Age-related variation in coagulation factors in non-valvular atrial fibrillation patients receiving direct oral anticoagulants.

Age is a significant risk factor for ischemic stroke. However, the influence of aging on coagulation parameters in non-valvular atrial fibrillation (NVAF) patients treated with direct oral anticoagulants (DOACs) remains unclear. A total of 775 samples were collected from 224 NVAF patients receiving apixaban, edoxaban or rivaroxaban. The samples were categorized into three age groups: (i) ≤ 64 years, (ii) 65-74 years, and (iii) ≥ 75 years (apixaban: N = 48, 108, 119; edoxaban: N = 63, 68, 126; rivaroxaban: N = 115, 90, 38, respectively). Coagulation parameters including fibrinogen (Fbg), factor II, factor V, factor VII, factor X, and D-dimer, were compared between the three age groups for each drug. The slopes in the correlation between drug concentrations and modified diluted prothrombin time (mdPT) were also assessed. Fbg and factor V increased with age, while factor II and factor X decreased. Factor VII and D-dimer showed no significant differences across age categories. The slope in response to drug concentrations was similar between the age groups. In NVAF patients treated with apixaban, edoxaban and rivaroxaban, some coagulation parameters exhibited age-related variation. However, the response of mdPT to drug concentration was consistent across age categories.

Investigation of real-world heparin resistance and anticoagulation management prior to cardiopulmonary bypass: report from a nationwide survey by the Japanese Association for Thoracic Surgery heparin resistance working group.

OBJECTIVE: Heparin resistance is often encountered during cardiopulmonary bypass. Heparin dose and activated clotting time target values for the initiation of cardiopulmonary bypass are not yet universally standardized; further no consensus exists on the management of heparin resistance. This study aimed to investigate the current real-world practice on heparin management and anticoagulant treatment for heparin resistance in Japan. METHODS: A questionnaire survey was conducted at medical institutions nationwide with which The Japanese Society of Extra-Corporeal Technology in Medicine members are affiliated, targeting surgical cases with cardiopulmonary bypass performed from January 2019 through December 2019. RESULTS: Among 69% (230/332) of the participating institutions, the criterion for heparin resistance was defined as "the target activated clotting time value not reached even with an additional dose of heparin administration". Cases of heparin resistance were reported in 89.8% (202/225) of the responded institutions. Of note, 75% (106/141) of the responded institutions reported heparin resistance associated with antithrombin activity ≥ 80%. Antithrombin concentrate was used in 38.4% (238/619 responses) or third dose of heparin in 37.8% (234/619 responses) for advanced heparin resistance treatment. Antithrombin concentrate was found to be effective in resolving heparin resistance in patients having normal, as well as lower antithrombin activity. CONCLUSION: Heparin resistance has occurred in many cardiovascular centers, even among patients with normal antithrombin activities. Interestingly, the administration of antithrombin concentrate resolved heparin resistance, regardless of the baseline antithrombin activity value.

[Suspected autoimmune coagulation factor IX deficiency difficult to diagnose due to concurrent lupus anticoagulant].

A woman in her 70s who was undergoing treatment for an overlap syndrome of autoimmune hepatitis and primary biliary cirrhosis developed persistent genital bleeding. Coagulation tests revealed a longer activated partial thromboplastin time, a 7% decrease in coagulation factor IX activity (FIX:C) and a FIX inhibitor (of 3 BU/ml). Lupus anticoagulant (LA), anticardiolipin antibody, and anti-ß2 glycoprotein I antibody were positive, and the activated partial thromboplastin time cross-mixing test suggested the presence of LA. Additionally, all intrinsic coagulation factors decreased, but activity of all factors except FIX showed dilution linearity, which suggested a false decrease in activity due to LA. Although definitive diagnosis was difficult due to concurrent LA, this case was strongly suspected to be autoimmune coagulation FIX deficiency complicated by LA. Bypass therapy was not performed because the patient had no anemia and was positive for LA, and immunosuppressive therapy with prednisolone was initiated immediately. Eleven weeks after diagnosis, FIX:C was 41% and zFIX inhibitor was less than 1 BU/ml, leading to remission.

Evaluation of newly-developed modified diluted prothrombin time reagent in non-valvular atrial fibrillation patients with direct oral anticoagulants: A comparative study with conventional reagents.

INTRODUCTION: A single assay to assess the effect of the direct FXa inhibitor is needed clinically because prothrombin (PT) assay is not yet sensitive enough for accurate evaluation. We developed modified diluted prothrombin time (mdPT) assay showing a high reactivity to direct FXa inhibitors based on prothrombin time (PT) reagent. The purpose of this study was to evaluate the reactivity of mdPT to direct FXa inhibitors comparing to that of commercial PT reagents and diluted prothrombin time (dPT). METHODS: The correlation and slopes of mdPT against the drug concentrations by anti-Xa assay were compared to those of the four commercial reagents of PT or dPT in 275, 257, and 243 clinical samples collected from non-valvular atrial fibrillation (NVAF) patients who are prescribed apixaban, edoxaban or rivaroxaban for stroke prevention, respectively. RESULTS: The correlation coefficient (95% confidence interval) of mdPT against apixaban, edoxaban, and rivaroxaban was 0.818 (0.775-0.854), 0.914 (0.892-0.932), and 0.814 (0.766-0.852), respectively. The slope (95% confidence interval) of mdPT for apixaban, edoxaban, and rivaroxaban was 0.0068 (0.0063-0.0075), 0.0076 (0.0072-0.0080), and 0.0072 (0.0065-0.0078), respectively, which were higher than that of four commercial PT and dPT reagents, ranging within 0.0006-0.0023, 0.0017-0.0038, and 0.0016-0.0057 (all, p < 0.001), respectively. CONCLUSION: Compared with other PT and dPT reagents, mdPT reagent showed sharper slope to all direct FXa inhibitors, and higher correlation to apixaban and comparable correlation to edoxaban and rivaroxaban. This new reagent is expected to be a coagulation screening assay having a consistently high response to any types of direct FXa inhibitors.

Bleeding After Gastric Endoscopic Submucosal Dissection Focused on Management of Xa Inhibitors.

Purpose: The use of direct oral Xa inhibitors (DXaIs) to prevent venothrombotic events is increasing. However, gastrointestinal bleeding, including that related to endoscopic resection, is a concern. In this study, we evaluated bleeding and coagulation times during the perioperative period of gastric endoscopic submucosal dissection (ESD). Materials and Methods: Patients who consecutively underwent gastric ESD from August 2016 to December 2018 were analyzed. Bleeding rates were compared among the 3 groups (antiplatelet, DXaIs, and control). DXaI administration was discontinued on the day of the procedure. Prothrombin time (PT), activated partial thromboplastin time, and the ratio of inhibited thrombin generation (RITG), which was based on dilute PT, were determined before and after ESD. Results: During the study period, 265 gastric ESDs were performed in 239 patients, where 23 and 50 patients received DXaIs and antiplatelets, respectively. Delayed bleeding occurred in 17 patients (7.4%) and 21 lesions (7.1%). The bleeding rate in the DXaI group was significantly higher than that in the other groups (30.4%, P<0.01), and the adjusted odds ratio of bleeding was 5.7 (95% confidence interval, 1.4-23.7; P=0.016). In patients using DXaIs, there was a significant (P=0.046) difference in the median RITG between bleeding cases (18.6%) and non-bleeding cases (3.8%). Conclusions: A one-day cessation of DXaIs was related to a high incidence of bleeding after gastric ESD, and monitoring of residual coagulation activity at trough levels might enable the predicted risk of delayed bleeding in patients using DXaIs.

Prevention of venous thromboembolism in pregnant women with congenital antithrombin deficiency: a retrospective study of a candidate protocol.

BACKGROUND: The best thromboprophylaxis for pregnant women with congenital antithrombin deficiency (CAD) is controversial. OBJECTIVE: To clarify the effectiveness of a protocol for venous thromboembolism (VTE) prevention in pregnant women with CAD. METHODS: Women at high risk of VTE were administered antithrombin concentrate and heparin after conception, whereas those at low risk of VTE were administered heparin alone until delivery. All women received antithrombin concentrate at delivery except for one who was diagnosed with CAD. RESULTS: Ten women had CAD, including one in the high-risk group and nine in the low-risk group. No women had VTE at delivery as per the protocol for VTE prevention. Almost all women had increased antithrombin activity before delivery followed by maintenance at ≥ 70% due to antithrombin concentrate administration. VTE prophylaxis during and after delivery was successful in all women with CAD. However, one woman in the low-risk group did not receive heparin and developed VTE induced by severe hyperemesis at 9 gestational weeks, before the diagnosis of CAD. Women in the high-risk group received antithrombin concentrate after delivery but had increased D-dimer levels at postpartum. CONCLUSIONS: Our protocol to prevent VTE in pregnant women with CAD is safe and effective.

Determination of diagnostic threshold in harmonization and comparison of clinical utility for five major antiphospholipid antibody assays used in Japan.

BACKGROUND: Anticardiolipin antibodies (aCL) and anti-ß2 -glycoprotein I antibodies (aß2 GPI) are essential in diagnosing antiphospholipid syndrome (APS) according to the international APS guideline. Five commercial assays for aCL and aß2 GPI are available in Japan, but their test results are quite discordant. For harmonization of diagnosing APS, upper reference limit (URL) and diagnostic accuracy of each assay were evaluated and compared by testing common sets of specimens across all assays. METHODS: We evaluated two manual and three automated assays for aCL and aß2 GPI of IgG- and IgM classes. 99%URL (the upper limit of reference interval: as per guideline) together with 97.5%URL were determined by testing sera from 198 to 400 well-defined healthy subjects. Both URLs were compared with the cutoff values, which were determined based on ROC analysis by testing 50 each of plasma specimens from patients with/without APS. Diagnostic accuracy was evaluated as area under curve (AUC) of the ROC curve. RESULTS: A variable degree of discrepancy between URLs and the cutoff values was observed, which was partly attributable to between-year assay variability. 97.5%URLs were set lower and closer to the cutoff values than 99%URLs. For all assays, diagnostic accuracies of both aß2 GPI-IgG and aCL-IgG were generally high (AUC: 0.84-0.93); whereas those for IgM-class assays were low (AUC: 0.57-0.67), implicating its utility is limited to rare IgG negative APS cases. CONCLUSION: To ensure harmonized APS diagnosis, the diagnostic thresholds of the five assays were evaluated by common procedures. Contrary to the guideline, 97.5%URL is rather recommended for diagnosing APS, which showed a closer match to the cutoff value.

Measurement of coagulation factor antibody levels is useful for diagnosis and determining therapeutic efficacy in hemorrhagic patients with autoantibodies to coagulation factor VIII and factor V: results from a single center in Japan.

Coagulation factor inhibitors (CFIs) sometimes cause fatal bleeding conditions. Determination of an inhibitor titer (INH-titer) using the Bethesda method is essential for diagnosing diseases associated with CFIs and examining the effects of immunosuppressive therapy. We reviewed 17 cases with CFIs (acquired hemophilia A, n = 11; FV inhibitor, n = 6) to examine the usefulness of determining quantities of an autoantibody to a coagulation factor (CF-IgG) by ELISA for diagnosis and therapeutic efficacy, as compared with INH-titer. One patient with an INH-titer and no evidence of CF-IgG was lupus anticoagulant (LA)-positive, and thus the positive INH-titer may have been a false positive caused by LA. Although INH-titer alone was insufficient to correctly identify patients with CFI, determination of CF-IgG appeared to be useful. In addition, even after INH-titer disappearance, hemorrhagic conditions recurred when CF-IgG was detected. These findings suggest that the presence of a clearance antibody against the coagulation factor might reduce the activity of that coagulation factor even after disappearance of the corresponding neutralizing antibody. Although the diagnosis and therapeutic efficacy can also be determined by INH-titer disappearance and improvement of corresponding coagulation factor activity, determination of CF-IgG by ELISA can improve the accuracy of these assessments.

[Antiphospholipid syndrome: diagnosis and management].

Antiphospholipid syndrome (APS) is an acquired thrombophilia associated with autoimmunity and is a syndrome that should always be considered when examining patients with thrombosis and pregnancy complications. As per the Sydney criteria, a diagnosis can be established with at least one clinical finding, such as arteriovenous thrombosis and the presence of at least one of the antiphospholipid antibodies (aPL), such as anticardiolipin antibodies (aCL). Moreover, phosphatidylserine-dependent anti-prothrombin antibody and anti-b2GPI-domain 1 antibody are correlated with APS manifestations and enable APS diagnosis. In addition to the inhibition of physiological coagulation inhibitors, such as protein C, the activation of vascular endothelial cells and complement activation by aPL is presumed to be the thrombus mechanism of APS. The mainstay of treatment is anticoagulant therapy with warfarin. For treating APS that has developed by arterial thrombosis, antiplatelet agent alone or in combination with warfarin is considered. In the triple positive aPL (aCL, anti-b2GPI antibodies, and lupus anticoagulant are detected at the same time) cases, sufficient anticoagulant therapy is required. Direct oral anticoagulants are not recommended in cases of triple positive aPL or arterial thrombosis. For patients with catastrophic APS, heparin therapy, plasmapheresis, and steroid pulse therapy are recommended.

International Council for Standardization in Haematology (ICSH) laboratory guidance for the verification of haemostasis analyser-reagent test systems. Part 2: Specialist tests and calibrated assays.

Before a new method is used for clinical testing, it is essential that it is evaluated for suitability for its intended purpose. This document gives guidance for the performance, verification and implementation processes required by regulatory and accreditation bodies. It covers the planning and verification of specialist haemostatic tests, including factor assays, D-dimers, direct anticoagulants and thrombophilia testing.

Survey of actual conditions of erythema marginatum as a prodromal symptom in Japanese patients with hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a rare but life-threatening condition. HAE types I and II (HAE-1/2) result from C1-inhibitor (C1-INH) deficiency. However, recent genetic analysis has established a new type of HAE with normal C1-INH (HAEnC1-INH). The mutations of factor XII, plasminogen, angiopoietin 1, and kininogen 1 genes may be the cause of HAEnC1-INH. Nevertheless, other causative molecules (HAE-unknown) may be involved. The Japanese therapeutic environment for HAE has been improving owing to the self-subcutaneous injection of icatibant, which was approved for the treatment of acute attack and enables early therapy. Erythema marginatum (EM) is a visible prodromal symptom which occasionally occurs prior to an angioedema attack; hence, recognizing the risk of an acute attack is important for early treatment. However, the detailed characteristics of EM remain unclear. In this study, we first investigated the clinical manifestations of EM in Japanese patients with HAE. METHODS: A 20-point survey was developed and distributed to 40 physicians to gather clinical data on EM from patients with HAE. RESULTS: Data on 68 patients with HAE (58 patients with HAE-1/2 and 10 patients with HAE-unknown) were collected. Of the patients with HAE-1/2, 53.4% experienced EM, whereas 43.1% did not. The forearm was the most frequent area of EM (64.5%), followed by the abdomen (29.0%) and upper arm and precordium (19.3%). Of the HAE-1/2 patients with EM, 41.9% always had angioedema following EM, while 29.0% always had colocalization of EM with angioedema. Moreover, 3.2% showed a correlation between the awareness of EM and severity of an angioedema attack. In 60.9% of HAE-1/2 patients with EM, the interval between the awareness of EM and appearance of angioedema was <3 h. Of the patients with HAE-unknown, 30.0% also experienced EM. CONCLUSION: We confirmed that more than one-half of Japanese patients with HAE-1/2 and one-third of those with HAE-unknown develop EM as the prodromal symptom of an angioedema attack. Physicians should communicate the significance of EM to patients with HAE to prepare them for possible imminent attacks.

Lupus anticoagulant hypoprothrombinemia syndrome associated with bilateral adrenal haemorrhage in a child: early diagnosis and intervention.

BACKGROUND: Lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS) is characterized by bleeding and thrombosis in patients with autoimmune diseases or infections. Paediatric LAHPS exhibits various degrees of bleeding, ranging from mild to severe; however, adrenal haemorrhage due to LAHPS and its long-term clinical course have not been sufficiently described. CASE PRESENTATION: A 9-year-old boy presented with prolonged abdominal pain and abnormal coagulation screening tests. The laboratory tests showed prolonged activated partial thromboplastin time and subsequently revealed the presence of lupus anticoagulant, anti-nuclear antibodies, and hypoprothrombinemia, leading to diagnosis of LAHPS. An enhanced computed tomogram demonstrated nodular lesions in the adrenal glands bilaterally, suggestive of adrenal haemorrhage. Laboratory and clinical manifestations exhibited life-threatening adrenal insufficiency that required hydrocortisone administration. The patient developed systemic lupus erythematosus, diagnosed 12 months later. CONCLUSIONS: This patient with LAHPS developed rare adrenal failure due to adrenal haemorrhage, a life-threatening event that should be recognized and treated early. In our case, renal dysfunction was also observed when systemic lupus erythematosus was diagnosed 1 year after LAHPS. Our case emphasizes that early recognition of adrenal failure and careful long-term observation is required in patients with autoantibodies.

Comparative evaluation of reagents for measuring protein S activity: possibility of harmonization.

Patients with congenital protein S (PS) deficiency show a hereditary predisposition for thrombosis, and PS deficiency is prevalent among Japanese populations. Diagnosis is based on symptoms of thrombosis and reduced PS activity. Three reagents that use different measurement principles for determining PS activity are available in Japan. This study aimed to confirm the possibility of harmonization of these three reagents to establish a universal standard for PS activity in Japanese populations. Commercial normal plasma and plasma samples obtained from healthy individuals and healthy pregnant women were tested at three facilities using three reagents for measuring PS: STA-Staclot Protein S (STA-PS), HemosIL Protein S (Clotting) (IL-PS), and a total PS assay (SNT-PS). The within-run precision of each reagent was good, as each had a coefficient of variation of ≤ 3.8%. The dilution linearity for each reagent was also good. The correlation coefficient was 0.94 for STA-PS vs. IL-PS, 0.93 for SNT-PS vs. STA-PS, and 0.90 for SNT-PS vs. IL-PS, indicating a good correlation. Although the three reagents available in Japan for measuring PS activity use different measurement methods, each showed good performance, and large differences were not observed between the obtained values. Harmonization among them appears possible.

Characterization of fibrin/fibrinogen degradation products reagents and their utility in critical care patients with enhanced fibrinolysis.

INTRODUCTION: Fibrin/fibrinogen degradation products (FDP) values reflect coagulation and fibrinolysis status, and FDP levels are helpful for diagnosis and classification of disseminated intravascular coagulation (DIC). FDP measurement has always played a key role in diagnosing DIC, a phenomenon that has recently gained renewed attention because of its occurrence in coronavirus disease 2019 (COVID-19) patients. Although the evaluation of FDP is crucial for the management of critical care, the variability among FDP reagents is unclear. In this study, we aimed to compare LIASAUTO P-FDP with three FDP reagents and investigate their characteristics. METHODS: In total, 172 plasmas samples were used in the correlation. The sample data were divided into three groups including negative, no and positive discrepancy based on the discrepancy percentages calculated from each correlation between LIASAUTO P-FDP and other three reagents. D-dimer, plasmin-α2 plasmin inhibitor complex (PIC), fibrin monomer complex (FMC), fibrinogen (Fbg) and Plasmin-α2 Plasmin Inhibitor (α2 PI) were measured and included in data analysis. RESULTS: The positive discrepancy groups showed higher D-dimer, PIC and FMC values than the negative discrepancy groups. The data indicated that LIASAUTO P-FDP had higher reactivity to D-dimer than other reagents and the values were elevated in the fibrinolysis-enhanced samples with various FDP fragments. CONCLUSION: LIASAUTO P-FDP displayed the reactivity towards various fibrin/fibrinogen degradation products, and it might be useful for DIC diagnosis because the fibrinolytic status differed in the DIC types and stages.

International Council for Standardization in Haematology (ICSH) laboratory guidance for the evaluation of haemostasis analyser-reagent test systems. Part 1: Instrument-specific issues and commonly used coagulation screening tests.

Before a new method is used for clinical testing, it is essential that it is evaluated for suitability for its intended purpose. This document gives guidance for the performance of verification, validation and implementation processes required by regulatory and accreditation bodies. It covers the planning and execution of an evaluation of the commonly performed screening tests (prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen assay), and instrument-specific issues. Advice on selecting an appropriate haemostasis analyser, planning the evaluation, and assessing the reference, interval, precision, accuracy, and comparability of a haemostasis test system are also given. A second companion document will cover specialist haemostasis testing.

Expert consensus regarding standardization of sample preparation for clotting time assays.

Accurate clotting time assay results are vital, as the test is employed to indicate the amount of oral anticoagulant to be prescribed, while it is also used for screening the hemorrhagic and thrombotic diseases. The procedure chosen for preparation of a patient blood sample including centrifugation can contribute to significant differences in the results obtained. Thus, for the purpose of proposing a standardized method to appropriately prepare blood samples prior to assay, the Japanese Society of Laboratory Hematology organized the Working Group for Standardization of Sample Preparation for Clotting Time Assays (WG). Following reviews of previously announced guidelines and original experimental results, consensus was obtained by the WG, with the main findings as follows. (1) The recommended anticoagulant in the blood collection tube is sodium citrate solution at 0.105-0.109 M (3.13-3.2%). (2) Whole blood samples should be stored at room temperature (18-25 ˚C) within 1 h of collection from the patient. (3) For plasma preparation, centrifugation at 1500 × g should be performed for at least 15 min or at 2000 × g for at least 10 min at room temperature. (4) After the plasma sample is prepared, it should be stored at room temperature and assayed within 4 h.

Microparticles and Nucleosomes Are Released From Parenchymal Cells Destroyed After Injury in a Rat Model of Blunt Trauma.

We investigated the relationships between circulating procoagulants and trauma severity, including cellular destruction, and the effects of thrombin generation on procoagulants in a rat blunt trauma model. The rats were subjected to tumbling blunt trauma, where they were tumbled for 0, 250, 500, or 1000 revolutions. Creatine kinase, nucleosome, and microparticle plasma levels increased gradually with trauma severity. Strong interrelationships were observed among creatine kinase, nucleosome, and microparticle levels. Time to initiation of thrombin generation shortened with increasing trauma severity. In accordance with trauma severity, prothrombin activity decreased, but the thrombin generation ratio increased. Time to initiation of thrombin generation and the thrombin generation ratio correlated with creatine kinase levels. In an in vitro study, a homogenized muscle solution, which included massive nucleosomes and microparticles, showed accelerated thrombin generation of plasma from healthy subjects. Procoagulants, such as microparticles and nucleosomes, are released from destroyed parenchymal cells immediately after external traumatic force, activating the coagulation cascade. The procoagulants shorten the time to initiation of thrombin generation. Furthermore, although coagulation factors are consumed, the thrombin generation ratio increases.