Relationship between plasma tissue Factor Pathway Inhibitor (TFPI) levels, thrombin generation and clinical risk of bleeding in patients with severe haemophilia A or B.

INTRODUCTION: Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor (TFPI) level. AIM: To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency. METHODS: Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT). RESULTS: Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was ≤ 4.9 $ \le 4.9$ , AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR. CONCLUSION: In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.

Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia.

BACKGROUND: Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients. METHODS: In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice. RESULTS: In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation (P=0.00013, P<0.0001, and P<0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; P=0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; P<0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets. CONCLUSIONS: ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD.

von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A-group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high-molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

Successful and safe response to ibrutinib alone in treating relapsed Waldenström macrogobulinemia and related acquired von Willebrand syndrome: an option to consider.

Ibrutinib, a first-class Bruton tyrosine kinase inhibitor, is known to be associated with adverse bleeding events and has been recently approved for the treatment of relapse Waldenström macroglobulinemia (WM). Here, we report the exhaustive clinical and biological follow-up of 2 patients treated by ibrutinib alone in the context of relapsed WM with an acquired von Willebrand syndrome (AVWS) complication. In two cases, ibrutinib has been shown to be quickly efficient and safe for treating both AVWS and its underlying condition the WM, without bleeding complications. Interestingly, ibrutinib treatment brings a rapid and extended over time normalization of von Willebrand factor clearance. These observations show that ibrutinib is a valuable therapeutic option in relapsed WM patients associated with AVWS and highlighting the need for further cohort studies with long-term follow-up of patients to confirm the efficacy and safety of a treatment by ibrutinib for WM patients with AVWS complication.

The role of tranexamic acid in the management of postpartum haemorrhage.

In the last decades, tranexamic acid (TXA) has emerged as an essential tool in blood loss management in obstetrics. TXA prophylaxis for postpartum haemorrhage (PPH) has been studied in double-blind, placebo-controlled, randomized clinical trials (RCTs). Given the small observed preventive effect, the systematic use of TXA for vaginal and/or caesarean deliveries remains controversial. The result of a pharmacokinetic modelling suggests that relative to intravenous administration, intramuscular administration may be an equally effective alternative route for preventing PPH and may enable access to this drug in low-resource countries. Prophylaxis is currently studied in high-risk populations, such as women with prepartum anaemia or placenta previa. TXA effectively reduces blood loss and PPH-related morbidity and mortality during active PPH, as demonstrated by high-grade evidence from large RCTs. The drug has a good safety profile: in most cases, only mild gastrointestinal or visual adverse events may be observed. TXA use does not increase the risk of serious adverse events, such as venous or arterial thromboembolism, seizures, or acute kidney injury. The TRACES in vivo analysis of biomarkers of TXA's antifibrinolytic effect have suggested that a dose of at least 1 g is required for the treatment of PPH. The TRACES pharmacokinetic model suggests that because TXA can be lost in the haemorrhaged blood, a second dose should be administered if the PPH continues or if severe coagulopathy occurs. Future pharmacodynamic analyses will focus on the appropriateness of TXA dosing regimens with regard to the intensity of fibrinolysis in catastrophic obstetric events.

Tranexamic acid dose-response relationship for antifibrinolysis in postpartum haemorrhage during Caesarean delivery: TRACES, a double-blind, placebo-controlled, multicentre, dose-ranging biomarker study.

BACKGROUND: The optimal dose of tranexamic acid to inhibit hyperfibrinolysis in postpartum haemorrhage is unclear. Tranexamic Acid to Reduce Blood Loss in Hemorrhagic Cesarean Delivery (TRACES) was a double-blind, placebo-controlled, randomised, multicentre dose-ranging study to determine the dose-effect relationship for two regimens of intravenous tranexamic acid vs placebo. METHODS: Women experiencing postpartum haemorrhage during Caesarean delivery were randomised to receive placebo (n=60), tranexamic acid 0.5 g (n=57), or tranexamic acid 1 g i.v. (n=58). Biomarkers of fibrinolytic activation were assayed at five time points, with inhibition of hyperfibrinolysis defined as reductions in the increase over baseline in D-dimer and plasmin-antiplasmin levels and in the plasmin peak time. RESULTS: In the placebo group, hyperfibrinolysis was evidenced by a mean increase over baseline [95% confidence interval] of 93% [68-118] for D-dimer level at 120 min and 56% [25-87] for the plasmin-antiplasmin level at 30 min. A dose of tranexamic acid 1 g was associated with smaller increases over baseline (D-dimers: 38% [13-63] [P=0.003 vs placebo]; plasmin-antiplasmin: -2% [-32 to 28] [P=0.009 vs placebo]). A dose of tranexamic acid 0.5 g was less potent, with non-significant reductions (D-dimers: 58% [32-84] [P=0.06 vs placebo]; plasmin-antiplasmin: 13% [18-43] [P=0.051]). Although both tranexamic acid doses reduced the plasmin peak, reduction in plasmin peak time was significant only for the 1 g dose of tranexamic acid. CONCLUSIONS: Fibrinolytic activation was significantly inhibited by a dose of intravenous tranexamic acid 1 g but not 0.5 g. Pharmacokinetic-pharmacodynamic modelling of these data might identify the best pharmacodynamic monitoring criteria and the optimal tranexamic acid dosing regimen for treatment of postpartum haemorrhage. CLINICAL TRIAL REGISTRATION: NCT02797119.

Cerebral Microbleeds During Transcatheter Aortic Valve Replacement: A Prospective Magnetic Resonance Imaging Cohort.

BACKGROUND: Cerebral microbleeds (CMBs) have been observed in healthy elderly people undergoing systematic brain magnetic resonance imaging. The potential role of acute triggers on the appearance of CMBs remains unknown. We aimed to describe the incidence of new CMBs after transcatheter aortic valve replacement (TAVR) and to identify clinical and procedural factors associated with new CMBs including hemostatic measures and anticoagulation management. METHODS: We evaluated a prospective cohort of patients with symptomatic aortic stenosis referred for TAVR for CMBs (METHYSTROKE [Identification of Epigenetic Risk Factors for Ischemic Complication During the TAVR Procedure in the Elderly]). Standardized neurologic assessment, brain magnetic resonance imaging, and analysis of hemostatic measures including von Willebrand factor were performed before and after TAVR. Numbers and location of microbleeds on preprocedural magnetic resonance imaging and of new microbleeds on postprocedural magnetic resonance imaging were reported by 2 independent neuroradiologists blinded to clinical data. Measures associated with new microbleeds and postprocedural outcome including neurologic functional outcome at 6 months were also examined. RESULTS: A total of 84 patients (47% men, 80.9±5.7 years of age) were included. On preprocedural magnetic resonance imaging, 22 patients (26% [95% CI, 17%-37%]) had at least 1 microbleed. After TAVR, new microbleeds were observed in 19 (23% [95% CI, 14%-33%]) patients. The occurrence of new microbleeds was independent of the presence of microbleeds at baseline and of diffusion-weighted imaging hypersignals. In univariable analysis, a previous history of bleeding (P=0.01), a higher total dose of heparin (P=0.02), a prolonged procedure (P=0.03), absence of protamine reversion (P=0.04), higher final activated partial thromboplastin time (P=0.05), lower final von Willebrand factor high-molecular-weight:multimer ratio (P=0.007), and lower final closure time with adenosine-diphosphate (P=0.02) were associated with the occurrence of new postprocedural microbleeds. In multivariable analysis, a prolonged procedure (odds ratio, 1.22 [95% CI, 1.03-1.73] for every 5 minutes of fluoroscopy time; P=0.02) and postprocedural acquired von Willebrand factor defect (odds ratio, 1.42 [95% CI, 1.08-1.89] for every lower 0.1 unit of high-molecular-weight:multimer ratio; P=0.004) were independently associated with the occurrence of new postprocedural microbleeds. New CMBs were not associated with changes in neurologic functional outcome or quality of life at 6 months. CONCLUSIONS: One out of 4 patients undergoing TAVR has CMBs before the procedure and 1 out of 4 patients develops new CMBs. Procedural or antithrombotic management and persistence of acquired von Willebrand factor defect were associated with the occurrence of new CMBs. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02972008.

Derivation and Validation of a Predictive Score for Respiratory Failure Worsening Leading to Secondary Intubation in COVID-19: The CERES Score.

Predictive scores assessing the risk of respiratory failure in COVID-19 mostly focused on the prediction of early intubation. A combined assessment of clinical parameters and biomarkers of endotheliopathy could allow to predict late worsening of acute respiratory failure (ARF), subsequently warranting intubation in COVID-19. Retrospective single-center derivation (n = 92 subjects) and validation cohorts (n = 59 subjects), including severe COVID-19 patients with non-invasive respiratory support, were assessed for at least 48 h following intensive care unit (ICU) admission. We used stepwise regression to construct the COVID endothelial and respiratory failure (CERES) score in a derivation cohort, and secondly assessed its accuracy for the prediction of late ARF worsening, requiring intubation within 15 days following ICU admission in an independent validation cohort. Platelet count, fraction of inspired oxygen, and endocan measured on ICU admission were identified as the top three predictive variables for late ARF worsening and subsequently included in the CERES score. The area under the ROC curve of the CERES score to predict late ARF worsening was calculated in the derivation and validation cohorts at 0.834 and 0.780, respectively. The CERES score is a simple tool with good performances to predict respiratory failure worsening, leading to secondary intubation, in COVID-19 patients.

ABO Blood Groups in Systemic Sclerosis: Distribution and Association with This Disease's Characteristics.

Systemic sclerosis (SSc) is an autoimmune disease associated with endothelial activation and fibrosis. Non-O blood group patients carry an increased risk of thrombosis, fibrosis and autoimmune diseases. The aim of our work was to evaluate the distribution of ABO groups in SSc patients and their association with the disease's characteristics. ABO groups were determined in 504 SSc patients (with 131 completed by a genotypic analysis). The distribution of ABO groups and their diplotypes in SSc patients was comparable to that of the general population, except for haplotypes O1 and B (65.6% vs. 61.6% and 8.8% vs. 5.8% in SSc patients vs. the general population, respectively, p = 0.01). The frequency of interstitial lung disease, pulmonary hypertension, calcinosis, digital ulcers, digestive diseases and venous thrombosis, and the Medsger score, were higher in non-O than in O-SSc patients, although they did not display statistical significance. Patients in the non-O group had higher levels of inflammation and endothelial activation biomarkers. In conclusion, the ABO blood group distribution of SSc patients did not differ significantly from that of the general population, but non-O blood groups were associated with inflammation and endothelial activation, and with a non-significant higher frequency of pulmonary and vascular complications in SSc.

Vascular Endothelial Damage in the Pathogenesis of Organ Injury in Severe COVID-19.

[Figure: see text].

Severe SARS-CoV-2 patients develop a higher specific T-cell response.

OBJECTIVES: Assessment of the adaptive immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for studying long-term immunity and vaccine strategies. We quantified IFNγ-secreting T cells reactive against the main viral SARS-CoV-2 antigens using a standardised enzyme-linked immunospot assay (ELISpot). METHODS: Overlapping peptide pools built from the sequences of M, N and S viral proteins and a mix (MNS) were used as antigens. Using IFNγ T-CoV-Spot assay, we assessed T-cell and antibody responses in mild, moderate and severe SARS-CoV-2 patients and in control samples collected before the outbreak. RESULTS: Specific T cells were assessed in 60 consecutive patients (mild, n = 26; moderate, n = 10; and severe patients, n = 24) during their follow-up (median time from symptom onset [interquartile range]: 36 days [28;53]). T cells against M, N and S peptide pools were detected in n = 60 (100%), n = 56 (93.3%), n = 55 patients (91.7%), respectively. Using the MNS mix, IFNγ T-CoV-Spot assay showed a specificity of 96.7% (95% CI, 88.5-99.6%) and a specificity of 90.3% (75.2-98.0%). The frequency of reactive T cells observed with M, S and MNS mix pools correlated with severity and with levels of anti-S1 and anti-RBD serum antibodies. CONCLUSION: IFNγ T-CoV-Spot assay is a reliable method to explore specific T cells in large cohorts of patients. This test may become a useful tool to assess the long-lived memory T-cell response after vaccination. Our study demonstrates that SARS-CoV-2 patients developing a severe disease achieve a higher adaptive immune response.

Coagulation biomarkers are independent predictors of increased oxygen requirements in COVID-19.

BACKGROUND: Hypercoagulability seems to contribute to SARS-CoV-2 pneumonia pathogenesis. However, age and metabolic syndrome are potential confounders when assessing the value of coagulation biomarkers' prediction of COVID-19 outcomes. We assessed whether coagulation biomarkers, including factor VIII (FVIII) and von Willebrand factor (VWF) levels, measured at time of admission, were predictive of COVID-19 adverse outcomes irrespective of age and major comorbidities associated with metabolic syndrome. METHODS: Blood was sampled at admission in 243 adult COVID-19 patients for analysis of coagulation biomarkers including FVIII and VWF on platelet-poor plasma. The association between baseline C-reactive protein (CRP), activated partial thromboplastin time ratio, prothrombin time ratio, D-dimers, fibrinogen, FVIII, VWF antigen (VWF:Ag), and FVIII/VWF:Ag ratio levels and adverse outcomes (increased oxygen requirements, thrombosis, and death at day 30) was assessed by regression analysis after adjustment on age, sex, body mass index (BMI), diabetes, and hypertension. RESULTS: In univariable regression analysis increased CRP (subdistribution hazard ratio [SHR], 1.68; 95% confidence interval [CI], 1.26-2.23), increased fibrinogen (SHR, 1.32; 95% CI, 1.04-1.68), and decreased FVIII/VWF:Ag ratio (SHR, 0.70; 95% CI, 0.52-0.96) levels at admission were significantly associated with the risk of increased oxygen requirement during follow-up. Leucocytes (SHR, 1.36; 95% CI, 1.04-1.76), platelets (SHR,1.71; 95% CI, 1.11-2.62), D-dimers (SHR, 2.48; 95% CI, 1.66-3.78), and FVIII (SHR, 1.78; 95% CI, 1.17-2.68) were associated with early onset of thrombosis after admission. After adjustment for age, sex, BMI, hypertension, and diabetes, these associations were not modified. CONCLUSION: Coagulation biomarkers are early and independent predictors of increased oxygen requirement in COVID-19 patients.

Emicizumab treatment: Impact on coagulation tests and biological monitoring of haemostasis according to clinical situations (BIMHO group proposals).

Emicizumab, a bispecific humanised monoclonal antibody restoring to some extent the function of activated FVIII deficient in haemophilia A, represents a major therapeutic advance in the management of haemophilia A patients. No dosage adjustment is required, which leads to a major change for patients used to regular biological monitoring which is particularly burdensome in the case of substitution therapy. In some circumstances, such as before an invasive procedure or in case of bleeding, biological monitoring will be necessary and emicizumab's interference with haemostasis tests, particularly those based on an activated partial thromboplastin times (aPTT), must be known to best interpret the tests and to select the most appropriate methods to guide therapy. The normalisation of aPTT in patients treated with emicizumab is not sufficient to consider haemostasis as normalised. In the event of administration of FVIII to a patient receiving emicizumab, the determination of FVIII should use a chromogenic method using non-human reagents. Coagulation global tests have been proposed to evaluate the biological response when using bypassing agents in patients treated with emicizumab, but the usefulness must be confirmed. The French group BIMHO presents proposals for biological monitoring of a patient treated with emicizumab according to clinical situations.

Factor VIII and IX assays for post-infusion monitoring in hemophilia patients: Guidelines from the French BIMHO group (GFHT).

Replacement therapy with plasma-derived or recombinant FVIII and FIX (pdFVIII/pdFIX or rFVIII/rFIX) concentrates is the standard of treatment in patients with haemophilia A and B, respectively. Measurement of factor VIII (FVIII:C) or factor IX (FIX:C) levels can be done by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). The French study group on the Biology of Hemorrhagic Diseases (a collaborative group of the GFHT and MHEMO network) presents a literature review and proposals for the monitoring of FVIII:C and FIX:C levels in treated haemophilia A and B patients, respectively. The use of CSA is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. Except for rFVIII-Fc, great caution is required when measuring FVIII:C levels by OSA in patients substituted by EHL-rFVIII. The OSA is recommended for the monitoring of patients treated with pdFIX or rFIX. Large discordances in the FIX:C levels measured for extended half-life rFIX (EHL-rFIX), depending on the method and reagents used, must lead to great attention when OSA is used for measuring FIX:C levels in patients substituted by EHL-rFIX. Data of most of recent studies, obtained with spiked plasmas, deserve to be confirmed in plasma samples of treated patients.

Multicentre pharmacokinetic evaluation of rFVIII-Fc (efmoroctocog alfa) in a real life and comparison with non-extended half-life FVIII concentrates.

The use of enhanced half-life (EHL) FVIII has improved the quality of prophylaxis in haemophilia A, but with a benefit that may vary from one patient to another. We analysed the pharmacokinetic data obtained with efmoroctocog alfa (rFVIII-Fc) in 114 patients and, in 47 cases, compared them to those previously measured with non-EHL FVIII. The in vivo recovery (IVR) of rFVIII-Fc measured with one stage clotting assay (OSA) and chromogenic assay (CSA) was 2.2 and 2.8 IU/mL per IU/kg, respectively. The median half-life (T1/2 ) of rFVIII-Fc was 14.5 hours whatever the FVIII:C assay used, but variable and correlated with preinfusion VWF:Ag levels (r = .76). Both IVR and T1/2 were lower in patients under 12 years old (2.4 IU/mL per IU/kg and 11.1 hours, respectively; CSA). PK study of rFVIII-Fc vs non-EHL FVIII showed a T1/2 ratio of 1.4 in favour of rFVIII-Fc, regardless of the patient's age. However the relative increase in T1/2 with rFVIII-Fc was lower than 30% in one-third of patients evaluated, particularly when the previous FVIII administered was a BHK-derived product. This study therefore suggests that analysis of individual PK profile in response to a specific FVIII concentrate is potentially useful before a switch in haemophilia A patients.

Acquired Hemophilia A in IgG4-Related Disease: Case Report, Immunopathogenic Study, and Review of the Literature.

We report the observation of a 75-year-old patient referred for cervical lymphadenopathies. A pre-lymphadenectomy blood work revealed an asymptomatic elevation of aPTT with low factor VIII (FVIII) levels and high anti-FVIII antibodies titers, consistent with acquired hemophilia A (AHA). Histological work-up of a cervical lymphadenopathy revealed benign follicular hyperplasia with IgG4+ lymphoplasmacytic infiltration; and serum IgG4 levels were markedly elevated, compatible with IgG4-related disease (IgG4-RD). He was successfully treated with a 9-month course of prednisone, secondarily associated with rituximab when an AHA relapse occurred. As this patient presented with an unusual association of rare diseases, we wondered whether there was a link between the two conditions. Our first hypothesis was that the anti-FVIII autoantibodies could be directly produced by the proliferating IgG4+ plasma cells as a result of broken tolerance to autologous FVIII. To test this assumption, we determined the anti-FVIII IgG subclasses in our patient and in a control group of 11 AHA patients without IgG4-RD. The FVIII inhibitor was mostly IgG4, with an anti-FVIII IgG4/IgG1 ratio of 42 at diagnosis and 268 at relapse in our patient; similar values were observed in non-IgG4-RD AHA patients. As a second hypothesis, we considered whether the anti-FVIII activity could be the result of a non-specific autoantibody production due to polyclonal IgG4+ plasma cell proliferation. To test this hypothesis, we measured the anti-FVIII IgG4/total IgG4 ratio in our patient, as well as in several control groups: 11 AHA patients without IgG4-RD, 8 IgG4-RD patients without AHA, and 11 healthy controls. We found that the median [min-max] ratio was higher in AHA-only controls (2.4 10-2 [5.7 10-4-1.79 10-1]), an oligoclonal setting in which only anti-FVIII plasma cells proliferate, than in IgG4-RD-only controls (3.0 10-5 [2.0 10-5-6.0 10-5]), a polyclonal setting in which all IgG4+ plasma cells proliferate equally. Our patient had intermediate ratio values (2.7 10-3 at diagnosis and 1.0 10-3 at relapse), which could plead for a combination of both mechanisms. Although no definitive conclusion can be drawn, we hypothesized that the anti-FVIII autoantibody production in our IgG4-RD AHA patient could be the result of both broken tolerance to FVIII and bystander polyclonal IgG4+ plasma cell proliferation.