Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure.

ABSTRACT: Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines.

Erratum to 'A comparative study in type 2 von Willebrand disease patients using four different platelet-dependent von Willebrand factor assays.' [Research and Practice in Thrombosis and Haemostasis Volume 7, Issue 3, March 2023, 100139].

[This corrects the article DOI: 10.1016/j.rpth.2023.100139.].

A comparative study in patients with type 2 von Willebrand disease using 4 different platelet-dependent von Willebrand factor assays.

Background: Several assays are now available to evaluate platelet-dependent von Willebrand factor (VWF) activity. Objective: To report the results obtained using 4 different assays in patients with von Willebrand disease (VWD) carrying variants mainly in the A1 domain, which is critical for VWF binding to glycoprotein Ib (GPIb) and ristocetin. Methods: We evaluated 4 different assays, 2 gain-of-function mutant GPIb binding (VWF:GPIbM) and 2 ristocetin cofactor (VWF:RCo) assays, in 76 patients with type 2 VWD. Patients and healthy controls were tested using VWF:GPIbM enzyme-linked immunosorbent assay (ELISA), VWF:GPIbM automated, VWF:RCo aggregometric, and VWF:RCo automated assays. Results: There was a good correlation (Pearson's r>0.82) and agreement (Bland-Altman plots assessment) between the 4 assays, although several outliers existed among the type 2B without high-molecular-weight multimers (HMWM). The VWF activity/VWF:antigen ratios, calculated for each assay, were used to establish the percentage of a correct diagnosis of type 2 (ratio<0.60) in these patients: VWF:RCo aggregometric, 2A(100%), 2M(78%), 2M/2A(100%), 2B(68%); VWF:RCo automated, 2A(88%), 2M(89%), 2M/2A(100%), 2B(63%); VWF:GPIbM ELISA, 2A(96%), 2M(67%), 2M/2A(67%), 2B(0%); VWF:GPIbM automated, 2A(73%), 2M(44%), 2M/2A(75%), 2B(84%). In type 2B patients with HMWM, all assays gave a ratio ≥0.60. Conclusion: The VWF:GPIbM-automated assay is the most effective to diagnose as type 2 the 2B variants, whereas the VWF:RCo assays are the most effective in detecting 2M and 2M/2A variants. The VWF:GPIbM ELISA greatly overestimates the activity of the type 2B patients lacking HMWM. In this study, the use of a VWF activity/VWF:antigen ratio cut-off of 0.70 halved the number of misdiagnosed patients.

von Willebrand factor neutralizing and non-neutralizing alloantibodies in 213 subjects with type 3 von Willebrand disease enrolled in 3WINTERS-IPS.

BACKGROUND: Type 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions. OBJECTIVE: The objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS. METHODS: An indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay. RESULTS: In total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14). CONCLUSIONS: Anti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used.

Genetic determinants of enhanced von Willebrand factor clearance from plasma.

BACKGROUND: Enhanced von Willebrand factor (VWF) clearance from plasma is associated with von Willebrand disease (VWD). However, the genetic background of this disease mechanism is not well defined. OBJECTIVE: To determine VWF variants that are associated with reduced VWF survival. METHODS: Two hundred fifty-four patients with VWD (type 1 = 50 and type 2 = 204) were investigated, and the results were compared with 120 healthy controls. The patients were comprehensively characterized for phenotypic and genetic features. The ratio of VWF propeptide (VWFpp)/VWF antigen (VWFpp ratio) was used to establish in each patient the VWF clearance state. RESULTS: Out of 92 variants associated with type 1 (7 were novel) and type 2 VWD, 19 had a VWFpp ratio ranging from 1.7 to 2.2, 24 had a VWFpp ratio between 2.3 and 2.9, and 24 variants had a ratio of ≥3. The VWFpp median ratio in healthy controls was 0.98 (0.55-1.6) so that a cut-off value of >1.6 was considered an indicator of accelerated VWF clearance from plasma. An enhanced VWF clearance was observed in 34% of type 1 cases, 100% of type 1 Vicenza cases, 81% of 2A cases, 77% of 2B cases, 88% of 2M cases, and 36% of 2N cases. CONCLUSIONS: An accelerated VWF clearance was found in most patients with type 2A, 2B, and 2M VWD, with a lower proportion of type 1 and 2N. Sixty-seven different variants alone or in combination with other variants were associated with an increased VWFpp ratio. The variants with the highest VWFpp ratio were mostly located in the D3-A1 VWF domains.

Successful Chemical Synovectomy in a Patient with Acquired von Willebrand Syndrome with Chronic Synovitis Due to Recurrent Knee Hemarthrosis: A Case Report.

Acquired von Willebrand syndrome (AVWS) is a rare, non-hereditary bleeding disorder related to heterogeneous medical conditions such as hematological malignancies and cardiovascular and autoimmune diseases. We describe the clinical course of a 62-year-old man with polycythemia vera who experienced post-traumatic knee and leg swelling due to hemarthrosis. He was treated at another center with low molecular weight heparin due to misdiagnosed deep vein thrombosis further exacerbating the ongoing bleeding. At our center, he was diagnosed with AVWS with reduced von Willebrand factor (VWF):GPIbR plasma activity and loss of high molecular weight multimers (HMWM). He was treated with compressive bandages with resolution. Five months later, on clinical recurrence of knee and leg swelling, knee ultrasound scan showed the presence of chronic synovitis and a hemorrhagic Baker's cyst with signs of rupture. The treatment consisted of chemical synovectomy with rifampicin and steroids preceded by systemic replacement therapy using plasma-derived factor VIII-VWF concentrate. At the end of the treatment cycle, our patient reported complete resolution of knee pain and restoration of joint range of motion and function. Ultrasound evaluation confirmed complete resolution of knee capsule distension and Baker's cyst. Hemarthrosis is an anecdotal presentation of AVWS and chemical synovectomy was successful in treating this complication. A multidisciplinary approach allowed an effective management of this rare complication.

Phenotypic and genetic characterizations of the Milan cohort of von Willebrand disease type 2.

von Willebrand disease (VWD) type 2 is caused by qualitative abnormalities of von Willebrand factor (VWF). This study aimed to determine the genotypic and phenotypic characterizations of a large VWD type 2 cohort from Milan. We included 321 patients (54% female) within 148 unrelated families from 1995 to 2021. Patients were fully characterized using laboratory phenotypic tests, and the genotypic diagnosis was confirmed by target genetic analysis using Sanger sequencing. Patients were diagnosed with type 2A (n = 98; 48 families), 2B (n = 85; 38 families), 2M (n = 112; 50 families), or 2N (n = 26; 12 families). Eighty-two unique VWF variants, including 8 novel variants, were found. The potential pathogenic effect of novel variants was assessed by in silico analysis. Most patients were heterozygous for a single variant (n = 259; 81%), whereas 37 cases (11%) had 2 variants (4 homozygous, 9 in trans, and 24 in cis). Twenty-five patients (8%) had ≥3 variants, mainly as a result of gene conversions. Among the 82 distinct variants identified, 5 different types, including missense (n = 64), gene conversion (n = 10), synonymous (n = 1), deletion (n = 4), and splice (n = 3), were observed. The results from this large cohort showed that VWD type 2 is invariably due to variants that do not prevent the synthesis of the protein, and a vast majority of patients (88%) had missense variants. Given the complexity of type 2 diagnosis and the necessity of performing several phenotypic tests, genetic analysis for patients suspected of having type 2 is beneficial to establish the correct diagnosis.

Increasing levels of von Willebrand factor and factor VIII with age in patients affected by von Willebrand disease.

Essentials VWF and FVIII increase with age in patients affected by VWD. VWF and FVIII increase in type 1 and in low levels of VWF patients. VWF and FVIII do not increase in type 1 Vicenza. FVIII increases in type 2 VWD patients. ABSTRACT: Background Increasing levels of von Willebrand factor (VWF) and factor VIII (FVIII:C) was associated with age in type 1 von Willebrand disease (VWD). Objectives To evaluate VWF and FVIII:C increase with age in a large group of patients with VWD and low levels of VWF, in whom levels were repeatedly measured. Methods Clinical charts from all patients evaluated at the A. Bianchi Bonomi Center between 1970 and 2018 were reviewed and data on VWF and FVIII:C collected. Patients affected by type 3, severe type 1 and 2N VWD were excluded. The repeated measurements were evaluated by linear mixed-effects models. A linear association between age and VWF/FVIII:C was shown after the age of 40 years in the linear mixed models and analyzed by calculating the regression slope coefficient (ß). Results A total of 617 patients were included in the study (314 type 2, 112 type 1, 181 low VWF levels), with a median age at first measurement of 28 years (interquartile range 14/42) and a mean follow-up of 16 years (standard deviation 11). VWF and FVIII:C increased with age in the whole group. The increase became linear after the age of 40 years (3.68 and 7.44 IU/dL per decade for VWF:activity and FVIII:C). In type 2, FVIII:C increased with age, whereas an increase of both VWF:activity and FVIII:C were shown in patients with type 1 VWD and low levels of VWF. Conclusions A differential increase of VWF and FVIII:C with age was shown among in different ages and types of VWD.

Rise of levels of von Willebrand factor and factor VIII with age: Role of genetic and acquired risk factors.

BACKGROUND: Von Willebrand factor (VWF) levels are regulated by genetic and acquired factors. The acquired factors are mostly related to age and could be mediators of the age effect on VWF levels. OBJECTIVES: To disentangle the role of genetic (sex, blood group) and acquired factors (comorbidities, body mass index, reduced kidney function, hormone use, and inflammation) in regulating von Willebrand factor antigen (VWF:Ag) and factor VIII activity (FVIII:C) levels in the normal population. METHODS: Analysis were performed in a large population sample (2923 individuals) from the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA study), after exclusion of individuals with active cancer and women who were pregnant or within nine months postpartum. The increase of VWF:Ag and FVIII:C with age was evaluated by linear regression after the age of 40 years. Analyses were adjusted for acquired factors and stratified for sex and blood group. RESULTS: VWF:Ag and FVIII:C increased with age: increase per decade of age for VWF:Ag 18 IU/dL (95%CI 15-20) and for FVIII:C 12 IU/dL (95%CI 10-14). After adjustment for acquired factors, the increase per decade was 13 IU/dL (95%CI 10-16) for VWF:Ag and 9 IU/dL (95%CI 6-11) for FVIII:C. The stratified analysis for blood group showed higher increase in the non-O group, but these differences were annulled after adjustment for acquired factors. CONCLUSIONS: VWF:Ag and FVIII:C increase with age. Carriers of blood group non-O present a steeper increase of VWF:Ag and FVIII:C with age, that is mediated by acquired factors.

How I treat gastrointestinal bleeding in congenital and acquired von Willebrand disease.

Gastrointestinal (GI) bleeding is distinctive of severe von Willebrand disease (VWD), generally arising in older patients; in most cases, blood transfusion and hospitalization are required. The presence of arteriovenous malformations is often described when endoscopic examinations are performed. Patients with congenital type 3, 2A, and 2B are those most frequently affected by this symptom, possibly due to the loss of high-molecular-weight multimers of von Willebrand factor (VWF). GI bleeding can also occur in patients affected by acquired von Willebrand syndrome. Endoscopic examination of the GI tract is necessary to exclude ulcers and polyps or cancer as possible causes of GI bleeding. In congenital VWD, prophylaxis with VWF/factor VIII concentrates is generally started after GI-bleeding events, but this therapy is not always successful. Iron supplementation must be prescribed to avoid chronic iron deficiency. Possible rescue therapies (high-dose statins, octreotide, thalidomide, lenalidomide, and tamoxifen) were described in a few case reports and series; however, surgery may be necessary in emergency situations or if medical treatment fails to stop bleeding. In this article, we present several clinical cases that highlight the clinical challenges of these patients and possible strategies for their long-term management.

Management of rare acquired bleeding disorders.

Autoantibodies toward clotting factors may develop in people suffering from autoimmune or neoplastic diseases, after drug intake or even in subjects without apparent conditions. They are more commonly directed against factor VIII (FVIII) or von Willebrand factor leading to acquired hemophilia A or acquired von Willebrand syndrome, respectively. Rarely, autoantibodies develop against other clotting factors, such as fibrinogen, FII, FV, FVII, FX, FXI, and FXIII. The clinical picture of an acquired bleeding disorder includes a wide spectrum of clinical manifestations ranging from minimal or no bleeding to life-threatening events. Patients with no previous personal or family history of bleeding may have sudden-onset hemorrhagic manifestations, sometimes fatal, especially if an early diagnosis is not made. On the other hand, some patients may not have hemorrhagic symptoms at onset, and their diagnosis can therefore be delayed. The laboratory diagnostic assessment is performed by screening coagulation tests followed by specific factor-level measurement and inhibitor-titrating assays. An early diagnosis of acquired coagulopathies is mandatory for starting the appropriate treatment aimed at both controlling the acute bleeding episode mainly using the bypassing agents, and eradicating the anticlotting factor autoantibody, using immunosuppressive treatment. Therefore, prompt intervention by an expert and a specialized center is needed for immediate recognition and treatment of the disease.

A two-step approach (Enzyme-linked immunosorbent assay and confirmation assay) to detect antibodies against von Willebrand factor in patients with Acquired von Willebrand Syndrome.

BACKGROUND: Acquired von Willebrand Syndrome is a rare bleeding disorder, which arises in individuals with no personal or family history of bleeding, associated with lymphoproliferative and myeloproliferative disorders or other diseases. AIM: To develop a two-step approach assay to detect autoantibodies against VWF and to verify their prevalence in AVWS. METHODS: AVWS definition: negative personal or family history of bleeding diathesis, VWF below normal range and recent history of bleeding manifestations. Twenty-three consecutive patients affected by AVWS were enrolled. An ELISA assay (first step) using recombinant VWF protein immobilized on plates and sheep/goat polyclonal anti-human IgG or IgM labelled with peroxidase was developed. A group of 40 healthy subjects was tested to calculate the floating cut point value. A confirmation assay (with addition of purified VWF vs buffer) was performed (second step). RESULTS: Twenty-one patients (93%) had an associated disease, two patients had idiopathic AVWS. Anti-VWF autoantibodies were detected in 9 patients (39%). Of these, eight (89%) had VWF:RCo levels <10%, but none of them resulted positive using Bethesda assay (neutralizing antibodies). The confirmation test confirmed the positive results obtained with ELISA and resulted negative in those patients with negative results and in the controls. CONCLUSION: With the present two-step approach assay nine out of 23 (39%) patients affected with AVWS resulted positive for anti-VWF autoantibodies. This ELISA assay might be used as an additional confirmation tool in the diagnostic procedure in patients affected by AVWS or in the follow-up of congenital and acquired patients exposed to replacement therapy.

Normal reference ranges of antithrombin, protein C and protein S: effect of sex, age and hormonal status.

INTRODUCTION: Antithrombin (AT), protein C (PC) and protein S (PS) deficiencies are risk factors for venous thromboembolism. Overlapping values between heterozygous carriers and normal individuals often make a correct classification of a deficiency difficult. The aim of this study was to investigate the effect of sex, age, menopause and hormone therapy on natural anticoagulant plasma levels in a large group of healthy individuals, and to evaluate the need of separate reference ranges. MATERIALS AND METHODS: AT and PC were measured with a chromogenic assay, antigenic free PS with an ELISA test. To evaluate the effect of sex, age, oral contraception, hormonal status (and their interaction) on AT, PC and PS levels, linear regression models were used. Biological relevance and the value of the normal deviate z were chosen as rules to decide for separate reference ranges. RESULTS: The study population consisted of 1837 healthy adult individuals (741 men, 1096 women), aged 18-85 years (median age: 44 years). In men AT levels decreased after the age of 50 years. Men had higher levels of PS than women, particularly at young ages. In women, after correction for menopause, only PC levels increased with age. Menopause affected AT and PS, but not PC levels. Oral contraceptive intake was associated with a decrease of AT and PS, and an increase of PC levels. CONCLUSIONS: For AT, PC and PS, sex- and age-specific normal reference ranges can be useful, in order to better discriminate true carriers of a natural anticoagulant deficiency.

Laboratory diagnostic outcome applying detection criteria recommended by the Scientific and Standardization Committee of the ISTH on Lupus Anticoagulant.

This study shows the diagnostic outcome of an APTT-based and two dRVVT-based commercial confirmatory integrated tests with the application of the recommendations by the Scientific and Standardization Committee (SSC) on Lupus anticoagulant (LA)/antiphospholipid syndrome (APS) of the International Society on Thrombosis and Haemostasis (ISTH) issued in 2009 concerning the cut-off values for the screening, mixing and confirmatory tests for the detection of LA and the mandatory need to perform mixing tests of patient plasma with pooled normal plasma. The study population included 565 patients collected from a large central coagulation laboratory, for which the attending physicians requested LA detection. One-hundred-six healthy subjects (HS) and 131 selected patients on oral anticoagulant therapy (OAT) were included as negative controls. The results suggest that the performance of mixing tests is indicated for those methods with relatively poor specificity, but is less needed for those methods with high specificity. Furthermore, the SSC recommendation to use normal mid-value (i.e. the 50th percentile of distribution of results from healthy subjects) as the cut-off to interpret results of confirmatory tests, showed a modest increase in LA detection rate (sensitivity) but at the expense of specificity, particularly in methods with low specificity.

Risk factors for postpartum hemorrhage in a cohort of 6011 Italian women.

INTRODUCTION: Postpartum hemorrhage is responsible for 25% of maternal pregnancy-related deaths and it is the first cause of maternal morbidity and mortality worldwide. OBJECTIVE: To define the prevalence of postpartum hemorrhage and associated risk factors after vaginal birth and to develop a risk model that improves postpartum hemorrhage prediction. PATIENTS AND METHODS: All women who underwent a vaginal delivery at the Obstetric Unit of a large University hospital in Milan (Italy) between July 2007 and September 2009 were enrolled. Postpartum hemorrhage was defined as ≥ 500 mL blood loss. A nomogram tailored to predict postpartum hemorrhage was developed, summarizing the impact of each covariate on the probability of postpartum hemorrhage. RESULTS: 6011 women were studied (24% had blood loss ≥ 500 mL and 4.8% ≥ 1000 mL). Nulliparity, episiotomy, retained placenta and high neonatal body weight were confirmed as risk factors for postpartum hemorrhage. The odds ratio of postpartum hemorrhage was 0.86 (95%CI 0.78-0.90) for each 1 gr/dL increase in ante-partum hemoglobin. An extensive internal validation of the developed nomogram demonstrated a good stability of the risk model. CONCLUSIONS: Low ante-partum hemoglobin is a new potentially modifiable risk factor for postpartum hemorrhage. A nomogram to predict the probability of postpartum hemorrhage is now available for external validation.