Long-term joint outcomes in adolescents with moderate or severe haemophilia A.

INTRODUCTION: Favourable joint outcomes are expected with modern haemophilia A (HA) management. Evaluation of long-term treatment outcomes is hampered by the delay between bleeding episodes during childhood and resulting joint outcomes in adulthood. AIM: To measure the long-term joint health of adolescents with moderate and severe HA, according to severity and inhibitor status. METHODS: Pilot cross-sectional study of five European PedNet centres in moderate and severe HA patients aged 10-19 years. Structured assessment of joint status by physical examination (HJHS) and ultrasound (HEAD-US). RESULTS: In total, 141 HA patients were evaluable, 100 without inhibitors (81 severe, 19 moderate HA), and 41 severe HA with current/past inhibitors. On physical examination, 12/81 (15%) of severe HA without inhibitors, 3/19 (16%) of moderate HA, and 13/41 (32%) of severe HA patients with inhibitors exhibited joint abnormalities. Inhibitor persistence, longer inhibitor duration, and a high peak inhibitor level were associated with impaired joint health. Ultrasound showed joint damage (bone or cartilage) in 13/49 (27%) of severe HA without inhibitors, 1/12 (8%) of moderate HA, and 10/28 (36%) of severe HA patients with inhibitors. A discordant ankle evaluation by ultrasound versus physical examination was present in 53/169 joints (31%). CONCLUSIONS: Most adolescents with severe or moderate HA show favourable joint health. Future research with combined ultrasound and/or MRI is needed to better understand joint outcomes in the remaining patients. Patents with inhibitors showed a two-fold increased proportion with joint deterioration. Ultrasound paired with physical examination increases sensitivity for detection of joint damage.

Anti-platelet antibody immunoassays in childhood immune thrombocytopenia: a systematic review.

BACKGROUND: In adult immune thrombocytopenia (ITP), an acquired autoimmune bleeding disorder, anti-platelet autoantibody testing may be useful as a rule-in test. Childhood ITP has different disease characteristics, and the diagnostic and prognostic value of anti-platelet antibody testing remains uncertain. OBJECTIVE: To systematically review the diagnostic accuracy of anti-platelet autoantibody testing in childhood ITP. METHODS: PubMed and EMBASE were searched for studies evaluating immunoassays in childhood ITP. Study quality was assessed (QUADAS2), and evidence was synthesized descriptively. RESULTS: In total, 40 studies (1606 patients) were identified. Nine studies reported sufficient data to determine diagnostic accuracy measures. Anti-platelet IgG antibody testing showed a moderate sensitivity (0·36-0·80 platelet-associated IgG [direct test]; 0·19-0·39 circulating IgG [indirect test]). In studies that reported control data, including patients with non-immune thrombocytopenia, specificity was very good (0·80-1·00). Glycoprotein-specific immunoassays showed comparable sensitivity (three studies) and predominantly identified IgG anti-GP IIb/IIIa antibodies, with few IgG anti-GP Ib/IX antibodies. Anti-platelet IgM antibodies were identified in a substantial proportion of children (sensitivity 0·62-0·64 for direct and indirect tests). CONCLUSION: The diagnostic evaluation of IgG and IgM anti-platelet antibodies may be useful as a rule-in test for ITP. In children with insufficient platelets for a direct test, indirect tests may be performed instead. A negative test does not rule out the diagnosis of ITP. Future studies should evaluate the value of anti-platelet antibody tests in thrombocytopenic children with suspected ITP.

Relative effects of plasma, fibrinogen concentrate, and factor XIII on ROTEM coagulation profiles in an in vitro model of massive transfusion in trauma.

Massive traumatic haemorrhage is aggravated through the development of trauma-induced coagulopathy, which is managed by plasma transfusion and/or fibrinogen concentrate administration. It is yet unclear whether these treatments are equally potent in ensuring adequate haemostasis, and whether additional factor XIII (FXIII) administration provides further benefits. In this study, we compared ROTEM whole blood coagulation profiles after experimental massive transfusion with different transfusion regimens in an in vitro model of dilution- and transfusion-related coagulopathy. Healthy donor blood was mixed 1 + 1 with six different transfusion regimens. Each regimen contained RBC, platelet concentrate, and either fresh frozen plasma (FFP) or Ringer's acetate (RA). The regimens were further augmented through addition of a low- or medium-dose fibrinogen concentrate and FXIII. Transfusion with FFP alone was insufficient to maintain tissue-factor activated clot strength, coincidental with a deficiency in fibrin-based clot strength. Fibrinogen concentrate conserved, but did not improve coagulation kinetics and overall clot strength. Only combination therapy with FFP and low-dose fibrinogen concentrate improved both coagulation kinetics and fibrin-based clot strength. Administration of FXIII did not result in an improvement of clot strength. In conclusion, combination therapy with both FFP and low-dose fibrinogen concentrate improved clotting time and produced firm clots, representing a possible preferred first-line regimen to manage trauma-induced coagulopathy when RBC and platelets are also transfused. Further research is required to identify optimal first-line transfusion fluids for massive traumatic haemorrhage.

Output control of da Vinci surgical system's surgical graspers.

INTRODUCTION: The number of robot-assisted surgeries performed with the da Vinci surgical system has increased significantly over the past decade. The articulating movements of the robotic surgical grasper are controlled by grip controls at the master console. The user interface has been implicated as one contributing factor in surgical grasping errors. The goal of our study was to characterize and evaluate the user interface of the da Vinci surgical system in controlling surgical graspers. MATERIALS AND METHODS: An angular manipulator with force sensors was used to increment the grip control angle as grasper output angles were measured. Input force at the grip control was simultaneously measured throughout the range of motion. Pressure film was used to assess the maximum grasping force achievable with the endoscopic grasping tool. RESULTS: The da Vinci robot's grip control angular input has a nonproportional relationship with the grasper instrument output. The grip control mechanism presents an intrinsic resistant force to the surgeon's fingertips and provides no haptic feedback. The da Vinci Maryland graspers are capable of applying up to 5.1 MPa of local pressure. CONCLUSIONS: The angular and force input at the grip control of the da Vinci robot's surgical graspers is nonproportional to the grasper instrument's output. Understanding the true relationship of the grip control input to grasper instrument output may help surgeons understand how to better control the surgical graspers and promote fewer grasping errors.

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B.

BACKGROUND: Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management. AIM: To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype. METHODS: Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories. RESULTS: FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories. CONCLUSION: FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.

Comparative analysis of spike-specific IgG Fc glycoprofiles elicited by adenoviral, mRNA, and protein-based SARS-CoV-2 vaccines.

IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps.

Biological stratification of clinical disease courses in childhood immune thrombocytopenia.

BACKGROUND: In childhood immune thrombocytopenia (ITP), an autoimmune bleeding disorder, there is a need for better prediction of individual disease courses and treatment outcomes. OBJECTIVE: To predict the response to intravenous immunoglobulins (IVIg) and ITP disease course using genetic and immune markers. METHODS: Children aged younger than 7 years with newly diagnosed ITP (N = 147) from the Treatment With or Without IVIG for Kids with ITP study were included, which randomized children to an IVIg or observation group. A total of 46 variables were available: clinical characteristics, targeted genotyping, lymphocyte immune phenotyping, and platelet autoantibodies. RESULTS: In the treatment arm, 48/80 children (60%) showed a complete response (platelets ≥100 × 109 /L) that lasted for at least 1 month (complete sustained response [CSR]) and 32 exhibited no or a temporary response (absence of a sustained response [ASR]). For a biological risk score, five variables were selected by regularized logistic regression that predicted ASR vs CSR: (1) hemoglobin; (2) platelet count; (3) genetic polymorphisms of Fc-receptor (FcγR) IIc; (4) the presence of immunoglobulin G (IgG) anti-platelet antibodies; and (5) preceding vaccination. The ASR sensitivity was 0.91 (95% confidence interval, 0.80-1.00) and specificity was 0.67 (95% confidence interval, 0.53-0.80). In the 67 patients of the observation arm, this biological score was also associated with recovery during 1 year of follow-up. The addition of the biological score to a predefined clinical score further improved the discrimination of favorable ITP disease courses. CONCLUSIONS: The prediction of disease courses and IVIg treatment responses in ITP is improved by using both clinical and biological stratification.

A clinical prediction score for transient versus persistent childhood immune thrombocytopenia.

Essentials There is a need for improved tools to predict persistent and chronic immune thrombocytopenia (ITP). We developed and validated a clinical prediction model for recovery from newly diagnosed ITP. The Childhood ITP Recovery Score predicts transient vs. persistent ITP and response to intravenous immunoglobulins. The score may serve as a useful tool for clinicians to individualize patient care. ABSTRACT: Background Childhood immune thrombocytopenia (ITP) is an autoimmune bleeding disorder. The prognosis (transient, persistent, or chronic ITP) remains difficult to predict. The morbidity is most pronounced in children with persistent and chronic ITP. Clinical characteristics are associated with ITP outcomes, but there are no validated multivariate prediction models. Objective Development and external validatation of the Childhood ITP Recovery Score to predict transient versus persistent ITP in children with newly diagnosed ITP. Methods Patients with a diagnosis platelet count ≤ 20 × 109 /L and age below 16 years were included from two prospective multicenter studies (NOPHO ITP study, N = 377 [development cohort]; TIKI trial, N = 194 [external validation]). The primary outcome was transient ITP (complete recovery with platelets ≥100 × 109 /L 3 months after diagnosis) versus persistent ITP. Age, sex, mucosal bleeding, preceding infection/vaccination, insidious onset, and diagnosis platelet count were used as predictors. Results In external validation, the score predicted transient versus persistent ITP at 3 months follow-up with an area under the receiver operating characteristic curve of 0.71. In patients predicted to have a high chance of recovery, we observed 85%, 90%, and 95% recovered 3, 6, and 12 months after the diagnosis. For patients predicted to have a low chance of recovery, this was 32%, 46%, and 71%. The score also predicted cessation of bleeding symptoms and the response to intravenous immunoglobulins (IVIg). Conclusion The Childhood ITP Recovery Score predicts prognosis and may be useful to individualize clinical management. In future research, the additional predictive value of biomarkers can be compared to this score. A risk calculator is available (http://www.itprecoveryscore.org).

A functional spleen contributes to afucosylated IgG in humans.

As a lymphoid organ, the spleen hosts a wide range of immune cell populations, which not only remove blood-borne antigens, but also generate and regulate antigen-specific immune responses. In particular, the splenic microenvironment has been demonstrated to play a prominent role in adaptive immune responses to enveloped viral infections and alloantigens. During both types of immunizations, antigen-specific immunoglobulins G (IgGs) have been characterized by the reduced amount of fucose present on N-linked glycans of the fragment crystallizable (Fc) region. These glycans are essential for mediating the induction of immune effector functions. Therefore, we hypothesized that a spleen may modulate humoral responses and serve as a preferential site for afucosylated IgG responses, which potentially play a role in immune thrombocytopenia (ITP) pathogenesis. To determine the role of the spleen in IgG-Fc glycosylation, we performed IgG subclass-specific liquid chromatography-mass spectrometry (LC-MS) analysis of Fc glycosylation in a large cohort of individuals splenectomized due to trauma, due to ITP, or spherocytosis. IgG-Fc fucosylation was consistently increased after splenectomy, while no effects for IgG-Fc galactosylation and sialylation were observed. An increase in IgG1- and IgG2/3-Fc fucosylation level upon splenectomy has been reported here for the first time, suggesting that immune responses occurring in the spleen may be particularly prone to generate afucosylated IgG responses. Surprisingly, the level of total IgG-Fc fucosylation was decreased in ITP patients compared to healthy controls. Overall, our results suggest a yet unrecognized role of the spleen in either the induction or maintenance of afucosylated IgG responses by B cells.

Diagnostic Accuracy in Acute Venous Thromboembolism: Comparing D-Dimer, Thrombin Generation, Overall Hemostatic Potential, and Fibrin Monomers.

Introduction For acute venous thromboembolism (VTE), a biomarker with higher specificity than D-dimer would be of great clinical use. Thrombin generation and overall hemostatic potential (OHP) reflect the hemostatic balance by globally assessing multiple coagulation factors and inhibitors. These tests discriminate between healthy controls and patients with a prothrombotic tendency but have yet to be established as clinical biomarkers of VTE. Objective This study compares endogenous thrombin potential (ETP) and OHP to D-dimer and fibrin monomers (FM) in outpatients with suspected VTE. Methods A cross-sectional diagnostic study where 954 patients with suspected pulmonary embolism or deep venous thrombosis were recruited consecutively from the medical emergency department at Karolinska University Hospital. D-dimer, FM, OHP, and ETP were analyzed in a subpopulation of 60 patients with VTE and 98 matched controls without VTE. VTE was verified either by ultrasonography or computed tomography and clinical data were collected from medical records. Results Compared with healthy controls, both VTE and non-VTE patients displayed prothrombotic profiles in OHP and ETP. D-dimer, FM, ETP area under the curve (AUC), and ETP T lag were significantly different between patients with VTE and non-VTE. The largest receiver-operating characteristic AUCs for discrimination between VTE and non-VTE, were found in D-dimer with 0.94, FM 0.77, and ETP AUC 0.65. No useful cutoff could be identified for the ETP or the OHP assay. Conclusion Compared with D-dimer, neither ETP nor OHP were clinically viable biomarkers of acute venous thrombosis. The data indicated that a large portion of the emergency patients with suspected VTE were in a prothrombotic state.

Evolution and Utility of Antiplatelet Autoantibody Testing in Patients with Immune Thrombocytopenia.

To this day, immune thrombocytopenia (ITP) remains a clinical diagnosis made by exclusion of other causes for thrombocytopenia. Reliable detection of platelet autoantibodies would support the clinical diagnosis, but the lack of specificity and sensitivity of the available methods for platelet autoantibody testing limits their value in the diagnostic workup of thrombocytopenia. The introduction of methods for glycoprotein-specific autoantibody detection has improved the specificity of testing and is acceptable for ruling in ITP but not ruling it out as a diagnosis. The sensitivity of these assays varies widely, even between studies using comparable assays. A review of the relevant literature combined with our own laboratory's experience of testing large number of serum and platelet samples makes it clear that this variation can be explained by variations in the characteristics of the tests, including in the glycoprotein-specific monoclonal antibodies, the glycoproteins that are tested, the platelet numbers used in the assay and the cutoff levels for positive and negative results, as well as differences in the tested patient populations. In our opinion, further standardization and optimization of the direct autoantibody detection methods to increase sensitivity without compromising specificity seem possible but will still likely be insufficient to distinguish the often very weak specific autoantibody signals from background signals. Further developments of autoantibody detection methods will therefore be necessary to increase sensitivity to a level acceptable to provide laboratory confirmation of a diagnosis of ITP.

Anti-platelet antibodies in childhood immune thrombocytopenia: Prevalence and prognostic implications.

BACKGROUND: Anti-platelet antibody testing may be useful for the diagnosis and management of childhood immune thrombocytopenia (ITP). OBJECTIVES: Here we aimed to assess the prevalence and prognostic significance of anti-platelet glycoprotein-specific IgM and IgG antibodies. METHODS: Children with newly diagnosed ITP were included at diagnosis and randomized to an intravenous immunoglobulins (IVIg) or careful observation group (TIKI trial). In this well-defined and longitudinally followed cohort (N = 179), anti-platelet glycoprotein-specific IgM and IgG antibodies were determined by monoclonal antibody-immobilization of platelet antigens. RESULTS: The dominant circulating anti-platelet antibody class in childhood ITP was IgM (62% of patients); but IgG antibodies were also found (10%). Children without IgM platelet antibodies were older and more often female. There was weak evidence for an association between IgM anti-GP IIb/IIIa antibodies and an increased bleeding severity (P = .03). The IgM and IgG anti-platelet responses partially overlapped, and reactivity was frequently directed against multiple glycoproteins. During 1-year follow-up, children with IgM antibodies in the observation group displayed a faster platelet recovery compared to children without, also after adjustment for age and preceding infections (P = 7.1 × 10-5 ). The small group of patients with detectable IgG anti-platelet antibodies exhibited an almost complete response to IVIg treatment (N = 12; P = .02), suggesting that IVIg was particularly efficacious in these children. CONCLUSIONS: Testing for circulating anti-platelet antibodies may be helpful for the clinical prognostication and the guidance of treatment decisions in newly diagnosed childhood ITP. Our data suggest that the development of even more sensitive tests may further improve the clinical value of antibody testing.

IgG-Fc glycosylation before and after rituximab treatment in immune thrombocytopenia.

The interactions of antibodies with myeloid Fcγ receptors and the complement system are regulated by an Asn297-linked glycan in the Fc portion of IgG. Alterations of serum IgG-Fc glycosylation have been reported in various autoimmune diseases, and correlate with treatment response and disease activity. We hypothesized that IgG-Fc glycosylation is altered in immune thrombocytopenia (ITP) and associates with response to anti-CD20 monoclonal antibody treatment (rituximab). IgG-Fc glycosylation was analyzed by liquid chromatography-mass spectrometry. We found that IgG-Fc glycosylation was identical between refractory ITP patients (HOVON64 trial; N = 108) and healthy controls (N = 120). Two months after rituximab treatment, we observed a shift in Fc glycosylation, with a mean 1.7% reduction in galactosylation for IgG1 and IgG4 and a mean 1.5% increase for bisection in IgG1, IgG2/3 and IgG4 (adjusted p < 1.7 × 10-3 and p < 2 × 10-4, respectively). Neither baseline nor longitudinal changes in IgG-Fc glycosylation after rituximab were associated with clinical treatment response. We conclude that IgG-Fc glycosylation in refractory ITP is similar to healthy controls and does not predict treatment responses to rituximab. The observed changes two months after treatment suggest that rituximab may influence total serum IgG-Fc glycosylation. Overall, our study suggests that the pathophysiology of refractory ITP may differ from other autoimmune diseases.

On the mechanical role of de novo synthesized elastin in the urinary bladder wall.

The urinary bladder wall (UBW), which is composed of smooth muscle, collagen, and elastin, undergoes profound remodeling in response to changes in mechanical loading resulting from various pathologies. In our laboratory, we have observed the production of fibrillar elastin in the extracellular matrix (ECM), which makes the UBW a particularly attractive tissue to investigate smooth muscle tissue remodeling. In the present study, we explored the mechanical role that de novo elastin fibers play in altering UBW ECM mechanical behavior using a structural constitutive modeling approach. The mechanical behavior of the collagen fiber component of the UBW ECM was determined from the biaxial stress-stretch response of normal UBW ECM, based on bimodal fiber recruitment that was motivated by the UBW's unique collagen fiber structure. The resulting fiber ensemble model was then combined with an experimentally derived fiber angular distribution to predict the biaxial mechanical behavior of normal and the elastin-rich UBW ECM to elucidate the underlying mechanisms of elastin production. Results indicated that UBW ECM exhibited a distinct structure with highly coiled collagen fiber bundles and visible elastic fibers in the pathological situation. Elastin-rich UBW ECM had a distinct mechanical behavior with higher compliance, attributable to the indirect effect of elastin fibers contracting the collagen fiber network, resulting in a retracted unloaded reference state of the tissue. In conclusion, our results suggest that the urinary bladder responds to prolonged periods of high strain by increasing its effective compliance through the interaction between collagen and de novo synthesized elastic fibers.

Genetic Variation in Low-To-Medium-Affinity Fcγ Receptors: Functional Consequences, Disease Associations, and Opportunities for Personalized Medicine.

Fc-gamma receptors (FcγR) are the cellular receptors for Immunoglobulin G (IgG). Upon binding of complexed IgG, FcγRs can trigger various cellular immune effector functions, thereby linking the adaptive and innate immune systems. In humans, six classic FcγRs are known: one high-affinity receptor (FcγRI) and five low-to-medium-affinity FcγRs (FcγRIIA, -B and -C, FcγRIIIA and -B). In this review we describe the five genes encoding the low-to-medium -affinity FcγRs (FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B), including well-characterized functionally relevant single nucleotide polymorphisms (SNPs), haplotypes as well as copy number variants (CNVs), which occur in distinct copy number regions across the locus. The evolution of the locus is also discussed. Importantly, we recommend a consistent nomenclature of genetic variants in the FCGR2/3 locus. Next, we focus on the relevance of genetic variation in the FCGR2/3 locus in auto-immune and auto-inflammatory diseases, highlighting pathophysiological insights that are informed by genetic association studies. Finally, we illustrate how specific FcγR variants relate to variation in treatment responses and prognosis amongst autoimmune diseases, cancer and transplant immunology, suggesting novel opportunities for personalized medicine.

Correlation of thromboelastography and thrombin generation assays in warfarin-treated patients.

Venous thromboembolism (VTE) affects approximately 1 per 1000 persons annually. Although patients are increasingly treated with direct oral anticoagulants, many patients continue to be anticoagulated with vitamin K antagonists (VKA). The most important adverse events during VKA treatment, bleeding and the risk of recurrent VTE, are difficult to predict. Global haemostatic assays, such as thrombin generation assays and the viscoelastic whole blood tests thromboelastography (TEG) and thromboelastometry (ROTEM), allow a comprehensive assessment of haemostasis and could potentially predict such side effects. In the present study we compared results from thrombin generation (Calibrated Automated Thrombogram and Innovance ETP assays) and TEG and ROTEM in 84 warfarin-treated patients with primary or recurrent VTE and 87 healthy controls. VKA treatment lead to lagtime prolongation and a lower overall thrombin production, which correlated strongly with INR (Pearson r = 0.89 and r = -0.85, respectively). The reduced thrombin generation of VKA-treated patients was accurately reflected by tissue-factor activated ROTEM (EXTEM) clotting time prolongation (vs. CAT lagtime, r = 0.87). Clot strength or clot formation kinetics were only weakly affected by thrombin generation. Intrinsic pathway activated TEG or ROTEM (INTEM) were not sensitive to the reduced thrombin generation. In conclusion, patients anticoagulated with VKA after VTE showed a reduced plasma thrombin generation that was accurately reflected by tissue factor activated ROTEM. ROTEM provided additional information to thrombin generation, including clot formation kinetics and strength.

Transient and chronic childhood immune thrombocytopenia are distinctly affected by Fc-γ receptor polymorphisms.

In childhood immune thrombocytopenia (ITP), anti-platelet autoantibodies mediate platelet clearance through Fc-γ receptor (FcγR)-bearing phagocytes. In 75% to 90% of patients, the disease has a transient, self-limiting character. Here we characterized how polymorphisms of FcγR genes affect disease susceptibility, response to intravenous immunoglobulin (IVIg) treatment, and long-term recovery from childhood ITP. Genotyping of the FCGR2/3 locus was performed in 180 children with newly diagnosed ITP, 22 children with chronic ITP, and 180 healthy control children by multiplex ligation-dependent probe amplification. Children with newly diagnosed ITP were randomly assigned to a single administration of IVIg or observation, and followed for 1 year (Treatment With or Without IVIg for Kids With ITP [TIKI] trial). We defined transient ITP as a complete recovery (≥100 × 109/L) 3 months after diagnosis, including both self-limiting disease/IVIg responders and chronic ITP as absence of a complete recovery at 12 months. ITP susceptibility, as well as spontaneous recovery and response to IVIg, was associated with the genetic variants FCGR2C*ORF and FCGR2A*27W and the FCGR2B promoter variant 2B.4. These variants were overrepresented in patients with transient (N = 131), but not chronic (N = 43), disease. The presence of FCGR2C*ORF predisposed to transient ITP with an odds ratio of 4.7 (95% confidence interval, 1.9-14.3). Chronic ITP was associated with a deletion of FCGR2C/FCGR3B (copy number region 1) with an odds ratio of 6.2 (95% confidence interval, 1.8-24.7). Taken together, susceptibility to transient and chronic ITP is distinctly affected by polymorphic variants of FCGR2/3 genes. Our data suggest that genotyping of the FCGR2/3 locus may be useful for prognosis and guidance of treatment decisions in newly diagnosed childhood ITP.

Whole blood ristocetin-activated platelet impedance aggregometry (Multiplate) for the rapid detection of Von Willebrand disease.

Von Willebrand disease (VWD) is the most common bleeding disorder, but no bedside tests specific for Von Willebrand factor are available. The objective of this study was to evaluate the diagnostic accuracy of whole blood ristocetin-induced platelet aggregometry (WB-RIPA) in VWD. WB-RIPA was performed in VWD patients (n=100) and healthy controls (n=17) using the Multiplate® platelet impedance aggregometry platform. The diagnostic properties of the test were described as sensitivity/specificity, positive and negative predictive value, and ROC area under the curve (AUC). Patients with VWD had impaired platelet aggregation by WB-RIPA. At a cut-off of 98 U, the test sensitivity and specificity of WB-RIPA for VWD was 0.95 and 0.53. A cut-off of 60 U provided a specificity of 1.00 with reduced sensitivity of 0.76. All patients with type 3 VWD and >90 % of patients with type 2 VWD were accurately distinguished from the controls. Incorrect classifications were attributable to patients with type 1 VWD, showing partly overlapping WB-RIPA results with healthy controls. Remarkably, these patients had lower bleeding scores and higher VWF activity than other type 1 VWD patients. Overall, WB-RIPA discriminated VWD patients from healthy controls accurately with a ROC AUC of 0.94. These results show that WB-RIPA is a promising diagnostic test for VWD, especially when timely results are required. Depending on the chosen test threshold, WB-RIPA could be clinically used as a rule out test, or to suggest patients in whom further testing for VWD is warranted.