Whole blood samples for faster real-time PCR analysis of thrombophilic mutations in SARS-CoV-2 virus positive patients.

High incidence of thrombosis and venous thromboembolism was reported in patients with COVID-19. In this study, we focused on analysis of thrombophilic mutations performed without a standard DNA extraction step. In one hundred of COVID-19 positive outpatients, real-time PCR for Leiden mutation in the FV gene and G20210A mutation in the FII gene was carried out from DNA extracts and modified whole blood samples, and their cycle threshold (Ct) values were evaluated. In the extracts, healthy homozygotes (wt/wt), heterozygotes (M/wt), and homozygous carriers of Leiden mutation (M/M) provided median Ct values of 18.5, 19.4/22.0, and 20.9. In the whole blood, Ct values were 25.3 (wt/wt), 24.8/27.2 (M/wt), and 26.9 (M/M). Median Ct values for G20210A in the extracts were 19.6 for homozygotes (wt/wt), and 19.7/20.4 for heterozygous carriers. The whole blood samples provided Ct values of 23.9 in healthy homozygotes and 26.3/27.2 in heterozygotes for G20210A mutation. No homozygous subjects for G20210A and no double heterozygotes (for Leiden and G20210A mutations) were found. Despite significant differences in the Ct values, genotyping showed complete result concordance of the DNA extracts and the whole blood samples. The integrity and amplificability of DNA molecules in the whole blood samples during 28 days of deep freezing, interrupted by four cycles of thawing, did not significantly change. In conclusion, we demonstrated a new protocol for the detection of the thrombophilic mutations via real time PCR on the modified whole blood of COVID-19 positive patients. The blood modification was reliable, easy, cheap, and saving costs and turnaround time of the whole laboratory process.

No Inhibitory Effect of Heparinized Blood on Real-Time PCR Analysis of Thrombophilic Mutations.

We compared the efficiency of real-time PCR analysis of FII (c.*97G>A, G20210A) and FV Leiden (c.1601G>A) thrombophilic mutations in the samples obtained from venous blood treated with various anti coagulant agents (EDTA, heparin, and sodium fluoride with potassium oxalate), or from clotted venous blood; one hundred samples of wild-type subjects were tested. Genomic DNA extracts and whole blood specimens modified by 90 °C heating were analysed by real-time PCR analysis; cycle threshold values were subsequently evaluated. Real-time PCR analysis for the FII gene assay performed in DNA extracts from EDTA blood samples revealed a median Ct value of 19.3. Similar Ct values were apparent in the DNA extracts obtained from the heparinized blood and sodium fluoride with potassium oxalatetreated samples: 18.5 and 18.9, respectively. Significantly higher Ct values were found in extracts from clotted blood with medians of 20.6 (tubes with inert separation gel) and 20.5 (tubes without the gel, both P < 0.001). The data on the FV real-time PCR analysis were very comparable to the FII assay. In the modified whole blood, the samples treated with heparin salts showed significantly lower Ct values (P < 0.001) in both assays when compared with the samples with EDTA, sodium fluoride with potassium oxalate, and with the samples with clotted blood. Our results indicate that real-time PCR analyses of thrombophilic mutations were not negatively influenced by the presence of heparin salts in collection tubes. Blood samples with various anticoagulants might be exchangeable for each other when DNA analysis of thrombophilic mutations is required.

Duplex ultrasound, clinical score, thrombotic risk, and D-dimer testing for evidence based diagnosis and management of deep vein thrombosis and alternative diagnoses in the primary care setting and outpatient ward.

Deep vein thrombosis (DVT) has an annual incidence of 0.2% in the urban population. First episodes of calf vein thrombosis (CVT) and proximal DVT are frequently elicited by risk factors, including varicose veins, cancer, pregnancy/postpartum, oral contraceptives below the age of 50 years, immobility or surgery. Leg pain and tenderness in the calf and popliteal fossa on physical examination may result from other conditions than DVT labeled as alternative diagnosis (AD) Congenital venous thrombophilia is present in every third first DVT, increased FVIII in every fourth first DVT, and FV Leiden/FII mutation in 40% of women on oral anticonceptive pill before reaching the menopause. Routine thrombophilia testing for FV Leiden/prothrombin mutation and FVIII as main risk factor for venous thrombosis is recommended. Primary superficial venous thrombosis (SVT) and DVT patients with a autosomal dominant family history of DVT are candidates for thrombophilia testing for congenital AT, PC and PS deficiency. The requirement for a safe diagnostic strategy of CVT and DVT should be based on an objective post-test incidence of venous thromboembolism (VTE) of less than 0.1% with a negative predictive value for exclusion of DVT of 99.9% during 3 months follow-up. Modification of the Wells score by elimination of the "minus 2 points" for AD is mandatory and will improve the diagnostic accuracy of CVT/DVT suspicion in the primary care setting and outpatient ward. The sequential use of complete DUS, ELISA D-dimer testing and modified clinical Wells' score assessment is safe and effective for the exclusion and diagnosis of CVT, DVT and AD. About 10% to 20% of patients with DVT develop overt post-thrombotic syndrome (PTS) at one year post-DVT, and both PTS and DVT recurrences further increase to about 30% during long-term follow-up. Objective risk stratification of PTS complications using DUS for recanalization and reflux and D-dimer testing will become an integral part in routine clinical practice to assess the optimal duration of wearing medical elastic stockings and anticoagulation for the prevention DVT recurrence as the best option to reduce the incidence and costs of suffering from irreversible PTS.

[The effectiveness of anagrelide treatment in patients with Ph negative myeloproliferative diseases: influence on the incidence of thrombosis in the data from the Registry of patients with essential thrombocythemia and thrombocythemia associated with other myeloproliferative diseases treated with Thromboreductin® to the end of 2012]. / Úcinnost lécby anagrelidem u nemocných s Ph negativními myeloproliferativními chorobami: ovlivnení výskytu trombózy ve výstupech Registru pacientu s dia-gnózou esenciální trombocytemie a trombocytemie provázející jiné myeloproliferativní onemocnení lécených Thromboreductinem® ke konci roku 2012.

In the Czech Republic, anagrelide (Thromboreductin®) [29] is used according to the recommendations of the Czech Working Group on Myeloproliferative Disorders (CZEMP) for treatment of thrombocythemia associated with Ph negative myeloproliferative disorders (MPDs). The patient data are collected in the Registry of patients with essential thrombocythemia (ET) and thrombocythemia associated with other MPDs treated with Thromboreductin®. At the end of 2012, the Registry contained data on 1,161 patients. Out of these, 1,159 patients with the dia-gnosis of a Ph negative MPD were evaluated. In 844 patients, precise WHO based dia-gnosis was known at start of therapy: 442 (52.4%) had ET, 108 (12.8%) had polycythaemia vera (PV) and 243 had primary myelofibrosis (PMF). The median age was 51 years at the time of diagnosis. At the time of the evaluation of the population, the median was 59 years. Every year, the proportion of patients newly treated with anagrelide as a firstline treatment in accordance with the CZEMP guidelines has been increasing. A growing proportion of patients has been treated with an additional cytoreducing drug, such as hydroxyurea and interferon. The majority of the patients received also an antiaggregant (or anticoagulant). More than a half of patients harbors the JAK2 mutation. A prompt decrease of platelet counts (as the response to Thromboreductin® treatment) was documented in most of the patients. After one year, 86.9% of patients had a full or partial response. In poorer responders, combination cytoreductive treatment was administered rather then the escalation of the Thromboreductin® dosage. There were 461 thrombotic manifestations in 363 patients and 61 haemorrhagic events in 57 patients recorded in the patients history. In the course of treatment (followup; F U), thrombosis was diagnosed only 179-times in 136 patients. There were more haemorrhagic events during F U: 109 events in 83 patients. Upon comparison of the number of events during F U to their numbers in history, we found a twofold decrease in arterial thrombosis, an almost twofold decrease in microvascular thrombosis and even a 6.6- fold decrease in venous thromboembolism events. Bleeding episodes increased 1.8-fold during F U. However, the vast majority of these hemorrhagic events were clinically insignificant. In conclusion, the treatment strategy according to the CZEMP guidelines incorporating anagrelide is highly effective in reducing the platelet counts, strongly prevents venous events, reduces arterial events, and leads to an increase of minor hemorrhages.

[Peripartal life-threatening hemorrhage - interdisciplinary consensus opinion]. / Peripartální zivot ohrozující krvácení - mezioborové konsenzuální stanovisko.

22 experts from the fields of gynecology and obstetrics, anesthesiology and resuscitation, intensive care, hematology and transfusion medicine has developed recommendations for diagnosis and procedure for life-threatening peripartum haemorrhage, which is still one of the most common causes of maternal mortality in childbirth. This guidelines, which is valid for the Czech Republic, supported by a total of 10 professional medical societies. There are based on new knowledge applicable at this time and is focused mainly on eliminating the most common causes of bleeding during delivery and prevention of haemorrhagic shock.

[Therapeutic approach to the bleeding in association with "old" and "new" anticoagulants]. / Terapeutický prístup ke krvácení v souvislosti se "starými" a "novými" antikoagulans.

There are a several new anticoagulants emerging in the clinical medicine in the last a few years. Rivaroxaban, an oral direct F Xa inhibitor has been already approved in more than 100 countries worldwide, and Dabigatran etexilate, an oral direct thrombin inhibitor is also routinely used in thromboprophylaxis of venous thromboembolism. These agents possess many of the characteristic of the "ideal" anticoagulant but a lack of specific antidotes may be a potentional disadvantage in the therapy of bleeding complications. We assume, this lack of antidote does not represent a significant problem. In comparison with established anticoagulants (unfractionated heparin - UFH, low molecular weight heparin - LMWH, vitamin K antagonists - VKAs), only UFH has a specific and affective reversal agent. LMWH does not have the full proven antidote and the reversal agents recommended for the effects of VKAs have several safety and practical limitations. In the article we propose the basic principles of the management of bleeding complications in association with the newer anticoagulans. To manage bleeding episodes, we recommend delaying the next dose or discontinuing of treatment, mechanical compression, surgical interventions and the administration of the blood products. In the case of life-threatening bleeding, Novosen - rF VII is indicated.

Significance and causes of abnormal preoperative coagulation test results in children.

To prevent bleeding related to adenoidectomy and tonsillectomy, coagulation screening tests were, until recently, performed routinely in the Czech Republic for all paediatric patients. The aim of this study was to evaluate benefit of preoperative coagulation screening tests in children. We retrospectively analysed laboratory and clinical data of children referred for abnormal preoperative coagulation test results (aPTT, PT) to the outpatient haematology clinic. A total of 274 paediatric patients were retrospectively evaluated due to abnormal preoperative coagulation tests results. In 140 of 274 patients (51.1%), coagulation tests were normal on repeated testing in a specialized haematology clinic. Ten patients had decreased factor XII. Five patients had a suspected bleeding disorder which was confirmed in two of them. One patient had low levels of von Willebrand factor, and one patient had mild factor VII deficiency. Both these patients had positive personal and/or family history of bleeding. Each case history was taken individually, without use of standardized questionnaires. Bleeding complications were not observed, and coagulation factor replacement was not needed perioperatively in our cohort. The majority of abnormal findings in aPTT and PT appeared only transiently. All the bleeding disorders found in our cohort of patients were mild in nature. Our findings provide supportive evidence for the current national Czech recommendation: laboratory coagulation screening should be performed only in patients with positive family and/or personal bleeding history.

[Diagnosis and treatment of BCR/ABL-negative myeloproliferative diseases--principles and rationale of CZEMP recommendations]. / Diagnostika a lécba BCR/ABL-negativních myeloproliferativních onemocnení--principy a výchdiska doporuceni CZEMP.

In 2009, the recommendations of the Czech Collaborative Group for Ph- Myeloproliferative Diseases (CZEMP) for diagnosis and treatment of BCR/ABL-negative myeloproliferative diseases (MPD), i.e., essential thrombocythemia (ET), polycythaemia vera (PV) and primary myelofibrosis (PMF) were updated and extended. The present article gives the rationale of the recommendations in full detail. The CZEMP diagnostic criteria for ET and PMF are based on histopathological (HP) findings, which must unconditionally be in line with the given clinical and laboratory characteristics of ET or of a certain stage of PMF, respectively. The platelet count is not decisive for diagnosis. In cases lacking an adequately taken and read HP finding, the Polycythemia Vera Study Group (PVSG) criteria are recommended. The diagnosis of typical PV is based on demonstration of the V617F mutation of the JAK2 gene along with a significant increase of red cell parameters. If these are close to borderline, the demonstration of increased total red cell mass (RCM) is required. In atypical cases lacking polyglobulia or elevated RCM, the HP picture of PV (in accordance with WHO description) plus JAK2 V617F mutation is satisfactory for diagnosis, or, in cases lacking JAK2 V617F mutation, the HP picture of PV along with polyglobulia (or increased RCM) is sufficient. The treatment principles of ET and other MPDs with thrombocythemia (MPD-T; i.e., the early stages of PMF and PV) are identical. The patients are stratified by their thrombotic risk (preceding thrombosis, another thrombophilic state, jAK2 mutation), presence of disease symptoms (mainly microcirculatory), platelet count and age. Only patients up to 65 years lacking the above mentioned risks with a platelet count < 1000 x 10(9)/l are considered as low-risk and do not demand cytoreducing therapy. The others are high-risk ones and have an indication for thromboreduction. In patients older than 65 years, the potentially leukemogenic drug hydroxyurea (HU) may be used. In the younger ones, the choice is between anagrelide (ANG) or interferon-alpha (IFN). In high-risk patients, the treatment goal is to maintain platelet counts below 400, and in low-risk ones, below 600 x 10(9)/l. In PV, polycythemia itself is another thrombotic risk factor. The condition is treated by bloodletting or erythrocytaphereses. If hematocrit levels < or =45 are not achieved, cytoreductive therapy using HU in patients over 65 years, or IFN in younger individuals is required. All patients with thrombocythemia in PV are high-risk and have an indication for cytoreduction. Acetylsalicylic acid is given to all patients with MPD-T with platelets < 1000 x 10(9)/l (at higher counts, hemorrhage is imminent), and to all individuals with PV, unless contraindication is present. In case of platelet count normalization, it may be withdrawn in cases of low-risk ET or PMF, not in JAK2+ PV. The treatment of advanced stages of PMF is symptomatic, with substitution of blood derivatives being the basis. The only curative treatment is allogeneic stem cell transplantation, which should not be indicated too early seeing to its risks, but also not too late--we must not allow transition into acute leukemia, which is heralded by blasts in the blood picture. The indication is the presence of any of the following criteria: values of hemoglobin < 10 g/dl, WBC < 4 x 10(9)/l and platelets < 100 x 10(9)/l, any percentage of blasts or > or = 10% immature granulocytes in the differential picture, >1 erythroblast per 100 cells--all at repeated examinations within at least a 2-month interval, and in addition, rapid progression of hepato-/splenomegaly, presence of general symptoms of the disease, portal hypertension and extensive swellings.

[Essential thrombocythaemia and other myeloproliferative disorders with thrombocythaemia treated with Thromboreductin. A report from the database of register for the 1st quarter of 2010]. / Esenciální trombocytemie a jiné myeloproliferate s trombocytemií lécené Thromboreductinem. Výstupy z databáze Registru k 1. ctvrtletí roku 2010.

In the Czech Republic, anagrelid is used according to the recommendations of the Czech Working Group on Myeloproliferative Disorders for treatment ofthrombocythaemias associated with chronic myeloproliferative disorders--mainly essential thrombocythaemia and, regularly, reports are being presented from the Register of Patients Treated with Thromboreductin, most recently last year (Vnitr Lék 2009; 55: I-XII). The Register commenced in 2005 and from then it aims to determine detailed clinical and laboratory profiles of the patients. The structure of the Register has changed significantly in the course of its existence, reflecting the reports from each of the analyses conducted so far. Also, the data entry in the database improves every year and it reaches 97% on some of the items. The longest evaluation period in some of the patients is 108 months. By April 2010, the Register database contained data on 717 patients. Of these, 672 patients with the diagnosis of a Ph-negative chronic myeloproliferative disorder were evaluated. This year's analysis included the patients with essential thrombocythaemia, polycythaemia vera and primary myelofibrosis only. The analysis included 418 women and 254 men with median age of50 years. Unlike the first years, 2/3 of the current sample are non pretreated patients, meaning that the patients reach the specialized centres early in their treatment. Also, patients, and the older patients in particular, are more frequently treated with combined regimens including Thromboreductin. We increasingly observe hypertension as one of the monitored risk factors preceding the disease and laboratory parameters showJAK2 mutation in more than a half of patients while some form ofthrombotic diathesis is found in the anamnesis of 7-10% of patients. Some bleeding is observed in 1-5% of the registered patients. In comparison to the previous years, this is a decrease in the prevalence of clinical symptoms prior to the disease onset; this is very likely associated with an earlier patient diagnosis within the asymptomatic phase of the disease. Therapeutically, we achieve a fast treatment response but there still are 16.3% of sufficient afterone year of treatment. Thromboreductin dose is increasing but even in this group it does not exceeds the mean of 2.38 mg per 24 hours. Complications are observed in 6.2% of patients in the first year of therapy, and ofthese, thrombotic events in about 2.5% and (small) bleeding complications in 4% of patients. The data suggest that we still do not reach treatment response in a certain proportion of patients after a year of their therapy. Even though the care results from the analysed data improve every year, the Register helps to uncover some issues that still remain, such as treatment intensification and other treatment modifications.

[Surgical interventions in patients with myeloproliferative disease--prophylaxis and treatment of haemostatic disorders]. / Intervencní zákroky u jedincu s myeloproliferativními chorobami--prevence a e lécba poruch hemostázy.

Despite many decades of laboratory and clinical research of pathogenesis bleeding and thrombosis and the search for the identification of the risk factors, the accomplished results are unsatisfactory in clinical praxis. In our article, we discuss incidence, pathogenesis, clinical manifestation and the risk factors for coagulopathy in patients with myeloproliferative disease. The surgical procedures are associated with higher risk of morbidity and mortality. The aim of the article is the formulation of the recommendations concerning management of surgical interventions with the goal to reduce the risk of bleeding and thrombotic diathesis.

[The results of patients with essentials thrombocythemia and other myeloproliferation-related thrombocythemia--a report of patients treated with Thromboreductin]. / Výsledky lécby nemocných s esentiální trombocytemií a dalsími myeloproliferacemi provázenými trombocytemií--zpráva z registru pacientu lécených Thromboreductinem.

The registry of patients treated with Thromboreductin (anagrelide) in the Czech Republic contains data concerning patients that have been treated using this drug since 2004. As of June 2009, the total number of patients was 549. The current analysis focused mainly on evaluation of anagrelide dosage needed to achieve a complete response in high-risk patients: reduction in platelet count to below 400 x 10(9)/l, which was also considered as reaching the therapeutic goal. The outcomes of the registry confirm that although anagrelide (Thromboreductin) is a very effective platelet-reducing agent, the administration of which is related to a low incidence of adverse effects and complications, the therapeutic goal is not achieved in all cases and or despite a quick treatment response, the therapeutic goal is achieved more slowly.

[Venous thromboembolism prophylaxis in internal medicine]. / Profvlaxe tromboembolické nemoci ve vnitrním lékarství.

Many of medical patients are significant risk of venous thromboembolism (VTE). VTE is the most common cause of preventable death in hospitalized patients. Prophylaxis is highly effective in reducing the risk of deep vein thrombosis and pulmonary embolism and should be used in most hospitalized patients. Various strategies improve adherence to evidence-based guidelines on the use of prophylaxis, including audit and feedback, and automatic reminders. The important clinical risk factors for PE (or venous thromboembolism VTE) include advanced age, general anaesthesia, prolonged immobility or paralysis, previous VTE, cancer, duration of surgery, orthopaedic surgery of lower limb leg, hip or pelvic fracture, major trauma, stroke, obesity, varicose veins, postoperative infection and heart failure. Medical patients ad bed rest or who are sick are in moderate risk of VTE and evidence based guidelines recommended thromboprophylaxis with low molecular weight heparin, or low dose of unfractionated heparin or Fondaparinux. For all situations both guidelines recommended against the use of aspirin for VTE prevention.

[Antithrombotic prophylaxis during pregnancy]. / Antitrombotická profylaxe v tehotenství.

Coagulation disturbances with their symptoms of thromboembolic--especially venous--disease play the most important role in the incidence of maternal mortality. The attention is focused on precise diagnostic procedures and on the treatment of all the disorders generally accompanied by coagulopathies. First-degree step is prevention of above mentioned complications, and the aim of its treatment is prevention of longterm complication or consequences. Actually is VTE resolved under the condition of the recommendation of 8th ACCP Conference.

[The profylaxis of venous thromboembolism in gynecology]. / Profylaxe venóizního tromboembolizmu v gynekologii.

The venous thromboembolism is a serious disease, but it is possible to some extend eliminate it by properthromboprophylaxis. The recommendations in gynecology result from guidelines not only in surgery and the internal medicine, but also from specific gynecologic conditions. The early and frequent mobilization is recommended for minor gynecological surgery and laparoscopic procedures. For major gynecological surgery and laparoscopic procedures in whom additional VTE risk factors are present, low molecular weight heparin is indicated. In major gynecological surgery for malignancy, low molecular heparin is needed in dose at least 4,000 antiXa IU. The prolonged prophylaxis for 28 days is also suitable in this setting.

[Budd-Chiari syndrome--hematologists' part in the multidisciplinary approach]. / Budd-Chiari syndrom--uloha hematologa v multidisciplinárním prístupu.

Budd-Chiari syndrome presents a serious disease with a complex etiology and clinical manifestations, which is associated with high morbidity and mortality. An early diagnosis and therapy is mandatory due to the severity of disease and therapy contains the treatment of underlying disorder, anticoagulation therapy and therapy of liver impairment. We discuss hematologists' contribution to the diagnostic approach and therapy of this syndrome.

[What is the current treatment of patients with essential thrombocytopenia and other myeloproliferations accompanied with thrombocythemia [corrected] and what can be the predictive sign of the risk of thrombosis in such patients--a report from the registry of patients treated by Thromboreductine]. / Jak lécíme nemocné s esenciální trombocytemií a dalsími myeloproliferacemi provázenými trombocytemií a co muze být prediktivní známkou rizika trombózy u techto nemocných--zpráva z registru pacientu lécených Thromboreductinem.

The registry of patients treated with Thromboreductine (anagrelid) in the contributing centres in the Czech Republic has been updated with data on the patients receiving this medication since 2004. The original purpose of the registry was to record responses to Thromboreductine therapy and adverse drug reactions in patients with essential thrombocytopenia. However, data on additional Ph negative myeloproliferations, as well as data on cytoreductive therapies other than exclusively that using Thromboreductine has also been recorded in the course of its compilation, including data on combined regimes. At present, the database contains data on 421 patients, and valid conclusions can be drawn if the level of data filling is enhanced. Evaluation has been currently focused on the analysis of the risk of development of clinical symptoms of thrombosis and on the standards of treatment from the viewpoint of the achieved treatment response. Analyses of data from the registry corroborate the special importance of the proof of JAK2 mutation, and of the test for factor V Leiden mutation, and of protein of S for the assessment of the risk of thromboembolic complications. The output of the analysis confirms that anagrelid is a very efficient thromboreductive agent the administration of which is associated with a low incidence of non-serious adverse effects (10.9%). However, in spite of a fast response to therapy, the therapeutic goal consisting in the reduction of the platelet count below 400 (or below 600) x 10(9)/l, i.e. the complete (or partial) treatment response, is relatively slow to achieve. This is likely to be due to lack of radical corrections in the dosage of the drug for different reasons.