Bleeding pattern and consumption of factor VIII concentrate in adult patients with haemophilia A without inhibitors in the Czech Republic between 2013 and 2021 (Czech National Haemophilia Programme registry data).

The manuscript provides an overview of treatment and its changes in adult patients with haemophilia A without inhibitors in the Czech Republic between 2013 and 2021 using data from the registry of the Czech National Haemophilia Programme (CNHP). Over a 9-year period, we focused on the reduction in the annual bleeding rate (ABR), joint bleeding rate (AJBR) and factor VIII consumption when patients with severe haemophilia A switched from on-demand treatment to prophylaxis. The ABR and AJBR include both patient-reported home treatment and treated hospitalisation episodes. All adult patients with severe haemophilia A were categorised into three groups according to the therapeutic regimen. The first group was patients on prophylaxis during the follow-up period, the second group consisted of patients on on-demand treatment, and the third group was patients who received both treatment regimens during follow-up. With an increase in the proportion of patients with severe haemophilia A on prophylaxis from 37 to 74% between 2013 and 2021, the ABR for all patients with severe haemophilia A decreased approximately 6.9-fold, and the AJBR decreased 8.7-fold. Expectedly, the factor consumption increased by approximately 68.5%. In the group of patients with severe haemophilia A who had switched from an on-demand to a prophylactic regimen, the total number of bleeding events decreased 3.5-fold, and the number of joint bleeding episodes decreased 3.9-fold. Factor VIII consumption increased by 78.4%. Our study supports a previously reported positive effect of prophylaxis on bleeding control. We believe that the substantial improvement in ABR justifies the increased treatment costs.

Antithrombin Deficiency: Frequency in Patients with Thrombosis and Thrombophilic Families.

PURPOSE: Antithrombin (AT) deficiency is a well-known inherited risk factor for venous thromboembolism (VTE). However, F V Leiden and F II20210a mutations have drawn much more attention in the recent years. Therefore, we have decided to analyze the frequency of antithrombin deficiency in different cohorts of patients and tried to formulate indications for its testing. RESULTS: Antithrombin deficiency was found in 4% of patients with recurrent VTE ≤ 50 years of age with, in 1% of patients with splanchnic vein thrombosis and in 2% of cases associated with combined oral contraceptives (COC) use or pregnancy. In patients with central venous thrombosis, antithrombin deficiency was not found. RECOMMENDATION: We consider antithrombin testing useful in patients with thrombosis occuring up to 45 years of age without any risk factors. Namely, females with VTE in pregnancy and puerperium should be tested as well as females with thrombosis on COC, if VTE occurred within the first year of their use. CONCLUSION: In spite of degressive interest in thrombophilia work up, we still consider antithrombin testing useful in defined clinical situations.

A rare case of non-traumatic spinal epidural hematoma in lumbar region associated with apixaban therapy.

Spontaneous spinal epidural hematoma (SSEH) is a very rare clinical entity with potential diagnostic difficulties and which can result in severe neurological deficit. The etiology of this rare condition is largely not known, but with potential predisposition in patients on anticoagulation medication. This includes the novel anticoagulants with direct inhibition of the factor Xa mechanism (DOACs). These medications are supposed to have more predictable pharmacokinetics with fewer severe haemorrhagic adverse events in comparison with standard warfarin therapy. However, in the last few years, an increasing number of case reports have been published of haemorrhage into the central nervous system. We present a case of non-traumatic spinal epidural hematoma in the lumbar region in a patient on chronic apixaban therapy. To the best of our knowledge, it is the first described SSEH in the lumbar region associated with apixaban therapy.

Ph- myeloproliferative neoplasms and the related risk factors for stroke occurrence: Results from a registry of patients treated with Anagrelide.

Arterial thrombosis is a common complication in patients with Ph- myeloproliferative neoplasms (MPN). We searched for the risk factors of stroke in MPN patients from anagrelide registry. We analyzed the potential risk factors triggering a stroke/TIA event in 249 MPN patients with previous stroke (n = 168) or Transient Ischemic Attack (TIA) (n = 140), and in 1,193 MPN control subjects (without clinical history of thrombosis). These patients were registered in a prospective manner, providing a follow-up period after Anagrelide treatment. The median age of the patients in the experimental group was of 56 years of age (ranging from 34-76) and of 53 years of age (ranging from 26-74) in the control group (p < 0.001). Using a multivariate model, we determined the following as risk factors: JAK2V617F mutation (OR 2.106, 1.458-3.043, p = 0.006), age (OR 1.017/year, 1.005-1,029, p = 0.006), male gender (OR 1.419, 1.057-1.903, p = 0.020), MPN diagnosis (OR for PMF 0.649, 0.446-0.944, p = 0.024), BMI (OR 0.687 for BMI > 25, 0.473-0.999, p = 0.05) and high TAG levels (OR 1.734, 1.162-2.586, p = 0.008), all of which were statistically significant for CMP development. Concerning the risk factors for thrombophilia, only the antiphospholipid syndrome (OR 1.994, 1.017-3.91, p = 0.048) was noteworthy in a stroke-relevant context. There was no significant difference between the blood count of the patients prior to a stroke event and the control group, both of which were under a cytoreductive treatment. We found that age, male gender, JAK2V617F mutation, previous venous thrombosis, and hypertriglyceridemia represent independent risk factors for the occurrence of a stroke in Ph- MPN patients.

Identifying risk factors and optimizing standard of care for patients with acquired haemophilia A: Results from a Czech patient cohort.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare autoimmune disorder, characterized by bleeds of varying severity caused by autoantibodies against factor VIII (FVIII). AIM: Identify risk factors associated with AHA-related deaths/relapses and assess the effect of increased corticosteroid doses. METHODS: AHA patients treated across two specialist centres in the Czech Republic, generally receiving first-line haemostatic therapy with rFVIIa and immunosuppression with corticosteroids/cyclophosphamide, were included. We analysed the association between early death (within 8 weeks of diagnosis [considered disease-related]) and age, malignancy, FVIII levels and bleeding severity. Risk factors associated with reduced 2-year survival and relapse incidence, and the effect of increased corticosteroid doses on early death and remission were also assessed. RESULTS: The demographics of the described cohort (n = 66) were similar to other AHA registries. Early death occurred in 20% of cases. Unlike age and malignancy, FVIII levels <1% and severe bleeding were associated significantly with early death (P = .010 and P = .046, respectively). Patients with underlying malignancy or requiring continued haemostatic therapy exhibited significantly decreased 2-year survival compared with those without these risk factors (P = .007 and P = .006, respectively). Patients with an underlying autoimmune disease relapsed significantly more than those without (P = .015). Higher corticosteroid doses were associated with a significantly increased incidence of early deaths (P < .001), but also with early remission (P < .001). CONCLUSION: Based on this rather large patient cohort, we were able to evaluate the significance of several risk factors associated with treatment outcomes in AHA and the effect of initial treatment with corticosteroids on survival and time to remission.

Ropeginterferon alfa-2b versus standard therapy for polycythaemia vera (PROUD-PV and CONTINUATION-PV): a randomised, non-inferiority, phase 3 trial and its extension study.

BACKGROUND: The PROUD-PV and CONTINUATION-PV trials aimed to compare the novel monopegylated interferon ropeginterferon alfa-2b with hydroxyurea, the standard therapy for patients with polycythaemia vera, over 3 years of treatment. METHODS: PROUD-PV and its extension study, CONTINUATION-PV, were phase 3, randomised, controlled, open-label, trials done in 48 clinics in Europe. Patients were eligible if 18 years or older with early stage polycythaemia vera (no history of cytoreductive treatment or less than 3 years of previous hydroxyurea treatment) diagnosed by WHO's 2008 criteria. Patients were randomly assigned 1:1 to ropeginterferon alfa-2b (subcutaneously every 2 weeks, starting at 100 µg) or hydroxyurea (orally starting at 500 mg/day). After 1 year, patients could opt to enter the extension part of the trial, CONTINUATION-PV. The primary endpoint in PROUD-PV was non-inferiority of ropeginterferon alfa-2b versus hydroxyurea regarding complete haematological response with normal spleen size (longitudinal diameter of ≤12 cm for women and ≤13 cm for men) at 12 months; in CONTINUATION-PV, the coprimary endpoints were complete haematological response with normalisation of spleen size and with improved disease burden (ie, splenomegaly, microvascular disturbances, pruritus, and headache). We present the final results of PROUD-PV and an interim analysis at 36 months of the CONTINUATION-PV study (per statistical analysis plan). Analyses for safety and efficacy were per-protocol. The trials were registered on EudraCT, 2012-005259-18 (PROUD-PV) and 2014-001357-17 (CONTINUATION-PV, which is ongoing). FINDINGS: Patients were recruited from Sept 17, 2013 to March 13, 2015 with 306 enrolled. 257 patients were randomly assigned, 127 were treated in each group (three patients withdrew consent in the hydroxyurea group), and 171 rolled over to the CONTINUATION-PV trial. Median follow-up was 182·1 weeks (IQR 166·3-201·7) in the ropeginterferon alfa-2b and 164·5 weeks (144·4-169·3) in the standard therapy group. In PROUD-PV, 26 (21%) of 122 patients in the ropeginterferon alfa-2b group and 34 (28%) of 123 patients in the standard therapy group met the composite primary endpoint of complete haematological response with normal spleen size. In CONTINUATION-PV, complete haematological response with improved disease burden was met in 50 (53%) of 95 patients in the ropeginterferon alfa-2b group versus 28 (38%) of 74 patients in the hydroxyurea group, p=0·044 at 36 months. Complete haematological response without the spleen criterion in the ropeginterferon alfa-2b group versus standard therapy group were: 53 (43%) of 123 patients versus 57 (46%) of 125 patients, p=0·63 at 12 months (PROUD-PV), and 67 (71%) of 95 patients versus 38 (51%) of 74 patients, p=0·012 at 36 months (CONTINUATION-PV). The most frequently reported grade 3 and grade 4 treatment-related adverse events were increased γ-glutamyltransferase (seven [6%] of 127 patients) and increased alanine aminotransferase (four [3%] of 127 patients) in the ropeginterferon alfa-2b group, and leucopenia (six [5%] of 127 patients) and thrombocytopenia (five [4%] of 127 patients) in the standard therapy group. Treatment-related serious adverse events occurred in three (2%) of 127 patients in the ropeginterferon alfa-2b group and five (4%) of 127 patients in the hydroxyurea group. One treatment-related death was reported in the standard therapy group (acute leukaemia). INTERPRETATION: In patients with early polycythaemia vera, who predominantly presented without splenomegaly, ropeginterferon alfa-2b was effective in inducing haematological responses; non-inferiority to hydroxyurea regarding haematological response and normal spleen size was not shown at 12 months. However, response to ropeginterferon alfa-2b continued to increase over time with improved responses compared with hydroxyurea at 36 months. Considering the high and durable haematological and molecular responses and its good tolerability, ropeginterferon alfa-2b offers a valuable and safe long-term treatment option with features distinct from hydroxyurea. FUNDING: AOP Orphan Pharmaceuticals AG.

Heparin-induced thrombocytopenia treated with fondaparinux: single center experience.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is the most frequent drug-induced, immune-mediated thrombocytopenia. It is associated with significant morbidity and mortality. Anticoagulation with heparin must be stopped immediately and replaced by some suggested alternative - lepirudin, danaparoid or argatroban. Fondaparinux has been also successfully used in HIT. METHODS: We present a cohort of 10 patients diagnosed with HIT and treated in a university hospital in a period of four years. Diagnosis was based on Keeling´s scoring system, screening immunologic test for HIT (STic EXPERT® HIT) and sandwich ELISA (detection of IgG/heparin-PF4 antibodies). While other alternative anticoagulants are not readily available in our hospital, we used fondaparinux in all cases. RESULTS: From 2014 to 2018, eight males and two females (mean age 67 years, range 46-86 years) were diagnosed with HIT in our hospital. This complication developed in 9 cases after low-molecular-weight heparin and in one after heparin flushes in hemodialysis. A drop-in platelet count developed in all patients, thrombotic complications in 7 and skin necrosis in 2 cases. Fondaparinux was used in all patients, including two cases with severe renal impairment, the dose was chosen individually. We observed complete platelet recovery in all cases. One patient died because of advanced malignancy, others did not have any complication. In 6 cases we switched to oral anticoagulation after platelet recovery. CONCLUSIONS: In our group of 10 HIT patients fondaparinux was shown to be both safe and effective, even in those with severe renal impairment. Additional studies are warranted to confirm this observation.

Treatment of polycythemia vera.

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by hematopoietic stem cell-derived clonal myeloproliferation resulting in erythrocytosis, leukocytosis and thrombocytosis. Survival is reduced compared with general population. Main reasons of death include thrombohemorrhagic complications, fibrotic progression and leuk-aemic transformation. Presence of Janus kinase (JAK2) gene mutations is a diagnostic marker and standard dia-gnostic criterion. World Health Organization 2016 diagnostic criteria focusing on hemoglobin levels, hematocrit, red cell mass and bone marrow morphology are mandatory. Therapeutic approach depends on stratification of patients according age and personal risk of thrombosis. Low-risk patients are treated first line with low-dose aspirin and phlebo-tomy. Cytoreduction is indicated in high-risk patients. Interferon-α has demonstrated efficacy in many clinical trials. Its pegylated form is well tolerated, enabling less frequent administration than standard interferon. Therefore it is therapy of choice based on Central European Myeloproliferative Neoplasm Organisation recommendation. Ropeginterferon α-2b has been shown to be more efficacious than hydroxyurea. Hydroxyurea is suspected of leukemogenic potential. JAK1/JAK2 inhibitor ruxolitinib is approved for hydroxyurea resistant/intolerant patients. Key words: diagnosis - polycythemia vera - therapy.

Investigation of congenital thrombophilic conditions: when, in whom, focusing on what or not at all?

Venous thromboembolism is a multifactorial disease. Inherited thrombophilia is linked with increased risk of VTE and we know about them more than 50 years. Through a robust thrombophilia work-up in the end of millenium, the criteria for testing have significantly gone down. It is associated with increased amount of information about clinical consequence of testing. We discuss current recommendations not only in the literature, but also in our clinic. Key words: criteria for testing - thrombophilia - venous thromboembolism.

Analysis of Risk Factors of Stroke and Venous Thromboembolism in Females With Oral Contraceptives Use.

Thrombotic diathesis has been a well-known complication of oral contraceptive use for more than 50 years. This is true not only for venous thrombosis but also for an arterial one. The etiology is usually multifactorial and depends on several additional risk factors. We analyzed the prevalence of inherited and acquired thrombophilia in a cohort of 770 females who had a thrombotic event in association with oral contraceptive use (700 women with venous thromboembolism [VTE], 70 with stroke). Moreover, we tried to identify additional risk factors. Inherited thrombophilia was found in 44.5% with higher frequency in the cohort with VTE (42%) than in females with stroke (24%). The most frequent finding was factor V Leiden. Cigarette smoking was significantly more frequent in the group with stroke (50% vs 25%). The prevalence of cigarette smoking in the group with VTE did not exceed the frequency in general population. Women on oral contraceptive pills have higher risk of venous as well as arterial thrombosis. The risk of venous thrombosis is increased in females with inherited thrombophilia, whereas those with some additional acquired risk factors (especially smoking) may be predisposed to arterial thrombosis. However, the absolute risk of thrombosis in healthy women is low, far less than the risk of unintended pregnancy. Moreover, the risk may be reduced by keeping some rules before the prescription of the pills, healthy life style, and a proper choice of contraception.

Fibrin Clot Architecture in Acute Ischemic Stroke Treated With Mechanical Thrombectomy With Stent-Retrievers　- Cohort Study.

BACKGROUND: The composition of intra-arterial clots might influence the efficacy of mechanical thrombectomy (MT) in ischemic stroke (IS) due to the acute occlusions within large cerebral arteries. The aims were to assess the factors associated with blood clot structure and the impact of thromboembolus structure on MT using stent-retrievers in patients with acute large artery IS in the anterior circulation.Methods and Results:In an observational cohort study, we studied the components of intra-arterial clots retrieved from large cerebral arteries in 80 patients with acute IS treated with MT with or without i.v. thrombolysis (IVT). Histology of the clots was carried out without knowledge of the clinical findings, including the treatment methods. The components of the clots, their age, origin and semi-quantitative graded changes in the architecture of the fibrin components (e.g., "thinning") were compared via neuro-interventional, clinical and laboratory data. The most prominent changes in the architecture of the fibrin components in the thromboemboli were associated with IVT (applied in 44 patients; OR, 3.50; 95% CI: 1.21-10.10, P=0.02) and platelet count (OR, 2.94; 95% CI: 1.06-8.12, P=0.04). CONCLUSIONS: In patients with large artery IS treated with the MT using stent-retrievers, bridging therapy with IVT preceding MT and higher platelet count were associated with significant changes of the histological structure of blood clots.

Real-life experience with the specific reversal agent idarucizumab for the management of emergency situations in dabigatran-treated patients: a series of 11 cases.

Non-vitamin K antagonist oral anticoagulants (NOACs) have a favorable benefit-risk profile compared with vitamin K antagonists. However, the lack of specific reversal agents has made the management of some patients receiving long-term treatment with NOACs problematic in emergency situations such as major bleeding events or urgent procedures. Idarucizumab, a fully humanized Fab antibody fragment that binds specifically and with high affinity to dabigatran, was recently approved for use in adult patients treated with dabigatran when rapid reversal of its anticoagulant effect is required. Clinical experience with idarucizumab is currently limited. We report 11 real-life clinical cases in which idarucizumab was used after multidisciplinary consultation in a variety of emergency situations including severe postoperative bleeding, emergency high-bleeding-risk surgery (hip/spine surgery and neurosurgery), invasive diagnostic testing (lumbar puncture), intracranial bleeding (pre-pontine subarachnoid hemorrhage and lobar intracerebral hemorrhage) and thrombolysis with recombinant tissue plasminogen activator for acute ischemic stroke. This case series illustrates the role of idarucizumab in improving patient safety in rare emergency situations requiring rapid reversal of the anticoagulant effect of dabigatran, while highlighting the importance of information and education about the availability and appropriate use of this recently approved specific reversal agent.

Thrombosis in thrombocythemic Ph- myeloproliferations is associated with higher platelet count prior to the event: results of analyses of prothrombotic risk factors from a registry of patients treated with anagrelide.

Controversies still exist regarding definition of the thrombotic risks in Ph- (BCR/ABL1-) myeloproliferative disorders with thrombocythemia (MPD-T). Platelet counts at diagnosis are currently not taken as a risk factor of thrombosis. In our cohort of 1179 patients with MPD-T, prospectively registered for anagrelide treatment, we found that the median platelet count prior to the thrombotic event was significantly higher than at time points without any ensuing thrombosis (453 vs. 400 × 10(9)/L, P < 0.001), albeit higher platelet counts at diagnosis tended to be connected with fewer thrombotic events (in contrast to WBC counts at diagnosis). The JAK2(V617F) mutation predicted both arterial and venous events, while age >65 yr, hypertension, diabetes mellitus, smoking, elevated triglyceride and homocysteine levels predicted arterial events only. For venous events, the specific thrombophilic risk factors (factor V 'Leiden' and others), antiphospholipid antibodies, and elevated factor VIII levels played a major role. During anagrelide treatment (± aspirin), we documented a decrease in both venous (6.7-fold) and arterial events (1.8-fold), while bleeding (mostly minor events) increased twofold compared to history. Our results suggest that keeping platelet counts at low levels may be a meaningful therapeutic measure to prevent thrombosis, although their counts at diagnosis lack any prognostic value.

Diagnosis, etiology and management of the Budd-Chiari Syndrome: a bloodcoagulation and hepatological study on the course of the disease treated with TIPS.

BACKGROUND: Budd-Chiari Syndrome (BCS) is characterized by obstruction of blood flow in hepatic veins. The aim of the study was to analyze diagnosis, etiology and management of BCS. METHODS: We analyzed 44 patients (32 females, 12 males, the mean age <35y of age) treated with TIPS. Ascites was found in 35 patients as the most frequent symptom. The median of total follow-up was 52 months. Non-covered (bare) or covered stent was inserted to all patients. Diagnosis of myeloproliferative neoplasm (MPN) was based on WHO criteria. Other inherited or acquired thrombophilia were assessed as well. Therapy of BCS was with regard to the etiology. RESULTS: The etiology of BCS was identified in 38 cases. Ph- MPN was found as the most common risk factor (50%, N.=22), especially polycythemia vera. JAK2V617F mutation was detected in the most of 22 MPN cases (82.5%). The second most common etiologic factor was inherited thrombophilia (18%, N.=8). In the non-covered (bare) stent group, a primary patency rates 52.9% in 1 year and 20% in 5 years after TIPS (Portasystemic Shunt, Transjugular Intrahepatic) creation. In the covered stent group the 1-year and 5-year primary patency rates were was 80% and 33.3% respectively. The average 5-year re-intervention rate per patient was 1.65 procedures in the bare stent group and 0.67 in the covered stent group. Re-interventions were more frequent in MPN patients. All patients were anticoagulated with heparin at the beginning, switched to vitamin K antagonist. On top of TIPS, anticoagulant and a vigorous therapy of underlying disorder are necessary. CONCLUSION: BCS is a serious and life-threatening disorder in MPD is a major cause of morbidity and mortality. Therapy requires a multidisciplinary approach. Insertion of TIPS dedicated covered stent is a very effective treatment in cases resistant to conservative approach with lower dysfunction rate and the number of re-interventions.

[Ph-negative myeloproliferative diseases with thrombocythemia in the context of Thromboreductin® treatment, data from registry 2013]. / Ph-negativní myeloproliferativní onemocnení s trombocytemií ve svetle úcinku lécby prípravkem Thromboreductin® v datech registru ke konci roku 2013.

Czech Working Group for Ph-negative Myeloproliferative diseases (CZEMP) recommends anagrelid (Thromboreductin®) for the treatment of Ph-negative chronic myeloproliferative disease (MPO) with thrombocythemia accompanying. To evaluate the efficacy of this treatment, the patient registry with essential thrombocythemia and/or thrombocytosis accompanying other Ph-negative myeloproliferative diseases was established. The beginnings of data collection go back to 2001, registry itself is maintained from 2005 and the aim is to archive the medical records with detailed physical and laboratory examination, safety patient profile included. The longest follow-up monitors 150 months period. Registry database contained 1,325 patients in the end of 2013, with an annual increase of anagrelid therapy as a drug of first choice in accordance with CZEMP guidelines approved by the Czech Society of Hematology of Czech Medical Association of J. E. Purkyne. Indication criteria contribute to this trend as anagrelid is the first choice agent in 65 years old patients, instead previous 60 years of age. Often, we can observe the combined treatment, especially, in older patients and in patients with primary myelofibrosis and polycythemia vera. There have been founded 543 thrombotic events in 413 patients and 63 bleeding events in 58 patients of study group by the end of 2013. During treatment, thrombosis was diagnosed 225 times in 171 patients and bleeding was observed 139 times in 104 patients. The therapeutic response is achieved after 3 months in 77% and after 6 months in 83% of subjects, but after 12 months, the treatment still fails in 12,5% of patients. It might be caused by slow titration of Thromboreductin®. One of the most important indicators of treatment success is the effect on clinical symptoms presentation, especially the occurrence of thrombotic events. The proof of a good treatment efficacy is demonstrated by 1.8 fold decrease in arterial thrombosis, more than 1.5 fold decrease in microvascular thrombosis and even 6.2 fold decrease in venous thromboembolism events. Bleeding is observed in about double more patients in comparison to the period before inclusion in the systematic monitoring, but the bleedings are clinically insignificant.Key words: anagrelid (Thromboreductin®) - Ph-myeloproliferative diseases - registry - thrombosis.

Preparing compound heterozygous reference material using gene synthesis technology: a model of thrombophilic mutations.

AIMS: The aim of our study is to present a novel approach for preparing a compound heterozygous reference material (hetRM) using gene synthesis technology with inverted insertion of wild-type and mutant fragments into a single cloning vector. Factor II (G20210A) and Factor V (G1691A Leiden) gene mutations were used as an experimental model. METHODS: During the gene synthesis, DNA fragments were aligned in the following order: G1691 FV wild-type forward strain, G20210 FII wild-type forward strain, 1691A FV mutant reverse strain, 20210A FII mutant reverse strain. The complete chain was inserted into a pIDT SMART cloning vector and amplified in an E. coli competent strain. For assessing hetRM characteristics and commutability, we used real-time PCR with subsequent melting curve analysis, real-time PCR with hydrolysis probes, allele-specific amplification, reverse hybridization, and dideoxynucleotide DNA sequencing. RESULT: All five methods yielded concordant results of DNA analysis of the hetRM. Differences in real-time PCR cycle threshold values after six-months of storage at -80 °C were not statistically significant from those obtained from freshly prepared hetRM aliquots, which is a good indication of their stability. CONCLUSION: By applying the procedures of gene synthesis and cloning technology, we prepared and verified a model genetic reference material for FII G20210A and FV G1691A testing with a compound heterozygous genotype. The hetRM was stable, commutable, and available in large quantities and in a wide concentration range.