ADAMTS13Ac). Their impact on mortality and morbidity is unclear. We conducted a prospective investigation of the VWF-ADAMTS13 axis in 292 adults (acute IS, n = 103; TIA, n = 80; controls, n = 109) serially from presentation until >6 weeks. The National Institutes of Health Stroke Score (NIHSS) and modified Rankin scale (mRS) were used to assess stroke severity. Presenting median VWF antigen (VWF:Ag)/ADAMTS13Ac ratios were: IS, 2.42 (range, 0.78-9.53); TIA, 1.89 (range, 0.41-8.14); and controls, 1.69 (range, 0.25-15.63). Longitudinally, the median VWF:Ag/ADAMTS13Ac ratio decreased (IS, 2.42 to 1.66; P = .0008; TIA, 1.89 to 0.65; P < .0001). The VWF:Ag/ADAMTS13Ac ratio was higher at presentation in IS patients who died (3.683 vs 2.014; P < .0001). A presenting VWF:Ag/ADAMTS13Ac ratio >2.6 predicted mortality (odds ratio, 6.33; range, 2.22-18.1). Those with a VWF:Ag/ADAMTS13Ac ratio in the highest quartile (>3.091) had 31% increased risk mortality. VWF:Ag/ADAMTS13Ac ratio at presentation of ischemic brain injury was associated with higher mRS (P = .021) and NIHSS scores (P = .029) at follow-up. Thrombolysis resulted in prompt reduction of the VWF:Ag/ADAMTS13Ac ratio and significant improvement in mRS on follow-up. A raised VWF:Ag/ADAMTS13Ac ratio at presentation of acute IS or TIA is associated with increased mortality and poorer functional outcome. A ratio of 2.6 seems to differentiate outcome. Prompt reduction in the ratio in thrombolysed patients was associated with decreased mortality and morbidity. The VWF:Ag/ADAMTS13Ac ratio is a biomarker for the acute impact of an ischemic event and longer-term outcome.

Congenital thrombotic thrombocytopenic purpura (cTTP) is an ultra-rare thrombomi-croangiopathy caused by an inherited deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13). There are limited data on genotype-phenotype correlation; there is no consensus on treatment. We reviewed the largest cohort of cTTP cases, diagnosed in the United Kingdom, over the past 15 years. Seventy-three cases of cTTP were diagnosed, confirmed by genetic analysis. Ninety-three percent were alive at the time of review. Thirty-six percent had homozygous mutations; 64% had compound heterozygous mutations. Two presentation peaks were seen: childhood (median diagnosis age, 3.5 years) and adulthood, typically related to pregnancy (median diagnosis age, 31 years). Genetic mutations differed by age of onset with prespacer mutations more likely to be associated with childhood onset (P = .0011). Sixty-nine percent of adult presentations were associated with pregnancy. Fresh-frozen plasma (FFP) and intermediate purity factor VIII concentrate were used as treatment. Eighty-eight percent of patients with normal blood counts, but with headaches, lethargy, or abdominal pain, reported symptom resolution with prophylactic therapy. The most common currently used regimen of 3-weekly FFP proved insufficient for 70% of patients and weekly or fortnightly infusions were required. Stroke incidence was significantly reduced in patients receiving prophylactic therapy (2% vs 17%; P = .04). Long-term, there is a risk of end-organ damage, seen in 75% of patients with late diagnosis of cTTP. In conclusion, prespacer mutations are associated with earlier development of cTTP symptoms. Prophylactic ADAMTS13 replacement decreases the risk of end-organ damage such as ischemic stroke and resolved previously unrecognized symptoms in patients with nonovert disease.

Background: Congenital Thrombotic Thrombocytopenic Purpura (TTP) is defined by severe ADAMTS13 activity (<10%) and absence of anti-ADAMTS13 antibodies, confirmed by mutational analysis. ADAMTS13 deficiency results in increased ultra large Von Willebrand Factor (VWF) multimers and disseminated microvascular ischemia. Current replacement therapy is primarily using plasma infusion (PI). Dose and PI interval are selected empirically for each patient. At present there is no method to assess disease and treatment response in patients with cTTP. Aims: We developed an innovative shear flow-based assay to assess cTTP, with normal platelet counts, receiving plasma infusion (PI) or intermediate purity factor VIII concentrate (BPL-8Y). Methods: A VenaFlux semi-automated microfluidic system provides high shear flow. Citrated whole blood was treated with DiOC6 for platelet fluorescence. Thrombus formation on collagen-coated microchannels was analysed by an epifluorescence microscope and an automated calculation of total surface coverage was developed. Surface coverage represented increasing thrombus formation (VWF-platelets). Total surface coverage within 180 seconds equalled 100% coverage. Normal range for surface coverage, using 50 normal controls, was 6–39%. cTTP samples were taken 30 minutes before and after prophylaxis (PI or BPL-8Y) and surface coverage determined. Further re-measurement was undertaken after initiation of antiplatelet therapy. Results: 21 confirmed cTTP patients: 18 female and 3 male, median age: 33 years (range 16–69). Median pre-treatment surface coverage was 89% (range 47–100%), with no significant difference considering mutation type (homozygous 98%, heterozygous 83%, p = 0.097) or age of presentation (childhood onset 73%, adult onset 90%, p = 0.19). PI improved surface coverage results (post PI coverage 44%, p = 0.0005). Antiplatelet therapy further improved median surface coverage, pre and post prophylaxis. Median pre-treatment surface coverage for patients receiving PI and antiplatelet therapy was 50% (p < 0.0001). This improvement was confirmed by the median post-treatment coverage (19% for patients on antiplatelets vs. 44% for patients not on antiplatelets, p = 0.0011). Post-treatment surface coverage returned to normal range in 100% patients who received PI and antiplatelet therapy compared to 50% in patients who received PI alone (p = 0.04). Summary/Conclusion: This advanced shear flow-based assay showed increased thrombi formation in cTTP patients, despite ‘remission’, confirming severe ADAMTS13 deficiency is associated with increased circulating ultra large VWF multimers and platelet thrombi. This assay provides additional information on treatment assessment in cTTP patients, optimised with regular PI and antiplatelet therapy, and it guides treatment.

Essentials Congenital thrombotic thrombocytopenic purpura (TTP) is primarily treated with plasma infusion. We present a pharmacokinetic analysis of ADAMTS-13 in six patients following plasma infusion. A median half-life of 130 h was demonstrated, ranging between 82.6 and 189.5 h. Investigation of interindividual clearance of ADAMTS-13 is necessary to optimize treatment. Background Congenital thrombotic thrombocytopenic purpura (TTP) is defined by persistent severe deficiency of ADAMTS-13 in the absence of anti-ADAMTS-13 inhibitory antibodies, confirmed by mutational analysis. Replacement of the missing protease prevents disease relapse, primarily using plasma infusion (PI). Objectives, patients and methods There is scant evidence regarding optimal dose and frequency of treatment, which tends to be empirically guided. We present a pharmacokinetic analysis of ADAMTS-13 in six patients with congenital TTP on established regimes following PI. Results We found a median clearance of 25.41 mL h(-1) and half-life of 130 h, ranging between 82.6 and 189.5 h (3.4-7.9 days, respectively). All patients reached baseline ADAMTS-13 level within 7-10 days post-plasma. Median ADAMTS-13 activity peak post-PI was 24.05 IU dL(-1). Variation was related to elimination rate, which, in turn, was affected by weight and metabolism, but not to von Willebrand factor antigen or activity levels. Using the pharmacokinetic parameters, we simulated individualized protocols based on PI dose or frequency to target hypothetical optimal plasma levels of ADAMTS-13 of 10 and 50 IU dL(-1), respectively. Results suggest a target trough ADAMTS-13 of 10 IU dL(-1) is feasible but 50 IU dL(-1) would not be achievable taking into account volume required. Conclusions Further work is needed to compare treatment of congenital TTP with PI vs. recombinant ADAMTS-13. PI may provide longer duration of ADAMTS-13 effect, but is limited by plasma volume required, whereas recombinant therapy can provide a higher ADAMTS-13 peak. We propose that investigation of interindividual clearance of ADAMTS-13 is necessary to optimize treatment and provide the rationale for dose and frequency of prophylaxis.

Plasma exchange (PEX) is a therapeutic procedure used to treat diseases caused by pathogenic antibodies or immune-complexes through the removal and the replacement of plasma. The frequency of complications and reactions associated with PEX are mild and of limited duration. In systemic autoimmune diseases and in a variety of other conditions, PEX might be use in association with intravenous immunoglobulin (IVIg) or therapeutic monoclonal antibodies for the management of acute or refractory patients to achieve a durable remission.

Rituximab (MabThera; Roche Pharmaceuticals) is a chimeric mouse-human monoclonal antibody against CD20. Once bound to CD20, rituximab affects B cell death, reducing pathogenic antibody production. Rituximab is used in a number of autoimmune diseases (Townsend et al, 2010) including Thrombotic Thrombocytopenic Purpura (TTP) (Scully et al, 2007). Rituximab has been shown to be effective and safe in acute immune TTP (iTTP) and pre-emptive therapy to prevent relapse (Westwood et al, 2017) by increasing ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif 13) activity through the reduction of anti-ADAMTS13 immunoglobulin (Ig)G antibody levels (Scully et al, 2007). The administration of rituximab may be associated with infusional reactions, including fever, chills, rigors and, less commonly, allergic anaphylactoid reactions. Infusional symptoms are most related to the first infusion of the drug (Kumar et al, 2012). Serum sickness is a classic immune complex (IC)-mediated type III delayed hypersensitivity reaction, initiated by the interaction of IgG or IgM with a soluble antigen leading to the formation of circulating immune complexes (Eggleton, 2001). Two forms of reactions have been identified, acute and delayed, both apparently related to the monoclonal components. It has been reported that delayed serum sickness usually occurs 10-14 days following antigen exposure or within a few days of secondary antigen exposure in the acute setting (Kumar et al, 2012). We have developed an assay to detect antibodies to rituximab, investigated in patients who received rituximab for iTTP from September 2015 to March 2017 and developed acute or delayed type serum sickness reactions following treatment with rituximab (Appendix S1). Sixty-four patients received rituximab for iTTP in our centre during the study period. There were 7 episodes defined as acute or delayed serum sickness reactions. Three patients received rituximab as part of first line therapy at presentation of TTP and four patients had previously had one acute TTP episode and received elective rituximab to prevent relapse. Three patients presented with acute serum sickness within a median of 10·5 h following rituximab infusion and four patients presented with delayed serum sickness reactions, within a median of 7 days of completing rituximab. Only the three patients with acute serum sickness had detectable antibodies to rituximab (Table 1). Further infusions of rituximab were avoided in those with acute serum sickness and positive antibodies to rituximab; they all received a humanised monoclonal antibody, ofatumumab, and completed therapy, with normalisation of ADAMTS13 activity. No reactions were documented with ofatumumab, except for a patient who developed a pruritic rash, resolving with a reduction in the infusional rate. One patient with delayed serum sickness and negative antibodies to rituximab received subsequent infusions of rituximab without further reactions. Nausea and vomiting multiple times Profuse diarrhoea Wide spread maculopapular rash on arms, legs and neck, no bruising Elective Ofatumumab (3 doses completed). Mild reaction during the 1st dose: pruritic rash on the chest and sore throat. 1: 375 mg/m2 × 4 2: 100 mg/m2 × 4 Facial swelling, sudden itch, shortness of breath and difficulty swallowing Full body rash Associated SLE and inflammatory arthritis Associated left MCA infarct and cardiac involvement during first TTP admission (2009) Laboratory investigations demonstrated a decrease of complement C3 and C4 in two patients, one with acute serum sickness and one with delayed serum sickness (C3: 0·68 and 0·53 g/l; C4: 0·04 and 0·10 g/l, respectively), while C3 and C4 were within normal ranges in the remaining 5 patients. We describe acute serum sickness in TTP patients receiving rituximab, confirmed by the presence of antibodies to rituximab. Anti-rituximab antibodies were only detected in the patients who presented with acute serum sickness during rituximab infusion. Acute serum sickness occurred significantly earlier within the second cycle (respectively after 6 h and 2 h) compared to the first cycle (after 24 h). It usually occurs within a few days of re-exposure. The classical clinical manifestations consist of fever, arthralgia, lymphadenopathy and skin eruption. Other symptoms reported were severe swelling, angioedema, tachycardia, breathlessness, diarrhoea and myalgia (Kumar et al, 2012). In our study, all the patients who presented with delayed serum sickness had undetectable anti-rituximab antibodies. Delayed serum sickness remains a clinical diagnosis based on the triad of fevers, rash and arthralgia. Serum sickness after administration of rituximab has been reported at a higher rate among patients with autoimmune diseases than among patients with lymphoma, suggesting its development is influenced by disease entity and concurrent therapies (Goto et al, 2009). Antigen-antibody complexes cause tissue damage on being deposited in target tissues, recruiting complement and activating mast cells and phagocytes. The pathophysiological mechanism should be caused by the presence of anti-rituximab antibodies. Some studies have reported that excess antigen may have bound all the anti-rituximab antibodies present in the circulation and, consequently, may have led to antigen-antibody complex formation and precipitation (Goto et al, 2009). Therefore, anti-rituximab antibodies may not be detectable because excessive amounts of antigen (i.e., rituximab) completely consumed any anti-rituximab antibodies present (Todd & Helfgott, 2007). It is not clear for how long anti-rituximab antibodies remain in the circulation. One case report reported that anti-rituximab antibodies were completely undetectable after two months (Goto et al, 2009). In our study, serial samples were available for 2 out of 3 patients with anti-rituximab antibodies, becoming undetectable at 1 and 6 months, respectively. Delayed type serum sickness reactions after rituximab have been described in other conditions, but a minority have detected antibodies to rituximab (Seror et al, 2007; Goto et al, 2009). The production of anti-rituximab antibodies has been described in pemphigus and systemic lupus erythematosus (SLE) (Saito et al, 2005; Lunardon & Payne, 2012). Rituximab may be more immunogenic in autoimmune disease because of the highly activated B lymphocyte status (Saito et al, 2005). In conclusion, with the increasing use of rituximab in autoimmune TTP, clinicians should be alert for acute or delayed serum sickness as possible adverse reactions and should consider the utility of testing for anti-rituximab antibodies to weigh the risks and benefits of continuing/reintroducing rituximab, given that re-exposure can result in more severe manifestations. In contrast, cases of delayed serum sickness were not associated with detection of anti-rituximab antibodies and further rituximab courses are possible. Measurement of anti-rituximab antibodies can guide the choice of alternative monoclonal anti-CD20 therapy to ensure continued TTP remission. None. C. Vendramin analysed the data and wrote the paper; M. Thomas, J.-P. Westwood, S. McGuckin and M. Scully collected data and reviewed the manuscript. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A diagnosis of thrombotic thrombocytopenic purpura (TTP) is confirmed by a severe deficiency (<10%) of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity. Autoantibodies to ADAMTS13 can be detected with a simplified enzyme-linked immunosorbent assay (ELISA). An alternative methodology is a Bethesda assay, which has never been formally assessed in TTP. This study aimed to investigate the inhibitory anti-ADAMTS13 antibody assay and determine if the Bethesda assay is advantageous compared with the ELISA, measuring total immunoglobulin G (IgG) antibodies to ADAMTS 13. The Bethesda method determines the neutralizing activity of anti-ADAMTS13 antibodies in pooled normal plasma. We selected six immune-mediated TTP (iTTP) patients with ADAMTS13 activity levels <10% and strong ADAMTS13 inhibitors by 50:50 mixing studies and analyzed anti-ADAMTS13 antibodies using the Bethesda and ELISA assays. ADAMTS13 activity was stable at room temperature, while a time-dependent decrease in activity was detected in assay conditions of 37°C. Adding 5 mM Ca2+ to citrated plasma prevented loss of ADAMTS13 activity with time. There was time dependence to the antibody-mediated inactivation, after 2-hour incubation. Two of the iTTP patients had no detectable ADAMTS13 antibodies by the Bethesda assay, but had high titer of anti-ADAMTS13 antibodies and low ADAMTS13 antigen levels. The Bethesda assay can only detect anti-ADAMTS13 antibodies that functionally inhibit ADAMTS13. The anti-ADAMTS13 IgG ELISA instead allows the rapid identification of total IgG autoantibodies, detecting both inhibitory and noninhibitory antibodies.

Abstract Introduction Congenital TTP (cTTP) is an ultra-rare disorder in which deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) results in circulating ultra large Von Willebrand Factor (VWF) multimers and subsequent microthrombi formation. Regular prophylactic therapy aims to improve outcomes from long-term complications, but also ongoing symptoms, including lethargy, headaches and abdominal pain, despite normal blood counts. Existing methods of quantifying ADAMTS13 activity lack the sensitivity to enable their use for evaluating treatment response in patients with cTTP. We present a novel flow-based assay with the aim of assessing treatment response, novel therapeutic options and analyzing the impact of different mutations on disease severity. Method A VenaFlux semi-automated microfluidic system was used to provide shear flow to mimic in vivo flow rates. Using whole blood, we analyzed platelet adhesion, aggregation and thrombi formation on microchannels coated with type I collagen and mounted onto the stage of an inverted epifluorescence microscope. Fresh, citrated whole blood was treated with DiOC6 to achieve platelet fluorescence and a macro on Image-Pro Premier was designed for automated calculation of total surface coverage. Surface coverage represented increasing thrombus formation with total coverage by thrombus within 180 seconds quantified as 100% coverage. Results were compared to a normal range developed using 43 normal controls (26=female, 17=male) with normal hemoglobin, platelet count and hematocrit. The surface coverage normal range was 6-39%. cTTP samples were analyzed for complete blood count, ADAMTS13 activity, VWF antigen, VWF activity and percentage surface coverage. Samples were taken 30 minutes before and after prophylactic treatment, either plasma infusion or BPL-8Y. Recombinant ADAMTS13 was added in-vitro on all pre-treatment samples with 15 minutes incubation time. Further re-measurement was undertaken after initiation of aspirin for at least ten days. Results Eighteen patients with cTTP confirmed by genetic analysis and ADAMTS13 levels <5 IU/dl were included (16 = female, 2 = male) with a median age of 33 (range: 15-69 years). Median VWF antigen levels: 114% (range: 54% - 276%, NR: 50-160%) and median VWF activity levels: 173% (range: 83% - 338%, NR: 50-187%). The median pre-treatment surface coverage was 90% (range 47% - 100%). There was no significant difference in surface coverage considering genetic mutation type (median coverage for homozygous patients 88%, heterozygous 67%, p=0.99), mutation location (pre-spacer mutation surface coverage 67%, post spacer mutation surface coverage 84%, p=0.84), or age of first symptom onset (childhood onset surface coverage 59%, adult onset 86%, p=0.19). Plasma infusion improved surface coverage results with pre treatment coverage of 90% compared to 44% post plasma infusion (p=0.0003). In vivo recombinant ADAMTS13 administration on pre prophylaxis samples, resulted in normalization of surface coverage in all patients (p<0.0001)(median post rADAMTS13 coverage 28%, range 3-39%). In patients initiated on aspirin, surface coverage had improved both pre and post prophylaxis. The median pre treatment surface coverage for patients on aspirin was 51% (vs. 90% pre treatment and no aspirin, p=0.004). This improvement persisted after treatment with post treatment surface coverage of 18% (vs. 44% post treatment but not on aspirin, p=0.003). 100% of patients who received aspirin saw surface coverage return to the normal range post treatment compared to 82% with plasma infusion alone (p=0.0195). Conclusion Plasma infusion and aspirin synergistically reduce surface coverage by thrombus in patients with cTTP, demonstrated on peak and trough samples. Furthermore, in vitro addition of recombinant ADAMTS13 completely normalized thrombus formation. There were no major differences in surface coverage by genetic mutation. The newly developed flow-based assay presented can be used to assess treatment options and efficacy in cTTP in addition to demonstrating cTTP disease pathophysiology that has not previously been identified. In combination with clinical symptoms it offers potential to improve and personalize treatment for patients with cTTP. Figure. Figure. Disclosures Liesner: Bayer: Consultancy, Research Funding; Sobi: Speakers Bureau; Roche: Research Funding; Baxalta: Consultancy, Research Funding; Novo Nordisk: Research Funding, Speakers Bureau; Octapharma: Consultancy, Other: Clinical study investigator for NuProtect Study (Octapharma sponsored), Research Funding, Speakers Bureau. Scully:Novartis: Honoraria, Other: Member of Advisory Board, Speakers Bureau.

AbstractImmune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by severe ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) deficiency, the presence of anti-ADAMTS13 autoantibodies and an open ADAMTS13 conformation with a cryptic epitope in the spacer domain exposed. A detailed knowledge of anti-ADAMTS13 autoantibodies will help identifying pathogenic antibodies and elucidating the cause of ADAMTS13 deficiency. We aimed at cloning anti-ADAMTS13 autoantibodies from iTTP patients to study their epitopes and inhibitory characteristics. We sorted anti-ADAMTS13 autoantibody expressing B cells from peripheral blood mononuclear cells of 13 iTTP patients to isolate anti-ADAMTS13 autoantibody sequences. Ninety-six B cell clones producing anti-ADAMTS13 autoantibodies were identified from which 30 immunoglobulin M (IgM) and 5 IgG sequences were obtained. For this study, we only cloned, expressed and purified the five IgG antibodies. In vitro characterization revealed that three of the five cloned IgG antibodies, TTP73–1, ELH2–1 and TR8C11, indeed recognize ADAMTS13. Epitope mapping showed that antibodies TTP73–1 and TR8C11 bind to the cysteine–spacer domains, while the antibody ELH2–1 recognizes the T2–T3 domains in ADAMTS13. None of the antibodies inhibited ADAMTS13 activity. Given the recent findings regarding the open ADAMTS13 conformation during acute iTTP, we studied if the cloned antibodies could recognize cryptic epitopes in ADAMTS13. Interestingly, all three antibodies recognize cryptic epitopes. In conclusion, we cloned three anti-ADAMTS13 autoantibodies from iTTP patients that recognize cryptic epitopes. Hence, these data nicely fit our recent finding that the conformation of ADAMTS13 is open during acute iTTP.

The British Journal of Haematology publishes original research papers in clinical, laboratory and experimental haematology. The Journal also features annotations, reviews, short reports, images in haematology and Letters to the Editor.

Immune-mediated Thrombotic Thrombocytopenic Purpura (TTP) is a life-threatening disorder caused by antibodies against ADAMTS13. From the United Kingdom TTP registry we undertook a prospective study investigating the impact of the presenting anti-ADAMTS13 IgG antibody and ADAMTS13 antigen on mortality. 312 episodes involving 292 patients over 87 months were included. 68% were female, median age 46 (range 11-88 years) and median presenting ADAMTS13 of <5% (range <5% – 18%). The mortality rate was 10.3% (n=32). 68% of patients had a raised troponin at presentation conferring a six-fold increase in mortality compared to those with normal troponin levels (12.1% vs. 2.0%, p=0.04). 24% had a reduced Glasgow Coma Score (GCS) at presentation with a nine-fold increase in mortality (20% vs. 2.2% for normal GCS at presentation, p<0.0001). Mortality increased with higher anti-ADAMTS13 antibody levels and lower ADAMTS13 antigen levels. Those with antibody levels in the upper quartile (antibody >77%) had a mortality of 16.9% compared to 5.0% for the lowest quartile (antibody <20%) (p=0.004). Those with an antigen level in the lowest quartile (antigen <1.5%) had a mortality of 18% compared to 3.8% for the highest quartile (antigen >11%) (p=0.005). The synergistic effect of anti ADAMTS13 IgG antibody in the upper quartile and ADAMTS13 antigen in the lowest quartile had the highest mortality of 27.3%. We conclude that both anti-ADAMTS13 IgG antibody and ADAMTS13 antigen levels correlate with outcome in TTP with increased cardiac and neurological involvement and increased mortality. For personal use only. on June 4, 2017. by guest www.bloodjournal.org From

BACKGROUND: Patients presenting with acute episodes of thrombotic microangiopathies (TMAs) require urgent access to plasma exchange (PEX). OctaplasLG, a solvent/detergent fresh-frozen plasma product that has undergone viral inactivation and prion reduction step, has been used in our institution since 2013, replacing Octaplas.STUDY DESIGN AND METHODS: We prospectively reviewed 981 PEX procedures where OctaplasLG was the replacement fluid in 90 patients admitted acutely with a TMA presentation within our institution from January 1, 2013, to December 31, 2015. We recorded citrate toxicities, plasma reactions, viral transfer, complications related to central venous catheter, and venous thrombotic events (VTEs).RESULTS: Citrate toxicities were 5.4%, plasma reactions were 2%, and all were classified as Grade 1 or 2. VTE had an incidence of 12.2%, although 50% of the episodes occurred in early remission when patients were not receiving PEX. No line insertions complications were recorded. Line-associated infections were 2.2%. Hepatitis B and C serology and human immunodeficiency virus (HIV) were checked on admission. There were four patients who may have had passive transient transfer of hepatitis B antibodies from pooled plasma. No hepatitis C or HIV viral transfer was documented after treatment and no seroconversion was detected after treatment.CONCLUSION: Our data have demonstrated that the incidence of complications during PEX is low and using OctaplasLG is comparable to the low incidence of reactions. No cases of anaphylaxis, transfusion-related acute lung injury, or fatal plasma reactions were seen. There was no evidence of viral transmission or seroconversion after treatment.

Immune-mediated thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder caused by antibodies against ADAMTS13. From the United Kingdom TTP registry, we undertook a prospective study investigating the impact of the presenting anti-ADAMTS13 IgG antibody and ADAMTS13 antigen on mortality. A total of 312 episodes involving 292 patients over 87 months were included; 68% were female, median age 46 (range, 11-88 years), and median presenting ADAMTS13 of <5% (range, <5%-18%). The mortality rate was 10.3% (n = 32); 68% of patients had a raised troponin at presentation conferring a sixfold increase in mortality compared with those with normal troponin levels (12.1% vs 2.0%, P = .04). Twenty-four percent had a reduced Glasgow Coma Score (GCS) at presentation with a ninefold increase in mortality (20% vs 2.2% for normal GCS at presentation, P < .0001). Mortality increased with higher anti-ADAMTS13 antibody levels and lower ADAMTS13 antigen levels. Those with antibody levels in the upper quartile (antibody >77%) had a mortality of 16.9% compared with 5.0% for the lowest quartile (antibody <20%) (P = .004). Those with an antigen level in the lowest quartile (antigen <1.5%) had a mortality of 18% compared with 3.8% for the highest quartile (antigen >11%) (P = .005). The synergistic effect of anti-ADAMTS13 IgG antibody in the upper quartile and ADAMTS13 antigen in the lowest quartile had the highest mortality of 27.3%. We conclude that both anti-ADAMTS13 IgG antibody and ADAMTS13 antigen levels correlate with outcome in TTP with increased cardiac and neurological involvement and increased mortality.