The Impact of Age and BMI on the VWF/ADAMTS13 Axis and Simultaneous Thrombin and Plasmin Generation in Hospitalized COVID-19 Patients

Aging and obesity independently contribute toward an endothelial dysfunction that results in an imbalanced VWF to ADAMTS13 ratio. In addition, plasma thrombin and plasmin generation are elevated and reduced, respectively, with increasing age and also with increasing body mass index (BMI). The severity risk of Corona Virus Disease 2019 (COVID-19) increases in adults older than 65 and in individuals with certain pre-existing health conditions, including obesity (>30 kg/m2). The present cross-sectional study focused on an analysis of the VWF/ADAMTS13 axis, including measurements of von Willebrand factor (VWF) antigen (VWF:AG), VWF collagen binding activity (VWF:CBA), Factor VIII antigen, ADAMTS13 antigen, and ADAMTS13 activity, in addition to thrombin and plasmin generation potential, in a demographically diverse population of COVID-19 negative (−) (n = 288) and COVID-19 positive (+) (n = 543) patient plasmas collected at the time of hospital presentation. Data were analyzed as a whole, and then after dividing patients by age (<65 and ≥65) and independently by BMI [<18.5, 18.5–24.9, 25–29.9, >30 (kg/m2)]. These analyses suggest that VWF parameters (i.e., the VWF/ADAMTS13 activity ratio) and thrombin and plasmin generation differed in COVID-19 (+), as compared to COVID-19 (−) patient plasma. Further, age (≥65) more than BMI contributed to aberrant plasma indicators of endothelial coagulopathy. Based on these findings, evaluating both the VWF/ADAMTS13 axis, along with thrombin and plasmin generation, could provide insight into the extent of endothelial dysfunction as well as the plasmatic imbalance in coagulation and fibrinolysis potential, particularly for at-risk patient populations.

Pregnancy onset congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome) mimicking HELLP syndrome: a case report

Objective Thrombotic thrombocytopenic purpura is a thrombotic microangiopathic condition characterized by hemolytic anemia, thrombocytopenia, neurologic abnormalities, fever and renal dysfunction. Thrombotic microangiopathies such as preeclampsia and HELLP syndrome are pregnancy-specific, whereas others such as thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome are not. In this report, we present a case at which we identified a novel mutation which led to a significant reduction of ADAMTS13 activity. Case(s) A nulliparous pregnant woman of 32-year-old presenting with epigastric pain, hypertension and low platelet count was first suspected of HELLP syndrome, but was diagnosed with congenital TTP after delivery. Conclusion HELLP syndrome co-existed with undiagnosed TTP in this case. We strive to have sufficient awareness in order to distinguish these two pathologies from each other on an antenatal basis, because the causes of the managements are entirely different.

Association between ADAMTS13 deficiency and cardiovascular events in chronic hemodialysis patients

A mild decrease of ADAMTS13 (a disintegrin and metalloprotease with thrombospodin type 1 motif 13) could attribute to stroke and coronary heart disease in general population. However, the role of ADAMTS13 in hemodialysis (HD) patients remains to be explored. This cross-sectional and observational cohort study enrolled 98 chronic HD patients and 100 normal subjects with the aims to compare the ADAMTS13 activity between chronic HD patients and normal subjects, and to discover the role of ADAMTS13 on the newly developed cardiovascular events for HD patients in a 2-year follow-up. Our HD patients had a significantly lower ADAMTS13 activity than normal subjects, 41.0 ± 22.8% versus 102.3 ± 17.7%, p < 0.001. ADAMTS13 activity was positively correlated with diabetes, triglyceride and hemoglobin A1c, and negatively with high-density lipoprotein cholesterol levels in HD patients. With a follow-up of 20.3 ± 7.3 months, the Cox proportional hazards model revealed that low ADAMTS13, comorbid diabetes, and coronary heart diseases have independent correlations with the development of cardiovascular events. Our study demonstrated that chronic HD patients have a markedly decreased ADAMTS13 activity than normal subjects. Although ADAMTS13 seems to correlate well with diabetes, high triglyceride and low high-density lipoprotein cholesterol levels, ADAMTS13 deficiency still carries an independent risk for cardiovascular events in chronic HD patients.

ADAMS-TS13 Inhibitor Level and Risk of Relapse in Acquired Idiopathic Thrombotic Thrombocytopenic Purpura

Introduction: Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy due to reduced activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, 13). This disorder can be due to a congenital deficiency state or be acquired (immune TTP (iTTP)) due to an antibody which either inhibits or causes clearance of ADAMTS13. The aim of our study was to determine whether ADAMTS13 inhibitor titer at initial presentation could serve as a predictor of refractory disease and relapse in iTTP. We also measured clinical outcomes across different gender and racial subgroups. Methods: The United States Thrombotic Microangiopathy (USTMA) iTTP registry was used to extract patient information for two academic institutions in Eastern North Carolina. Descriptive statistics were used to analyze the data. The first iTTP episode recorded in the data base was used as the index episode. All patients included in the final analysis had an ADAMTS13 activity of &lt;10%. An inhibitor level of 5 Bethesda units was arbitrarily chosen as the cutoff between low (&lt;5) and high (&gt;/5) inhibitor level. Response time was defined as the number of days of plasma exchange (PEX) required to achieve a platelet count of 150,000 for two consecutive days. Relapse was defined as occurrence of a new episode of iTTP 30 days after achievement of response. Refractory disease was defined as persistence of thrombocytopenia or absence of a sustained platelet count increment or platelet counts of &lt; 50,000 despite 4-7 days of plasma exchanges and steroid treatment. Rituximab resistance was defined as lack of platelet recovery to more than 150,000 within 11 to 14 days of administration of the first dose of Rituximab. Results: A total of 161 patients with iTTP were identified. Ten patients had ADAMTS13 activity &gt;10% and 15 patients did not have a reported inhibitor level. These subjects were not included in the final analysis. The cohort had 28% male (n =38/136) and 72% (n=98/136) female patients. There were more African American patients 73% (n=99/136) than Caucasians 24% (n=32/136). There were also 2 Hispanic, 1 Native American and 2 patients with unidentified race. Median ADAMTS3 inhibitor titer was 1.05 (Range 0-87). Forty three patients with ADAMTS13 activity &lt;10 % had an inhibitor level of 0 (i.e undetectable).They were included in the low inhibitor group. Overall, 88% patients (n=120/136) had low inhibitor level and only 12% (n=16/136) had a high inhibitor. Thirteen percent females (n=13/98) and 8% (n=3/38) males had a high inhibitor level (p=0.387). Fourteen percent (n=14/99) African Americans and 6 % (n=2/32) Caucasians had a high inhibitor, p=0.23. In the low inhibitor group 30% (n=36/120) patients suffered at least one episode of relapse whereas 31% (n=5/16) had relapsed in the high inhibitor group. The median time to response was 6 days (range 1-76) in the low inhibitor group and 7 days (range 4-20) in the high inhibitor group (p=0.61). While looking at the various subgroups, median time to response for males was 6 days (range 4-21), females 6 days (range 1-76) , African Americans 6 days (range 3-29) , and Caucasians 6 days (range 1-76). The frequency of refractory disease was 31 % (n=5/16) in the high inhibitor group and 29% (n=34/119) in the low inhibitor group. At the time of enrollment in the registry, Rituximab was not a part of first line therapy. Only 26 out of 136 patients had received Rituximab. In the low inhibitor group 5 patients displayed Rituximab resistance whereas there were no patients in the high inhibitor group with Rituximab resistance. Conclusion: When evaluating patients presenting with iTTP in two centers in North Carolina, no correlation was found between a high inhibitor levels of &gt;/ 5 Bethesda units and risk of relapse or refractory disease. A larger study is needed to evaluate this further. Disclosures No relevant conflicts of interest to declare.

Recombinant ADAMTS13 for Patients with Sickle Cell Disease: Design of a Phase 1 Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics Study

Background Sickle cell disease (SCD) is an autosomal recessive hemoglobinopathy associated with chronic hemolysis and vaso-occlusive crises (VOCs) resulting in pain, organ damage, and a shortened lifespan. Current treatment options are limited, and many individuals with SCD continue to experience VOCs despite receiving therapy. Although the precise cause of VOCs is not clear, evidence suggests that cell adhesion is involved. Von Willebrand factor (VWF) is a multimeric glycoprotein that mediates the adhesion of platelets to each other and to other cell types, including vascular endothelium and leukocytes. An emerging hypothesis is that VWF contributes to the pathophysiology of VOCs through the formation of hyper-adhesive ultra-large VWF multimers. VWF activity is regulated by the metalloprotease ADAMTS13, which specifically cleaves ultra-large VWF multimers in an extended conformation. Patients with SCD have been shown to have higher levels of VWF multimers and lower levels of ADAMTS13 activity during VOCs. This imbalance could be caused either by the increased generation and release of ultra-large VWF multimers or by the inhibition of ADAMTS13 activity by plasma free hemoglobin or thrombospondin-1. Increasing the plasma concentration of ADAMTS13 using a recombinant ADAMTS13 (rADAMTS13; TAK-755, Takeda Development Center Americas, Inc., Lexington, MA, USA) may be therapeutically beneficial by enhancing cleavage of ultra-large VWF multimers. Here, we report the design and enrollment status of the Recombinant ADAMTS13 In Sickle Cell Disease (RAISE-UP) study (NCT03997760), the first clinical study of a recombinant ADAMTS13 in patients with SCD. Study Design and Methods This phase 1, randomized, double-blind, placebo-controlled, multicenter, ascending single dose study will assess the safety (including immunogenicity), tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of rADAMTS13 in patients with SCD. This study is planned to be conducted in 2 parts (part A and part B). Here we present the study design for part A which is being conducted initially and will enroll approximately 20 patients aged between 18 and 65 years with a documented history of SCD (HbSS or HbSβ 0 thalassemia). Concurrent treatment with a stable dose of hydroxyurea is allowed. Exclusion criteria include an acute VOC in the preceding 21 days and a blood transfusion either within the last 30 days or on ≥2 occasions in the last 90 days. Ethics committee approval and patient consent were obtained. Patients will be randomized 3:1 to receive a single intravenous infusion of either rADAMTS13 or placebo in 3 sequential dose cohorts. Patients in cohort 1 (n=4) will receive a 40 IU/kg dose, cohort 2 (n=8) will receive an 80 IU/kg dose, and cohort 3 (n=8) will receive a 160 IU/kg dose (Figure). In cohorts 2 and 3, 6 patients will receive rADAMTS13 and 2 patients will receive placebo. The first 3 patients enrolled in each cohort will be dosed with a separation time of at least 14 days. Enrollment into the next higher dose cohort will only be allowed following review of safety data and authorization by a dose escalation committee. Enrollment will be paused if anaphylaxis, binding or inhibitory antibodies, a life-threatening condition, or death are reported. All patients will complete an end-of-study visit on day 28 following infusion. Primary safety endpoints include adverse events, serious adverse events (SAEs), adverse changes in vital signs and laboratory parameters, and incidence of binding and inhibitory antibodies against rADAMTS13 occurring during the study. A secondary objective is to assess the PK of single-dose rADAMTS13 in each dose cohort, including an assessment of ADAMTS13 antigen and ADAMTS13 activity. Secondary PD objectives are to assess the effect of rADAMTS13 on VWF and platelet count and to study the correlation of plasma free hemoglobin and thrombospondin-1 with rADAMTS13 activity and VWF. Enrollment has been completed for cohort 1. In the review of safety data by the dose escalation committee, no drug-related SAEs were reported and no binding or inhibitory antibodies to ADAMTS13 were observed. On the basis of these findings, cohort 2 has been opened for enrollment. Figure 1 Figure 1. Disclosures Patwari: Takeda Development Center Americas, Inc.,: Current Employment. Nguyen: Takeda Development Center Americas, Inc.,: Current Employment. Bhattacharya: Takeda: Current equity holder in publicly-traded company; Takeda Development Center Americas, Inc.: Current Employment. Jain: Takeda Development Center Americas, Inc.,: Current Employment; Takeda: Current equity holder in publicly-traded company.

Single B Cell Immunoglobulin Sequencing Identifies Distinct Features of Monoclonal Antibodies in Patients with Immmune Thrombotic Thrombocytoepenic Purpura

Introduction. Immune thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal blood disorder, resulting from autoantibodies against ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor. However, the structural feature, binding epitope, and the mechanism of action of these autoantibodies in patients with acute iTTP are not fully understood. Methods. To further understand the pathogenesis of iTTP, single B cell immunoglobulin (Ig) sequencing using 10xChromium in 4 patients experiencing an acute episode of iTTP was performed; the expression and preliminary functional characterizations of selected clones were also carried out. Results. Approximately 2,631 viable and fluoresceinated ADAMTS13 labeled B cells (e.g., 7AAD -CD19 +CD20 +ADAMTS13 +) were sorted out from peripheral blood mononuclear cells of four patients with acute iTTP. These enriched ADAMTS13 antibody-producing B cells were then used for single cell analysis using 10xGenomics 5'-VDJ kit following the manufacturer's instruction. The single-cell gene expression libraries and VDJ libraries were constructed and sequenced by Hiseq at 20,000 reads/cell for gene expression and 5,000 reads/cell for VDJ sequences. Sequencing FASTQ files were mapped and counted by running through the Cell Ranger pipeline, and the final data were then further analyzed by the Loupe browser. We showed for the first time that the most frequent VJ combinations in the anti-ADAMTS13 IgG were: IGHV4-39:ILGJ4, IGHV3-48:ILGJ4, IGLV1-44:ILGLJ2, GLV5-45:ILGLJ3, IGLV2-14:ILGJ2, and IGLV3-21:ILGJ3 as shown in Figure 1. Of the top ten clones, the most frequently observed CDR3 (complementarity-determining region-3) sequences of these antibodies were CARDQLGISETQGSDLW on the heavy chain and CVIWHNSAWVF on the light chain as shown in Figure 2 and Table 1. The variable region sequences from the heavy and the light chains of Ig molecules were cloned into a human IgHG1 and a human IgL vector, respectively, which was then cotransfected in HEK293 cells. Western blotting, ELISA, immunoprecipitation, and functional assays were used to determine the expression and the function of human monoclonal IgG antibodies. Our preliminary results demonstrated the human monoclonal IgG antibodies bound and/or inhibited plasma ADAMTS13 activity. Conclusions. We conclude that there is clonal expansion of ADAMTS13 antibody producing B cells in acute iTTP and the cloned human monoclonal antibodies using the single B cell sequencing approach are functional. Our ongoing analysis on the structural and functional relationship of a large number of isolated human monoclonal antibodies may shed new light on the pathogenesis of iTTP. These antibodies may be useful to explore structural elements required for allosteric regulation of ADAMTS13 activity. Figure 1 Figure 1. Disclosures Zheng: AJMC: Honoraria; Clotsolution: Other: Co-founder; Takeda: Consultancy, Honoraria; Sanofi-Genzyme: Honoraria, Speakers Bureau; Alexion: Speakers Bureau.

Relapsed Refractory Acquired Thrombotic Thrombocytopenic Purpura (aTTP) Following COVID-19 Vaccination

Introduction: Acquired thrombotic thrombocytopenic purpura (aTTP) due to an acquired deficiency in the enzyme ADAMTS13 leads to ultra-large von Willebrand multimers, thrombocytopenia and microangiopathic hemolytic anemia. Complications include microvascular and macrovascular thrombosis. We present an unusual case of a patient with a history of refractory aTTP who experienced relapsed aTTP following COVID-19 vaccine. Case Description: A 57-year-old African-American male with a history of refractory aTTP experienced a relapse following 3 years of remission after receiving COVID-19 vaccination. The patient was initially diagnosed with aTTP in 2016, after presenting with symptoms of dark urine, mild headaches and transient episodes of aphasia and paresthesia. Due to symptoms and persistently low ADAMTS13 levels, he required prolonged and extensive treatment including over 5 weeks of daily therapeutic plasma exchange (TPE), followed by gradual reduction in frequency of TPE sessions, as well as trials of rituximab, eculizumab, steroids, mycophenolate mofetil and bortezomib. Ultimately, he achieved remission after 9 months of intermittent TPE, 3 months of weekly bortezomib 1 mg/m 2, mycophenolate mofetil up-titrated to 1,750 mg twice daily, and then slowly tapered off over a 2-year period. The patient was doing well for 3 years without manifestations of aTTP (2 years off all therapeutics), until he developed a petechial rash 7 weeks after receiving the second dose of the Moderna COVID-19 vaccine. He was found to have acute thrombocytopenia with platelets of 38 x 10 9/L (normal range 135-317 x 109/L), from a baseline of 200-300 x 10 9/L. He was referred to the emergency department, where additional labs were notable for mildly elevated LDH of 508 U/L (normal range 122-222 U/L), hemoglobin of 12.4 g/dL (normal range 13.2-16.6 g/dL), creatinine at baseline, and peripheral blood smear showing 1-3 schistocytes per high-powered field. ADAMTS13 activity level was t &lt;5% (normal &gt;/= 70%), with positive ADAMTS13 inhibitor screen and titer of 1.5 (normal &lt;0.4), consistent with relapsed aTTP. The patient was admitted to the hospital, and initiated on daily TPE, with steroids and diphenhydramine prior to each TPE session. He quickly improved with TPE alone , but given his history of refractory aTTP, he was discharged on weekly rituximab for 4 weeks and caplacizumab 11 mg daily for 30 days. His platelets remained stable within the upper limit of normal during his 30 day course of caplacizumab. However, 3 weeks after completion of caplacizumab, he had an acute drop in his platelets to 23 x 10 9/L. His ADAMTS13 level was again found to be &lt;5%, and inhibitor level was the highest that it had ever been at 11.4. He was again hospitalized and underwent 8 sessions of daily TPE, as well as re-initiation of caplacizumab, mycophenolate mofetil 500 mg bid (with increasing taper), and a prednisone taper. Intravenous Cyclophosphamide 750 mg/m 2 was also added every 3 weeks. With this regimen, patient's platelet count normalized and remain stable, and his ADAMTS13 activity level has reached 52-59%. Discussion: Cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) have been described as a complication following vaccination with formulations containing replication-defective adenoviral vectors (AstraZeneca-Oxford and Johnson&Johnson COVID-19 vaccines)(Arepally and Ortel 2021, Simpson, Shi et al. 2021). VITT and aTTP are both immune-mediated, however, VITT is distinct and pathogenically linked to autoimmune heparin-induced thrombocytopenia (HIT), given the presence of anti-platelet factor 4 antibodies in these patients, whereas aTTP is due to reduction in ADAMTS13 level, secondary to an antibody inhibitor of ADAMTS13 (Arepally and Ortel 2021). Recently, cases have been reported of de novo aTTP developing shortly after COVID-19 vaccination with all available vaccines, except the Moderna (mRNA-1273) vaccine (Al-Ahmad, Al-Rasheed et al. 2021, de Bruijn, Maes et al. 2021, Maayan, Kirgner et al. 2021, Ruhe, Schnetzke et al. 2021, Waqar, Khan et al. 2021, Yocum and Simon 2021). Additionally, cases of relapsed aTTP have been described following only the BNT162B2 (Pfizer-BioNTech) vaccine (Maayan, Kirgner et al. 2021, Sissa, Al-Khaffaf et al. 2021). This is the first case, to our knowledge, reported in the literature of aTTP following vaccination with Moderna's mRNA-1273 vaccine. Disclosures No relevant conflicts of interest to declare.

Neutrophil Extracellular Traps (NETs) Contribute to the Formation of Microvascular Thrombosis in Immune Thrombotic Thrombocytopenic Purpura

Introduction. Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal blood disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from immunoglobulin (Ig) G-mediated inhibition of plasma ADAMTS13 activity. However, severe ADAMTS13 deficiency is necessary but not sufficient to cause acute iTTP. An environmental factor such as infection or acute inflammation may be necessary to trigger the acute onset of the disease. We and others have previously reported that plasma markers of neutrophil activation and neutrophil extracellular traps (NETs) formation are significantly elevated in patients with acute iTTP, which returns to normal during remission. However, the pathogenetic role of NETs in acute iTTP is not fully understood. Methods and results. Using flow cytometry, microfluidic shear-based assay, and confocal imaging analysis, we determined the in vivo NETosis in blood samples obtained from patients with acute episode of iTTP and ex vivo NETs formation, as well as the therapeutic efficacy of DNase I on thrombus formation under flow. We showed that by flow cytometry that only very few CitH3+/MPO+ positive neutrophils were present in the healthy donor blood. This population of cells dramatically increased after being stimulated with a bacterial toxin (i.e., Shigatoxin-2) at ~100 ng/mL for 15 min. Importantly, the number of CitH3+/MPO+ positive neutrophils in the sample obtained from a patient with acute iTTP was ~1,000 times higher than that in the healthy controls (Fig. 1), suggesting a massive NETosis in patients with acute iTTP. Microfluidic shear-based assay and confocal imaging analysis further confirmed a dramatic increase in adhesion and aggregation of murine platelets (stained with Alexa647 anti-CD41) and neutrophil (stained with Hoechst), as well as formation of NETs (stained with Syto green) following a perfusion of an Adamts13 -/- murine whole blood (anti-coagulated with thrombin inhibitor, PPACK) under arterial shear (15 dyne/cm 2) over a stimulated murine endothelial surface. Interestingly, an addition of DNase I (100 U/mL) significantly reduced the overall surface coverage of platelets and neutrophils on the murine endothelial surface under the same conditions (Fig. 2). Conclusions. These results demonstrate for the first time NETosis and NETs formation are common in patients with acute iTTP and in Adamts13 -/- mice after being stimulated with shigatoxin; DNase I appears to be highly efficacious eliminating the NETs and platelet/neutrophil-dominant thrombosis under arterial flow. Our findings support the pathogenetic role of NETs in the onset and progression of iTTP, and the therapeutic potential of DNase I in such a fatal disease. Figure 1 Figure 1. Disclosures Zheng: Alexion: Speakers Bureau; Sanofi-Genzyme: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria; Clotsolution: Other: Co-founder; AJMC: Honoraria.

Real-World Variability in ADAMTS13 Assay Methods May Impact upon Follow up for Patients with Thrombotic Thrombocytopenic Purpura (TTP)

Introduction ADAMTS13 activity (ADAMTS13:Ac) measurement is an important diagnostic marker in acute TTP (defined as a level of &lt;10%). Increasing numbers of commercial assays have been developed (ELISA, FRET and chemiluminesence) for diagnostic purposes. The International Council for Standardization in Haematology have not made specific recommendations on ADAMTS13 assay selection in a recent publication in 2020. ADAMTS13:Ac has traditionally been used for diagnostic purposes but can also be useful for monitoring disease status and relapse risk. Patients between levels of 10-50% may require increased monitoring and subsequently treatment to avoid frank relapse, with an inverse relationship between ADAMTS13:Ac levels and intensity of clinical monitoring. We seek to compare the differences between different commercial assays available to measure the ADAMTS13:Ac in patients who have a history of TTP and whether this influences clinical practice. Methods Patients with an acute diagnosis of TTP as an inpatient or outpatient surveillance with past history of TTP were reviewed at Guy's and St Thomas' Hospital, London, UK, and samples were taken to measure ADAMTS13:Ac as part of disease monitoring between February and July 2021. A comparison was performed between an ELISA (Technozym ADAMTS13 activity, Technoclone) and FRET (Technofluor, Technoclone), with all samples processed in parallel. With no international consensus, broadly, levels of &lt;10% were defined as either active TTP or relapse; patients with 10-30% required more intensive outpatient monitoring to detect for relapse and &gt;50% was defined as within the normal range. Results 47 samples from 31 known or newly diagnosed TTP patients were processed over a six month period. Intra- and inter-assay CV for both ELISA and FRET on laboratory testing was as expected from manufacturers guidelines. Figure 1 plots the ADAMTS13:Ac ELISA vs FRET, with a Pearson correlation of p=0.949 across all samples. Ranges were for ELISA (0 - 109.3) and FRET (1.6 - 130.3). Mean ADAMTS13:Ac difference was 16±13.5%, with consistent positive bias in favour of the FRET assay. Higher levels demonstrated greater variability as demonstrated in Table 1, with the mean difference between ELISA and FRET increasing with increasing levels. Based on the above definitions as in the methods, there were 14/47 (29%) samples which had discordant results between the ELISA and FRET methods: 3 discordant samples on diagnosis of relapse, 8 discordant for where the ELISA detected levels &lt;30% and FRET &gt;30%; and 2 discordant for where ELISA &lt;50% and FRET &gt;50%. Conclusion Real-world clinical testing demonstrates substantial variability within a single manufacturer between two separate methods and demonstrates an impact on patient follow up and implications in decisions on follow-up intervals and treatment. There was a consistent positive bias in ADAMTS13:Ac by FRET measurement as compared with the ELISA. Greater consistency was demonstrated at the lower end of testing, where there was good concordance at levels of &lt;10%, confirming that both assays were effective at determining a diagnosis or relapse of TTP. Further interrogation of other commercial platforms is warranted to establish the variability of results to inform on clinical practice in the monitoring of patients with TTP. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.