Markers of refractory primary immune thrombocytopenia.

Refractory immune thrombocytopenia (ITP) is a challenging disease that can be defined by refractoriness to second-line treatments. In this review, we list and comment available evidence about clinical and biological factors associated with refractoriness to splenectomy, thrombopoietin receptor agonists (TPO-RAs), rituximab and fostamatinib, as well as those associated with multirefractory ITP (active disease with failure of rituximab, TPO-RAs and splenectomy).

Complement activation negatively affects the platelet response to thrombopoietin receptor agonists in patients with immune thrombocytopenia: a prospective cohort study.

Increased platelet destruction is central in the pathogenesis of immune thrombocytopenia. However, impaired platelet production is also relevant and its significance underlies the rationale for treatment with thrombopoietin receptor agonists (TPO-RAs). Previous studies have associated enhanced complement activation with increased disease severity. Additionally, treatment refractoriness has been demonstrated to resolve by the administration of complement-targeted therapeutics in a subset of patients. The association between complement activation and the platelet response to TPO-RA therapy has previously not been investigated. In this study, blood samples from patients with immune thrombocytopenia (n = 15) were prospectively collected before and two, six and 12 weeks after the initiation of TPO-RA therapy. Plasma levels of complement degradation product C4d and soluble terminal complement complexes were assessed. Patients with significantly elevated baseline levels of terminal complement complexes exhibited more often an inadequate platelet response (p = .04), were exclusively subjected to rescue therapy with intravenous immunoglobulin (p = .02), and did not respond with a significant platelet count increase during the study period. C4d showed a significant (p = .01) ability to distinguish samples with significant terminal complement activation, implying engagement of the classical complement pathway. In conclusion, elevated levels of complement biomarkers were associated with a worse TPO-RA treatment response. Larger studies are needed to confirm these results. Biomarkers of complement activation may prove valuable as a prognostic tool to predict which patients that potentially could benefit from complement-inhibiting therapy in the future.

What is the context?Primary immune thrombocytopenia (ITP) is a potentially serious illness associated with an increased risk of bleeds. Manifestations range from confined skin bruising to life-threatening intracranial hemorrhages.It is an acquired immune disorder characterized by increased destruction and impaired production of platelets.Treatments aim at suppressing the destruction and supporting the production of platelets.Thrombopoietin receptor agonists (TPO-RA) are medically approved platelet growth factors that contribute to the generation of new platelets.The complement system is an evolutionary preserved part of innate immunity.Previous studies have indicated that complement activation may be an important contributor to disease and that the administration of complement-inhibiting therapy improves the platelet count in a subset of patients with primary ITP.What is new? The potential association between complement activation and a poor platelet response to TPO-RA therapy in primary ITP has not been previously studied.In fifteen patients with primary ITP starting TPO-RA therapy, we prospectively followed the platelet response and levels of complement biomarkers for 12 weeks.We showed that patients with high levels of complement biomarkers exhibited a worse treatment response during the study period.What is the impact?Our results suggest that levels of complement biomarkers may be valuable to predict which patients with treatment-refractory ITP that potentially could benefit from complement-inhibiting therapy in the futureLarger studies are needed to confirm our results.

SARS-CoV-2 vaccines are not associated with hypercoagulability in apparently healthy people.

Background: SARS-CoV-2 adenoviral vector DNA vaccines have been linked to the rare but serious thrombotic postvaccine complication vaccine-induced immune thrombotic thrombocytopenia. This has raised concerns regarding the possibility of increased thrombotic risk after any SARS-CoV-2 vaccines. Objectives: To investigate whether SARS-CoV-2 vaccines cause coagulation activation leading to a hypercoagulable state. Methods: This observational study included 567 health care personnel; 521 were recruited after the first dose of adenoviral vector ChAdOx1-S (Vaxzevria, AstraZeneca) vaccine and 46 were recruited prospectively before vaccination with a messenger RNA (mRNA) vaccine, either Spikevax (Moderna, n = 38) or Comirnaty (Pfizer-BioNTech, n = 8). In the mRNA group, samples were acquired before and 1 to 2 weeks after vaccination. In addition to the prevaccination samples, 56 unvaccinated blood donors were recruited as controls (total n = 102). Thrombin generation, D-dimer levels, and free tissue factor pathway inhibitor (TFPI) levels were analyzed. Results: No participant experienced thrombosis, vaccine-induced immune thrombotic thrombocytopenia, or thrombocytopenia (platelet count <100 × 109/L) 1 week to 1 month postvaccination. There was no increase in thrombin generation, D-dimer level, or TFPI level in the ChAdOx1-S vaccine group compared with controls or after the mRNA vaccines compared with baseline values. Eleven of 513 (2.1%) participants vaccinated with ChAdOx1-S had anti-PF4/polyanion antibodies without a concomitant increase in thrombin generation. Conclusion: In this study, SARS-CoV-2 vaccines were not associated with thrombosis, thrombocytopenia, increased thrombin generation, D-dimer levels, or TFPI levels compared with baseline or unvaccinated controls. These findings argue against the subclinical activation of coagulation post-COVID-19 vaccination.

ß-Amyloid in Cerebrospinal Fluid: How to Keep It Floating (Not Sticking) by Standardization of Preanalytic Processes and Collection Tubes.

BACKGROUND: Phosphorylated tau (pTau), total tau (tTau), and ß-amyloid (Aß) are established cerebrospinal fluid (CSF) biomarkers used to help diagnose Alzheimer disease. Preanalytic workups of CSF samples lack harmonization, making interlaboratory comparison of these biomarkers challenging. The Aß adsorbs to sample tubes, yielding underestimated concentrations, and may result in false Alzheimer disease diagnosis. Our primary aim was to compare Aß recovery across multiple polypropylene tubes and to test the stability of tTau, pTau, and Aß in the best performing tube. METHODS: Eight polypropylene tubes were tested using 3 CSF pools with Aß concentrations <500, 500-1000, and >1000 ng/L. All samples were analyzed in duplicate. Tubes were cut open to assess their different infrared adsorption spectra. Freshly drawn CSF from 14 patients was distributed into 4 Sarstedt 5-mL (no. 63.504.027; Sar5CSF) tubes, left at room temperature for up to 7 days, and analyzed for pTau, tTau, and Aß by ELISA. RESULTS: Two Sarstedt 5-mL tubes and a Sarstedt 10-mL (Sar10CSF) tube showed significantly higher Aß recovery at all 3 concentrations compared with the 5 other tubes. The infrared adsorption spectra of Sar10CSF and Sar5CSF tubes were practically identical, unlike the other tubes. No significant loss of pTau, tTau, and Aß was observed in CSF left at room temperature for up to 7 days (P > 0.05). CONCLUSIONS: Recovery of Aß from Sar5CSF tubes is equivalent to Aß recovery from Sar10CSF tubes. Levels of pTau, tTau, and Aß were stable for at least 7 days at room temperature but not at 37 °C.

Markers of endothelial cell activation and neutrophil extracellular traps are elevated in immune thrombocytopenia but are not enhanced by thrombopoietin receptor agonists.

INTRODUCTION: Patients with immune thrombocytopenia (ITP) are at increased risk of thrombosis, which seems to be further enhanced by treatment with thrombopoietin-receptor-agonists (TPO-RAs). The underlying mechanisms of thrombosis in ITP are not fully understood. Endothelial cell activation and neutrophil extracellular traps (NETs) play important roles in thrombosis, however, their roles in ITP itself, or in TPO-RA-treatment, have not yet been fully explored. We aimed to investigate whether endothelial cell activation and NETs are involved in the hypercoagulable state of ITP, and whether TPO-RA-treatment enhances endothelial cell activation and NET formation. MATERIAL AND METHODS: We measured markers of endothelial cell activation including intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and thrombomodulin in 21 ITP patients, and E-selectin in 18 ITP patients. Markers of NET formation, citrullinated histone H3-DNA (H3Cit-DNA) and cell-free DNA (cfDNA), were measured in 15 ITP patients. All markers were measured before, and 2 and 6 weeks after initiation of TPO-RA-treatment in ITP patients, and in matched controls. RESULTS: Higher levels of ICAM-1, thrombomodulin, and H3Cit-DNA were found in ITP patients, both before and after TPO-RA-treatment, compared with controls. No differences were found for VCAM-1, E-selectin or cfDNA. TPO-RA-treatment did not further increase markers of endothelial cell activation or NET formation. CONCLUSIONS: This study showed that ITP patients have increased endothelial cell activation and NET formation, both of which may contribute to the intrinsic hypercoagulable state of ITP. TPO-RA-treatment, however, did not further increase endothelial cell activation or NET formation indicating that other drug-associated prothrombotic mechanisms are involved.

Increased microvesicle-associated thrombin generation in patients with immune thrombocytopenia after initiation of thrombopoietin receptor agonists.

Immune thrombocytopenia (ITP) patients have thrombocytopenia and increased bleeding risk, but, conversely, they also have increased thrombotic risk which appears to be exacerbated by thrombopoietin-receptor agonist (TPO-RA)-treatment. Microvesicles (MVs) released from activated/apoptotic cells are prothrombotic due to exposure of phosphatidylserine (PS) and tissue factor (TF). MVs are increased in ITP patients, but their prothrombotic effect, before and during treatment with TPO-RAs, is unclear.We studied the effect of TPO-RAs on the procoagulant activity of MVs in 11 ITP patients, before, and two and six weeks after initiation of treatment, and in 15 healthy controls. MV-associated PS-activity, TF-activity and the capacity of isolated MVs and plasma to generate thrombin in a phospholipid-dependent manner were measured.Before treatment with TPO-RAs, prothrombotic markers in ITP patients were comparable to levels found in healthy controls. After both two and six weeks of TPO-RA-treatment, ITP patients had higher MV-associated PS-activity and phospholipid-dependent thrombin generation in plasma than controls. In addition, ITP patients had increased phospholipid-dependent MV-associated thrombin generation two weeks after initiation of TPO-RA-treatment compared with controls and pre-treatment levels. MV-associated TF-activity was low in controls and in ITP patients before and after initiation of TPO-RA-treatment.In conclusion, TPO-RAs increase phospholipid-dependent MV-associated thrombin generation in ITP patients. This could contribute to or exacerbate a pre-existing hypercoagulable state. Phospholipid-dependent thrombin generation generated by isolated MVs, or measured directly in plasma, may be potential tools that could help in the risk-assessment of future thromboembolic events in ITP patients, both before and after initiation of TPO-RA-treatment.

Effect of thrombopoietin receptor agonists on markers of coagulation and P-selectin in patients with immune thrombocytopenia.

Thrombopoietin-receptor-agonists (TPO-RA) are effective treatments of immune thrombocytopenia (ITP). Previous long-term TPO-RA clinical trials have shown that thrombotic events occurred in 6% of TPO-RA-treated ITP patients. To explore the increased risk of thrombosis, the effects of TPO-RA on markers of coagulation and P-selectin were studied. The study comprised two ITP cohorts and controls. Cohort 1 included 26 patients with sequential samples acquired before and during treatment with TPO-RA. Cohort 2 included a single sample in 18 patients on TPO-RA for more than one year. Thrombin generation (endogenous thrombin potential (ETP)) prothrombin fragments 1 + 2 (F1+2), D-dimer, and plasminogen-activator-inhibitor-1 (PAI-1) were measured as well as soluble P-selectin (sP-selectin). Sequential expression of encoding genes for P-selectin (SELP) and PAI-1 (SERPINE1) was determined in four patients in cohort 1. Significantly higher levels of F1+2, D-dimer, and PAI-1 were found in ITP patients before TPO-RA treatment and in patients on long-term TPO-RA treatment than in controls. Pre-treatment levels of sP-selectin did not differ from controls. Analysis of longitudinal trends showed an increase in platelet count, sP-selectin, and PAI-1 after initiation of TPO-RA, followed by gradual decline. Platelet count and sP-selectin remained at higher levels throughout the study, whereas PAI-1 did not. Levels of other studied parameters did not show significant changes after initiation of treatment. Expression of SELP was up-regulated after initiation of TPO-RA, while the expression of SERPINE1 showed no significant changes. In conclusion, elevated pre-treatment levels of F1+2, D-dimer and PAI-1 are compatible with ITP being an intrinsically pro-thrombotic condition. After TPO-RA treatment, there were no significant changes in markers of coagulation activation or fibrinolysis, except for an initial increase in PAI-1 and a significant increase in sP-selectin both of which may contribute to increased thrombotic risk associated with TPO-RA treatment in ITP.

Prevalence and severity of interstitial lung disease in mixed connective tissue disease: a nationwide, cross-sectional study.

BACKGROUND: Mixed connective tissue disease (MCTD) is an immune-mediated, systemic disorder of unknown cause. OBJECTIVE: To assess the prevalence, pattern and severity of interstitial lung disease (ILD) in a cross-sectional study of the nationwide, Norwegian MCTD cohort. METHODS: 126 patients with MCTD were systematically examined for ILD by high-resolution CT (HRCT), pulmonary function tests (PFT), 6 min walk test (6MWT) and by the New York Heart Association (NYHA) functional classification of dyspnoea. The extent and type of HRCT lung abnormalities were scored according to the CT criteria of ILD recommended by the Fleischner Society. RESULTS: All 126 patients were Caucasian, 75% women. At the time of the cross-sectional ILD study, the patients had a mean disease duration of 9.0 years. 52% of the patients had abnormal HRCT findings, most commonly reticular patterns consistent with lung fibrosis (35%). Lung fibrosis was quantified as minor in 7%, moderate in 9% and severe in 19% of the patients. Fibrosis was uniformly concentrated in the lower parts of the lungs and was not associated with smoking. Patients with severe lung fibrosis had lower PFT values, shorter 6MWT and a higher mean NYHA functional class. After a mean 4.2 years' follow-up, overall mortality was 7.9%. Mortality in patients with normal HRCT was 3.3%, as compared with 20.8% in patients with severe lung fibrosis (p<0.01). CONCLUSIONS: Severe lung fibrosis is common in MCTD, has an impact on pulmonary function and overall physical capacity and is associated with increased mortality.