A Functional Assay for the Determination of Heparin-Induced Thrombocytopenia via Flow Cytometry.

Heparin-induced thrombocytopenia (HIT) is a life-threatening complication of heparin therapy (both unfractionated heparin and low-molecular-weight heparin). In our study, we examined a group of 122 patients with suspected HIT. The samples of all patients were analyzed in the first step using an immunoassay (ID-PaGIA Heparin/PF4, Hemos1L-Acustar HIT IgG, ZYMUTEST HIA Monostrip IgG) to detect the presence of antibodies against heparin-PF4 complexes (platelet factor 4). When the immunoassay was positive, the sample was subsequently analyzed for HIT with a functional flow cytometry assay, the HITAlert kit, the purpose of which was to demonstrate the ability of the antibodies present to activate platelets. A diagnosis of HIT can be made only after a positive functional test result. In this article, we present an overview of our practical experience with the use of the new functional method of analysis, HIT, with flow cytometry. In this work, we compared the mutual sensitivity of two functional tests, SRA and the flow cytometry HITAlert kit, in patients perceived as being at risk for HIT. This work aims to delineate the principle, procedure, advantages, pitfalls, and possibilities of the application of the functional test HITAlert using flow cytometry.

DNA Polymorphisms in Pregnant Women with Sticky Platelet Syndrome.

Sticky platelet syndrome (SPS) is a thrombophilia caused by the increased aggregability of platelets in response to the addition of low concentrations of epinephrine (EPI) and/or adenosine diphosphate (ADP). Some of the single nucleotide polymorphisms (SNP), alleles and haplotypes of platelet glycoprotein receptors were proved to have a role in the etiology of thrombotic episodes When comparing SPS and the control group, in VEGFA rs3025039, the p value for both CC vs. TT and CT vs. TT analyses was <0.001. Interestingly, no minor TT genotype was present in the SPS group, suggesting the thrombotic pathogenesis of recurrent spontaneous abortions (RSA) in these patients. Moreover, we found a significant difference in the presence of AT containing a risky A allele and TT genotype of ALPP rs13026692 (p = 0.034) in SPS patients when compared with the controls. Additionally, we detected a decreased frequency of the GG (CC) genotype of FOXP3 rs3761548 in patients with SPS and RSA when compared with the control group (p value for the CC (GG) vs. AA (TT) 0.021). This might indicate an evolutionary protective mechanism of the A (T) allele in the SPS group against thrombotic complications in pregnancy. These results can be used for antithrombotic management in such pregnant patients.

How can Secondary Thromboprophylaxis in High-Risk Pregnant Patients be Improved?

Low-molecular-weight heparin (LMWH) is suggested for thromboprophylaxis in pregnant women with previous venous thromboembolism (VTE). Anyway, there is only limited amount of studies evaluating the effect of LMWH on hemostatic parameters during pregnancy of patients with previous VTE and the need of secondary thromboprophylaxis. We therefore provide results of prospective and longitudinal assessment of changes in hemostasis in high-risk pregnant women at four times during pregnancy (T1-T4) and one time after the postpartum period (T5) used for individualized modification of thromboprophylaxis. In this study, the results of coagulation factor VIII (FVIII) and protein S (PS) activity, ProC Global ratio and anti-Xa activity were evaluated. Despite the thromboprophylaxis, an increased predisposition to thromboembolic complications was detected (significant increase in FVIII activity and decrease in PS function, ProC Global ratio not normalized even after the postpartum period - p < .0001 between controls and T5 for PS and ProC Global). These results indicate that hemostasis may not be restored even 6 to 8 weeks after delivery and pose the question when is it safe to withdraw the anticoagulant thromboprophylaxis in high-risk patients with prior VTE.

A new decade awaits sticky platelet syndrome: where are we now, how do we manage and what are the complications?

INTRODUCTION: Sticky platelet syndrome is a less known platelet function disorder with a familiar occurrence and likely genetic background. Clinically, it is characterized by an increased risk of venous and arterial thromboembolic events and obstetric placenta-mediated complications. The increased aggregation after low-dose ADP and/or epinephrine is its distinctive laboratory feature. Though described for almost 40 years, several issues regarding its etiology, involved pathomechanisms, genetic background, optimal diagnostic and treatment approach remain controversial. AREAS COVERED: The work aims to summarize published studies, the actual definition of the syndrome, and point out its drawbacks. A literature search on Medline, Embase, and archives from EHA congresses was performed (terms: 'sticky platelet syndrome' - 'platelet hyperreactivity' - 'platelet hyperaggregability'). The authors added in their unpublished data. The introductory overview of the present understanding is followed by the discussion of the pathophysiologic, diagnostic, and therapeutic problems. EXPERT OPINION: Despite the growing evidence provided by case reports and series, the lack of robust studies limits the decision-making on diagnostics and management. The diagnostic issues, particularly the standardization of light transmission aggregometry, represent the crucial problem for the broader acceptance of the syndrome.

PLAIN LANGUAGE SUMMARY Sticky platelet syndrome is aplatelet function disorder. It is associated with an increased risk of venous thromboembolism, arterial thrombosis, and obstetric placenta-mediated complications. Increased aggregation after low-dose ADP and/or epinephrine is the defining laboratory feature. Furthermore, afew studies report the familiar occurrence with possible genetic background. Several issues regarding the syndrome remain controversial: its exact etiology, genetics, optimal diagnostic, and treatment approach. These uncertainties provide ground for debate of the syndrome as aunique clinical entity. The review has two goals. Firstly, it summarizes the published studies and the actual definition of the syndrome. Secondly, it tries to point out the open pathophysiologic, diagnostic, and therapeutic problems.

Multimer Analysis of Von Willebrand Factor in Von Willebrand Disease with a Hydrasys Semi-Automatic Analyzer-Single-Center Experience.

von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder. This disorder develops as a result of defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for VWF-related disorders requires the assessment of both VWF level and VWF activity, the latter requiring multiple assays. As an additional step, an evaluation of VWF structural features by multimer analysis is useful in selective investigations. Multimer analysis is also important for the selection of a suitable VWF therapy preparation (desmopressin, VWF/FVIII concentrate, recombinant VWF) and the determination of the correct dose for the patient. Based on clinical and laboratory findings, including the analysis of VWF multimers, we classified our patients into individual types of VWD. Our study group included 58 patients. The study group consisted of 66% (38 patients) with VWD type 1, 5% (3 patients) with VWD type 2, 7% (4 patients) with VWD type 3, 5% (3 patients) with mixed type 1/2A VWD, and 17% (10 patients) comprising an unclassified group. In this article, we provide an overview of our practical experience using a new complementary method-the analysis of von Willebrand factor multimers with a semi-automatic analyzer Hydrasys 2 scan. We explain the principle, procedure, advantages, and pitfalls associated with the introduction of the VWF multimer analysis methodology into standard VWD diagnostics.

How Can Rotational Thromboelastometry as a Point-of-Care Method Be Useful for the Management of Secondary Thromboprophylaxis in High-Risk Pregnant Patients?

Thromboprophylaxis with low-molecular-weight heparin (LMWH) for patients with a history of venous thromboembolism (VTE) is suggested. Rotational thromboelastometry (ROTEM®) represents an innovative point-of-care method enabling the complex and quick evaluation of hemostasis. However, there are only episodic cases of its use for hemostasis assessment and guidance of LMWH in pregnancy. Therefore, we provide the results of unique prospective and longitudinal monitoring of hemostasis in high-risk pregnant women, which we used for the individualized optimalization of secondary thromboprophylaxis. According to the shortening of clot formation time (CFT) in EXTEM (p = 0.0007 from the 26th gestational week vs. controls) and INTEM (p = 0.002 from the 35th gestational week), increase in alpha angle (AA) in EXTEM, INTEM, and HEPTEM, and the persistence of increase in maximum clot firmness (MCF) in EXTEM, INTEM, and HEPTEM (p < 0.001 from the 26th and 35th gestational week vs. controls for EXTEM and INTEM, p = 0.0012 from the 26th gestational week in HEPTEM), LMWH dose was modified. Even after the postpartum period, AA in EXTEM was steeper than in controls (p = 0.0007), indicating that hemostasis is not fully normalized after 6-8 weeks following delivery. Therefore, ROTEM may be a useful tool for the individual evaluation of the termination of anticoagulant thromboprophylaxis.

Use of Fibrinogen Determination Methods in Differential Diagnosis of Hypofibrinogenemia and Dysfibrinogenemia.

BACKGROUND: Fibrinogen plays an important role in hemostasis. The normal concentration of fibrinogen in blood plasma is between 1.8 - 4.2 g/L. Decreased fibrinogen levels are observed in congenital afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, disseminated intravascular coagulation, fibrinolytic therapy, some more severe hepatic parenchymal disorders, and increased blood loss. Elevated fibrinogen levels occur in inflammatory diseases and neoplastic diseases, in pregnancy, and postoperative conditions. Functional fibrinogen measurement is also one of the basic coagulation screening tests. The fibrinogen antigen assay is used to distinguish between qualitative and quantitative fibrinogen disorders. METHODS: The aim of the study was the use of fibrinogen determination methods in differential diagnosis of hypofibrinogenemia and dysfibrinogenemia, statistical evaluation and determine the relationship of fibrinogen Clauss assay, prothrombin time (PT) derived fibrinogen assay, and fibrinogen antigen in the group of 60 patients with congenital fibrinogen disorders (n = 40 dysfibrinogenemia; n = 20 hypofibrinogenemia). RESULTS: The results measured by the PT-derived fibrinogen assay were approximately four times higher compared to the fibrinogen Clauss assay in the group of patients with dysfibrinogenemia. In patients with hypofibrinogenemia, there is a correlation (r = 0.9016) between the fibrinogen Clauss assay and PT-derived fibrinogen assay with a statistical significance of p < 0.0001. Using a linear or quadratic interpolation function, we were able to determine the fibrinogen Clauss assay and the fibrinogen antigen assay before analysis. CONCLUSIONS: The higher level of the PT-derived fibrinogen assay compared to the fibrinogen Clauss assay in the group of patients with dysfibrinogenemia may pose a greater risk to asymptomatic patients who require diagnosis and treatment in case of bleeding. The fibrinogen value using the PT-derived fibrinogen assay could erroneously give a normal level. The use of the interpolation function is important to estimate the value of fibrinogen activity and antigen before the analysis itself by the Clauss assay or analysis by the fibrinogen antigen assay.

Sticky Platelet Syndrome: 35 Years of Growing Evidence.

Since the identification of antithrombin deficiency by Egeberg in 1956, ongoing research in prothrombotic defects continues to progress. Interestingly, past research has predominantly focused on coagulation factors and not on other components of the hemostatic system. The possible role of platelet function defects in the development of thrombotic events was suggested for the first time in the late 1970s, when an increased platelet adhesiveness and aggregation after epinephrine (EPI) and adenosine diphosphate (ADP) was found in a group of patients with unexplained transient ischemic attack. Clinical evidence for other types of thrombotic events (e. g. myocardial infarction, ischemic stroke, optic neuropathy, and pregnancy-related complications) with similar laboratory findings was provided by several authors in the 1980s and 1990s, with Drs. Mammen and Bick undertaking key research. The familial occurrence was noted as well, and the term sticky platelet syndrome was introduced by Holliday in 1983 to describe the defect. The term in our present understanding describes a thrombophilic qualitative platelet disorder characterized by increased in vitro platelet aggregation after the addition of very low concentrations of ADP and/or EPI and an increased risk of thromboembolic (predominantly arterial) events. Although now recognized for 35 years, significant issues, namely its etiology, inheritance, epidemiology, and diagnostics, remain a matter of vigorous debate. The aim of this review is to summarize the history, key works, and present understanding of the syndrome and to outline present-day diagnostic and clinical problems and controversies.

Monitoring of Hemostasis and Management of Anticoagulant Thromboprophylaxis in Pregnant Women with Increased Risk of Fetal Loss.

Physiological prothrombotic changes during pregnancy and the postpartum period, along with other preexisting maternal risk factors, increase the risk of both venous thromboembolism (VTE) and adverse pregnancy outcomes. Pregnancy complications that develop due to placental insufficiency as a result of inappropriate activation of coagulation are present in more than 5% of pregnancies and can contribute to significant maternal morbidity and mortality. Therefore, anticoagulant prophylaxis in women with congenital and acquired thrombophilic conditions should be actively considered. According to the Guidelines of American College of Chest Physicians, the use of low-molecular-weight heparin is suggested for prophylaxis of VTE and pregnancy complications in high-risk pregnant women. However, personalized refinements of such thromboprophylaxis remains unspecified, despite the necessity of better targeted recommendations for life-threatening conditions. We, therefore, review the possibilities of longitudinal monitoring and comprehensive assessment of changes in hemostasis in the group of high-risk pregnant women, which can then be used for early prediction and individualization of the optimal anticoagulant thromboprophylaxis of pregnancy complications. Simultaneously, we present our single-center experience with such monitoring and our first series of results.

Sticky platelet syndrome: an important cause of life-threatening thrombotic complications.

Sticky platelet syndrome (SPS) is a prothrombotic thrombocytopathy with familial occurrence, characterized by hyperaggregability of platelets in response to adenosine diphosphate (ADP), epinephrine (EPI) or both. The syndrome has been identified in approximately 21% of unexplained arterial thrombotic episodes, regarded to be the most common thrombophilia in arterial thrombosis and 13.2% of unexplained venous thromboembolism (VTE). The relatively young age at the first manifestation, relation to fertility and pregnancy, seriousness of the symptoms, easy and effective management of the disorder indicate to the necessity to take it into account in the differential diagnosis of the underlying cause of the thrombotic event. As the various localizations of the thrombosis in SPS have been reported, its management often requires a multidisciplinary approach. This review deals with the clinical aspects of thrombophilia, its etiopathogenesis, diagnosis as well as novel advances in the treatment and outlines the challenges for the further research.

The use of intravenous immunoglobulin in immune tolerance induction in inherited haemophilia A: a single-centre experience and a review of literature.

The immune tolerance induction is the treatment of choice for the eradication of factor VIII inhibitors, a serious complication of inherited haemophilia A. Despite the preferred treatment of patients with good prognosis and testing of different regimens, the immune tolerance is achieved in 70-80%. Several modifications of regimens including the addition of immunomodulatory agents were proposed in order to improve immune tolerance induction (ITI) outcome. Intravenous immunoglobulin has complex immunomodulatory properties and has been used in immune tolerance induction since the introduction of Malmö regimen in the 1980s. The aim of the work is to evaluate the published evidence of its use in ITI in haemophilia A. In addition, the authors' own experience with a high-dose regimen using human plasma-derived factor VIII containing von Willebrand factor and pulsed IVIg is reported. The course of three patients with severe inherited haemophilia A and inhibitor (all high responders, two with poor prognosis, one with rescue treatment) is described. At least partial success was achieved in all patients and no adverse events attributable to intravenous immunoglobulin were observed. Despite the limited evidence, the addition of intravenous immunoglobulin appears to be a promising treatment modification for patients with poor prognosis or previous failure of immune tolerance induction.

Sticky platelet syndrome: history and future perspectives.

The sticky platelet syndrome (SPS) is a thrombophilic qualitative platelet disorder with familial occurrence and autosomal dominant trait, characterized by increased in vitro platelet aggregation after low concentrations of adenosine diphosphate and/or epinephrine. Its clinical manifestation includes arterial thrombosis, pregnancy complications (fetal growth retardation and fetal loss), and less often venous thromboembolism. SPS was considered to be a rare thrombophilic disorder, but it can be found relatively often as a cause of unexplained thrombosis, particularly among patients with arterial thrombosis such as stroke. The syndrome was recognized as a distinct disorder in 1983 by Holiday and further characterized in the 1980s and 1990s, with Mammen and Bick providing the key findings. Although recognized for more than 30 years, significant issues, namely the syndrome's etiology, inheritance, and epidemiology, remain unclear. The aim of the first part of this review is to summarize the previous 35 years of the research into, and to provide a brief historical account of, SPS. The history section is focused particularly on the work of two most prominent investigators: Eberhard F. Mammen and Rodger L. Bick. The second part summarizes the present understanding of the syndrome and outlines unresolved issues and the trends in which the future research is likely to continue.

Sticky platelet syndrome.

Sticky platelet syndrome (SPS) is a thrombophilic thrombocytopathy with familial occurrence and autosomal dominant trait, characterized by an increased in vitro platelet aggregation in response to low concentrations of adenosine diphosphate (ADP) and/or epinephrine (EPI). According to aggregation pattern, three types of the syndrome can be identified (hyperresponse after both reagents, Type I; EPI alone, Type II; ADP alone, Type III). Clinically, the syndrome is associated with both venous and arterial thrombosis. In pregnant women, complications such as fetal growth retardation and fetal loss have been reported. The first thrombotic event usually occurs before 40 years of age and without prominent acquired risk factors. Antiplatelet drugs generally represent adequate treatment. The use of other antithrombotics is usually ineffective and may result in the recurrence of thrombosis. In most patients, low doses of antiplatelet drugs (acetylsalicylic acid, 80-100 mg/d) lead to normalization of hyperaggregability. Combination of SPS with other thrombophilic disorders has been described. Despite several studies investigating platelet glycoproteins' role in platelets' activation and aggregation, the precise defect responsible for the syndrome remains unknown. The aim of this review is to summarize authors' own experience about SPS and the clinical data indexed in selected databases of medical literature (PubMed and Scopus).

The prevalence of the platelet glycoprotein VI polymorphisms in patients with sticky platelet syndrome and ischemic stroke.

INTRODUCTION: The aim of the study was to evaluate the genetic variability of the GP6 gene in patients with sticky platelet syndrome (SPS), a disorder characterized by platelet hyperaggregability, and thus to identify the genetic changes of the glycoprotein VI with possible relation to the platelet hyperaggregability. PATIENTS AND METHODS: Seventy-one patients with SPS, clinically manifested as ischemic stroke, and 77 controls without SPS and with negative personal history of thromboembolic events were involved. SPS was diagnosed by platelet aggregometry (PACKS-4 aggregometer, Helena Laboratories) according to the method and criteria described by Mammen and Bick. Seven single-nucleotide polymorphisms (SNPs) of the GP6 gene (rs1654410, rs1671153, rs1654419, rs11669150, rs1613662, rs12610286, and rs1654431) were evaluated with the use of restriction-fragment-length polymorphism analysis. RESULTS: All allele and genotype frequencies were comparable between both SPS patients and the control group with no statistically significant differences. The haplotype analysis showed a higher occurrence of the one major haplotype (TTGTGA, 0.228 vs. 0.174; odds ratio (OR) 1.421; confidence interval (CI) 0.799-2.526) and two minor haplotypes (CGATAA, 0,026 vs. 0,006; OR 4.117; CI 0.443-38.25; TTGTGG, 0.018 vs. 0.009; OR 2.107; CI 0.259-17.12) in patients with SPS. None of haplotype differences was statistically significant. However, both the allele G of SNP rs12610286 (P = 0.029; OR 2.411; CI 1.134-5.123) and one major haplotype (TTGTGA; P = 0.012; OR 2.749; CI 1.223-6.174) were found significantly more frequent in patients with SPS type I in comparison with controls. CONCLUSION: Our results, especially higher occurrence of four haplotypes in SPS patients, can support an idea that variability of the GP6 gene may be associated with the platelet hyperaggregability in SPS.

Successful immune tolerance induction consisting of high-dose factor VIII rich in von Willebrand factor and pulsed intravenous immunoglobulin: a case report.

INTRODUCTION: The development of factor VIII inhibitors is a serious complication of replacement therapy in patients with congenital hemophilia A. Immune tolerance induction has been accepted as the only clinically proven treatment allowing antigen-specific tolerance to factor VIII. However, some of its issues, such as patient selection, timing, factor VIII dosing, use of immunosuppressive or immunomodulatory procedures, still remain the subject of debate. CASE PRESENTATION: A case of a 3-year-old Caucasian boy with severe congenital hemophilia A, intron 22 inversion of the F8 gene and high-titer inhibitor, who underwent an immune tolerance induction according to the modified Bonn regimen (high doses of plasma-derived factor VIII rich in von Willebrand factor and pulsed intravenous immunoglobulin) is presented. The treatment lasted for 13 months and led to the eradication of inhibitor. CONCLUSION: Addition of intravenous immunoglobulin did not negatively affect the course of immune tolerance induction and led to the rapid eradication of factor VIII inhibitor.

Is Gas6 protein associated with sticky platelet syndrome?

The aim of this study was to detect the prevalence of the polymorphisms of growth arrest-specific gene 6 (Gas6; Gas6 c. 834 + 7G > A) in patients with sticky platelet syndrome (SPS). Sticky platelet syndrome is a hereditary, autosomal dominant thrombophilia characterized by platelet hyperaggregation after low concentrations of platelet inducers-adenosine diphosphate (ADP) and epinephrine (EPI). The cause of SPS still remains unknown, but in recent years it was suggested that Gas6 protein may have a potential role in the pathogenesis of SPS. To assess the Gas6 polymorphisms (Gas6 c. 834 + 7G > A), 128 patients with SPS were included in the study and examined by polymerase chain reaction (PCR) method. GG genotype was detected in 63 (49.2%) patients, GA genotype in 53 (41.4%) patients, and AA genotype in 12 (9.4%) patients. The results in controls did not differ significantly compared to patients with SPS. Our findings did not prove allele A to be less associated with thrombosis and that ''prothrombotic'' allele G may be associated with higher risk of thrombosis. We cannot support the idea that Gas6 protein and Gas6 polymorphisms may be associated with thrombosis in SPS.

Circulating vascular endothelial growth factor in the normo- and/or microalbuminuric patients with type 2 diabetes mellitus.

Relationship between serum vascular endothelial growth factor (VEGF) level and parameters of endothelial injury and/or dysfunction in patients with diabetes mellitus type 2 with or without microalbuminuria was investigated. Eighty-four diabetic patients were divided in two subgroups (42 each): normoalbuminuric (NAU) and microalbuminuric (MAU). Forty-two blood donors were in control group. Serum VEGF and plasma von Willebrand factor, soluble thrombomodulin, plasminogen activator inhibitor 1, thrombin-activatable fibrinolysis inhibitor (TAFI) and tissue plasminogen activator (t-PA) were measured using enzyme-linked immunosorbent assay in all subjects. VEGF was significantly higher in NAU compared to controls. The difference between MAU and controls was not statistically significant, but there was a trend toward significance. Only TAFI correlated with VEGF in MAU. An observed significant increase of serum VEGF level already in NAU suggests that serum VEGF could be a sensitive predictor of endothelial dysfunction in type 2 diabetes.