Determining Thrombogenicity: Using a Modified Thrombin Generation Assay to Detect the Level of Thrombotic Event Risk in Lupus Anticoagulant-Positive Patients.

The aim of this study was to determine the thrombogenicity of lupus anticoagulant (LA) antibodies using a modified thrombin generation assay (TGA) with the addition of activated protein C (APC) in a group of 85 patients with LA-positive samples. Of these, 58 patients had clinical manifestations of antiphospholipid syndrome (APS) according to the Sydney criteria classification, i.e., each patient had thrombosis or foetal loss, and 27 patients did not show any clinical manifestations of APS. A comparison of the two groups' TGA results revealed statistically significant differences (Fisher's test p = 0.0016). The group of patients exhibiting clinical manifestations of APS showed higher thrombogenicity in 56.9% of patients, while the group of patients not yet exhibiting clinical manifestations of APS showed higher thrombogenicity in 25.9% of patients. There were no significant differences in the specificity of the TGA test between the groups of patients exhibiting similar clinical manifestations. Receiver operating characteristic curve analysis showed a more significant relationship (p = 0.0060) for TGA than for LA titre (p = 0.3387). These data suggest that the determination of LA thrombogenicity with the TGA assay leads to an increased prediction of the manifestation of a thromboembolic event. Our findings appear to be particularly relevant for the prediction of thrombotic events in patients with laboratory-expressed APS and no clinical manifestations.

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.

Determination of Thrombogenicity Levels of Various Antiphospholipid Antibodies by a Modified Thrombin Generation Assay in Patients with Suspected Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a hypercoagulable state accompanied by the presence of heterogeneous antiphospholipid antibodies (aPL), which nonspecifically affect hemostasis by the presence of lupus anticoagulans (LA), anticardiolipin antibodies (aCL), antibodies against ß2-glycoprotein-I (anti-ß2GPI), but also non-criteria antibodies such as antibodies against ß2-glycoprotein-I domain I (anti-DI), anti-phosphatidylserine/prothrombin (anti-PS/PT), anti-annexin V, and many others. The main target of the antibodies is the activated protein C (APC) system, the elimination of which can manifest itself as a thrombotic complication. The aim of this study was to determine the thrombogenicity of antibodies using a modified protein C-activated thrombin generation assay (TGA) on a group of 175 samples suspected of APS. TGA was measured with/without APC and the ratio of both measurements was evaluated (as for APC resistance), where a cut-off was calculated ≤4.5 (90th percentile) using 21 patients with heterozygous factor V Leiden mutation (FV Leiden heterozygous). Our study demonstrates the well-known fact that multiple positivity of different aPLs is a more severe risk for thrombosis than single positivity. Of the single antibody positivity, LA antibodies are the most serious (p value < 0.01), followed by aCL and their subgroup anti-DI (p value < 0.05). Non-criteria antibodies anti-annexin V and anti-PT/PS has a similar frequency occurrence of thrombogenicity as LA antibodies but without statistical significance or anti-ß2GPI1 positivity. The modified TGA test can help us identify patients in all groups who are also at risk for recurrent thrombotic and pregnancy complications; thus, long-term prophylactic treatment is appropriate. For this reason, it is proving increasingly beneficial to include the determination antibodies in combination with modified TGA test.

COVID-19 in 96 Patients With Hematologic Disease: The First Single-center Experience From the Czech Republic.

BACKGROUND: Coronavirus disease 2019 (COVID-19) represents an important infectious complication associated with high mortality rates in patients with hematologic diseases. There have not been published any epidemiologic studies from Czech Republic so far. PATIENTS AND METHODS: This study is the first analysis of patients with hematologic malignancies and bone marrow failure syndromes treated at single hematology center in the Czech Republic between March 1 and December 31, 2020, in whom COVID-19 infection was confirmed. RESULTS: The sample comprised 96 patients aged 26 to 84 years (median, 66.0 years). At the time of their COVID-19 diagnosis, 75 patients (78.1%) were treated for hematologic diseases. Twenty-seven patients (28.1%) in the sample had complete remission (CR) of their hematologic disease. They were nonsignificantly more likely to have asymptomatic to moderate COVID-19 infection than those who failed to achieve CR (74.1% vs. 56.5%; P = .06). A more severe course of the infection was significantly correlated with older age (P = .047). Lung involvement was also statistically significantly associated with older age (P = .045). Over the study period, a total of 15 patients died. Age greater than 60 years was significantly associated with deaths from COVID-19 (P = .036), with failure to achieve CR having a statistically nonsignificant impact on mortality (P = .22). CONCLUSION: These results confirm the prognostic significance of age for achieving treatment response of hematologic disease as well as the severity and mortality of COVID-19 in hematology patients.

The role of new technologies in myeloproliferative neoplasms: Application of next-generation sequencing in myelofibrosis.

INTRODUCTION: Driver mutations in Philadelphia chromosome-negative myeloproliferative neoplasms are well known. In the past, whole-genome sequencing identified nondriver mutations in other genes, potentially contributing to evolution of malignant clones. METHODS: Next-generation sequencing was used to assess the presence of any mutations in 14 candidate genes at the point of diagnosis and the resultant impact on the clinical course of the disease. RESULTS: The study analysed 63 patients with myelofibrosis (MF). Nondriver mutations were detected in 44% of them. The most frequently affected genes were ASXL1 (27%), TET2 (11%) and SF3B1 (6%). The frequency of such mutations was highest in primary MF (59%) and lowest in the prefibrotic phase of primary MF (21%). Patients with prognostically unfavourable sequence variants in genes had significantly worse overall survival (53 vs 71 months; HR = 2.77; 95% CI 1.17-6.56; P = .017). CONCLUSION: In our study, multivariate analysis proved DIPSS to be the only significant factor to predict patient survival. DIPSS contains all of the important clinical and laboratory factors except genetic changes. Stratification of patients according to DIPSS is still beneficial although there are newer and improved scoring systems like GIPSS or MIPSS70. Assessing subclonal mutations in candidate genes during diagnosis may aid in the identification of high-risk MF patients and is therefore relevant for making a prediction for overall survival more accurate.

Current Promising Biomarkers and Methods in the Diagnostics of Antiphospholipid Syndrome: A Review.

Antiphospholipid syndrome (APS) is a hypercoagulation condition associated with the incidence of heterogenic antiphospholipid antibodies (aPLs), which non-specifically affect hemostasis processes. APS is clinically manifested by recurrent arterial and venous thromboses and reproduction losses. The aPL antibodies, which may induce clinical manifestations of APS, include criteria antibodies anti-cardiolipin, anti-ß2-glycoprotein-I, and lupus anticoagulant, but also non-criteria antibodies, for example anti-ß2-glycoprotein-I domain I, anti-phosphatidylserine/prothrombin, anti-annexin V, and many others. APS occurs mostly in patients of younger and middle age, most frequently in females. Laboratory diagnostics of APS are quite difficult, as they include a wide spectrum of examining methods, which are based on various principles of detection and are performed using various laboratory techniques. The objective of the review is to describe the current state of potentially examined biomarkers and methods in APS diagnostics. The aforementioned biomarkers are lupus anticoagulant, anti-ß2-glycoprotein-I, anti-cardiolipin, anti-ß2-glycoprotein-I domain I, anti-phosphatidylserine/prothrombin, anti-ß2-glycoprotein-I IgA, anti-cardiolipin IgA, anti-annexin V and II, anti-prothrombin, anti-cardiolipin/vimentin, anti-protein S/protein C, and antibodies against phospholipid antigens for whose diagnostics we may use some of the methods established for a long time and some of the modern methods-the coagulation method for the determination of lupus anticoagulant (LA), enzyme-linked imunosorbent assay (ELISA), chemiluminescence analysis (CLIA), multiplex fluorescence flow immunoassay (MFFIA), fluorescence enzyme immunoassay (EliA), line immunoassay (LIA), multiline dot assay (MLDA), and thin-layer chromatography (TLC). Conclusion: Antibodies against phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, cardiolipin/vimentin complex, and annexin V are currently the most studied new markers. However, these assays have not been standardized until now, both from the laboratory and clinical point of view. In this review we summarize the evidence of the most studied aPL markers and their potential clinical significance in seronegative APS (SN-APS).

The Modern Pneumatic Tube System Transports with Reduced Speed Does Not Affect Special Coagulation Tests.

Pneumatic tube transport systems (PTS) for delivery of patient samples to a hemostasis laboratory are often used to reduce turnaround time for vital analyses. PTS in our hospital has the ability to regulate the transport speed in the range of 3-6 m/s with acceleration control technology. We evaluated the effects of PTS transport for routine coagulation tests, platelet function tests and special global coagulation tests. Duplicate samples were collected from 29 patients and 40 healthy individuals. One sample was sent using PTS and the other was carried by personnel to the lab for determination of protrombin time, activated partial thromboplastin time, trombin time, fibrinogen, antitrombin and thrombin generation test. Platelet function was measured by means of a Apact 4004® analyzer using the inductors (ADP, Arachidonic acid and Epinephrine). Samples transported using PTS with normal transport speed 6 m/s does not affect basic coagulation tests (PT, aPTT, FIB, TT and AT), but TGT has significantly altered. The use of PTS with controlled acceleration regulated the increase in thrombin generation from 10% to 3%, which is not statistically signifiant. The use of PTS with controlled acceleration did not show a significant difference even with the highly sensitive method of platelet aggregation. We conclude that PTS with acceleration control with transport speed from 3 to 6 m/s does not affect to platelet activity as measured by LTA and also global coagulation test - TGT. The advantage of PTS transport is very rapid assessment laboratory testing. From the above validation study, it is clear that PTS should always be validated for specialized laboratory methods and appropriately adapted to specific transport conditions.

Anti-domain 1 ß2 glycoprotein antibodies increase expression of tissue factor on monocytes and activate NK Cells and CD8+ cells in vitro.

BACKGROUND: ß2-Glycoprotein I (ß2GPI) represents the major antigenic target for antiphospholipid antibodies (aPL), with domain 1 (D1) being identified as a risk factor for thrombosis and pregnancy complications in APS. We aimed to analyse the ability of aPL, and particularly anti-D1 ß2GPI, to stimulate prothrombotic and proinflammatory activity of immune cells in vitro. METHODS: Peripheral blood mononuclear cells (PBMCs) from 11 healthy individuals were incubated with: (1) "anti-D1(+)"-pooled plasma derived from patients suspected of having APS contained anticardiolipin antibodies (aCL), lupus anticoagulant (LA), anti-ß2GPI and anti-D1 ß2GPI; (2) "anti-D1(-)"-pooled plasma from patients suspected of having APS contained aCL, LA, anti-ß2GPI, and negative for anti-D1 ß2GPI; (3) "seronegative"-negative for aPL. RESULTS: The presence of anti-D1(+) and anti-D1(-) plasma resulted in increased HLA-DR and CD11b on monocytes. While only anti-D1(+) plasma markedly increased the percentage and median fluorescence intensity (MFI) of CD142 (tissue factor, TF) on monocytes in comparison with those cultured with anti-D1(-) and seronegative plasma. Anti-D1(+) plasma resulted in increased percentage and MFI of activation marker CD69 on NK and T cytotoxic cells. Expression of IgG receptor FcγRIII(CD16) on monocytes and NK cells was down-regulated by the anti-D1(+) plasma. CONCLUSIONS: Taking together, our study shows the ability of patient-derived aPL to induce immune cell activation and TF expression on monocytes. For the first time, we demonstrated the influence of anti-D1 ß2GPI on the activation status of monocytes, NK and cytotoxic T cells. Our findings further support a crucial role of D1 epitope in the promotion of thrombosis and obstetrical complications in APS.

Multianalyte Determination of NOACs Using LC-MS/MS and Comparison with Functional Coagulation Assays.

BACKGROUND: Detection of new oral anticoagulant (NOAC) levels by screening, special and global tests, and liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) is important in clinical situations when the cause of bleeding needs to be determined. METHODS: We compared a routine coagulation test, special function test for NOACs, global coagulation test, and an LC-MS/MS method that enables simultaneous determination of apixaban, dabigatran and rivaroxaban in human plasma within one analysis to determine the optimal indication of the comparison methods, including their limitations and interferences. RESULTS: This study was conducted on a set of blood samples from 116 patients treated with NOACs. The results of both specific dilute thrombin time (dTT) tests for dabigatran provided the same results as the activated partial thromboplastin time (aPTT) screening test in comparison with LC-MS/MS as a reference. The dTT assay HemosIL® showed better results for low concentrations when compared to LC-MS/MS than dTT HYPHEN® as HemosIL® uses a non-linear calibration curve. Results of the specific anti-Xa assay yielded better results than the prothrombin time test in comparison with LC-MS/MS as a reference, especially for apixaban, but also for rivaroxaban. Our LC MS/MS method is simply feasible, but only in a specialized laboratory. The method is easy-to-use for the simultaneous determination of all dabigatran, apixaban and rivaroxaban by LC-MS/MS within three minutes with a concentration range of 1 to 500 µg/L without dilution. CONCLUSIONS: In the normal practice of the coagulation laboratory, it is advisable to use specific tests for NOAC determination as screening and global assays are not sufficiently specific. The dTT test is the optimal choice for dabigatran determination and for xabans to determine anti-Xa activity. The LC-MS/MS method is suitable as an arbitration method for serious conditions.

Polymorphism of the Fcγ Receptor II as a Possible Predisposing Factor for Heparin-Induced Thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) represents a serious complication of heparin treatment. IgG antibodies binding platelet factor 4 (PF4) and heparin trigger the clinical manifestations of HIT. However, only a portion of the antibodies have the ability to activate platelets, and these can be identified by a platelet aggregation test (functional testing). However, this expression has been detected to have a molecular cause, which is a mutation of FcγRIIa. The FcγRIIa receptor is responsible for the activation of platelets by antibodies in HIT. METHODS: To determine HIT, impedance aggregometry using the Multiplate analyzer (MEA) as heparin-induced aggregation technique and the Technozym HIT IgG ELISA test were used. The MEA method uses sensitization of donor platelets with patient plasma in the presence of heparin at a concentration of 0.5 IU/mL. The results were compared with the ELISA test. Mutation of FcγRHa was assessed using the asymmetric real-time PCR method that is based on the reaction with two hybridization probes and melting curve analysis. RESULTS: Examined were 100 patients at a clinically intermediate and higher risk of HIT according to the 4T's score. All samples were examined by the ELISA test and MEA, with positive samples being further confirmed by high-concentration heparin. In the group of patients, 10.0% were positive by MEA as compared with 4% determined by ELISA. The results of genetic analysis of FcγRIIa did not provide statistically significant differences between positive patients found by the functional test as well as the ELISA test and seronegative patients. CONCLUSIONS: The genetic mutation FcγRIIa is a predisposing factor for manifestation of HIT in the form of thrombocytopenia, but the process of seroconversion apparently needs another inducing factor. Therefore, the examination of mutations can be classified as predisposing factors rather than to confirm the diagnosis of HIT.

Refractory immune thrombocytopenia. Successful treatment with repeated cyclosporine A: two case reports.

Treatment of chronic, severe refractory immune thrombocytopenia after splenectomy is difficult. Only less data exist on clinical use of cyclosporine A (CyA) in the management of refractory ITP. In this report, we describe two cases in which standard immunosuppressive therapy, other immunosuppression including cyclosporine A or splenectomy had no therapeutic effect. Even after splenectomy, recommended procedures were inefficient and critical thrombocytes count persisted. After repeated administration of cyclosporine A which had been ineffective prior to splenectomy; however, both patients achieved long-term complete remission of the ITP. Side effects of CyA were moderate. The presented cases have confirmed the potential therapeutic effect of CyA in refractory post-SE ITP.

Efficacy of rituximab in primary immune thrombocytopenia: an analysis of adult pretreated patients from everyday hematological practice.

Although rituximab has seen increasing use in the treatment of immune thrombocytopenia (ITP) for many years, its therapeutic role in this disease remains unclear. We retrospectively analyzed data of all patients with ITP treated with rituximab (375 mg/m(2) once weekly for four consecutive weeks) and consecutively entered the findings into the databases of six large academic centers in the Czech Republic. A total of 114 patients were included in the analysis. All of the patients received rituximab as a second or additional line of therapy. The overall response rate (ORR) after rituximab therapy was 72 % [48 % complete response (CR), 24 % partial response (PR)] at month 6, and 69 % (45 % CR, 24 % PR) at month 12. For the group of patients with newly diagnosed (acute) ITP, the results of treatment were significantly better than for the group of patients with persistent or chronic ITP; nonetheless, this group of patients was far too small (n = 18) for our findings to be generalized. Multivariate analysis revealed that the ORR was significantly influenced primarily by the number of therapies prior to rituximab (the more previous therapies, the worse treatment response). The results of our analysis "from everyday hematological practice" confirm the high efficiency of rituximab treatment in pretreated adult patients with ITP.

The pathophysiology of endothelial function in pregnancy and the usefulness of endothelial markers.

AIM: The aim of this study was to assess coagulation markers of endothelial damage and examine new markers of endothelial activation such as matrix metalloproteinases (MMPs) in a group of healthy pregnant women. Matrix metalloproteinase (MMP)-2, in particular, plays a major role in the degradation of the extracellular matrix confirming its essential function in both the survival (angiogenesis) and death of endothelial cells. Detection of specific coagulation factors, mainly released from the vascular endothelium such as vWF, sTM (soluble thrombomodulin) and ePCR (endothelial protein C receptor) and factors dependent on endothelial activation such as t-PA and PAI-1, could provide information on possible endothelial dysfunction and help differentiate pregnant patients with an altered thrombotic state. METHODS: Healthy pregnant women underwent complete assessment for endothelial damage (as vWF, vWF activity, sTM, ePCR, EMP, MMP-2, MMP-9 and TIMP-2) using the ELISA and other methods. RESULTS AND CONCLUSIONS: The results show that endothelial activation during pregnancy is different from that in other pathological conditions involving endothelial damage and typically characterized by higher levels of both coagulation endothelial markers and MMPs. In pregnancy, changes in extracellular matrix composition and matrix metalloproteinase activity also occur and promote vascular remodeling but, only in the uterus. Predisposing risk factors for epithelial dysfunction, and vascular mediators associated with vascular remodeling must be assessed from concentrations in whole blood. The levels of MMPs are not increased in the circulation and the local situation in the uterus cannot be monitored this way. However, MMP-2 processes and modulates the functions of many other vasoactive and pro-inflammatory molecules including adrenomedullin, big endothelin-1, calcitonin gene-related peptide, CCL7/MCP-3, CXCL12/SDF-1, galectin-3, IGFBP-3, IL-1 Beta, S100A8, and S100A9. These molecules represent new potential molecular markers of endothelial damage during pregnancy.

Precursor T-lymphoblastic lymphoma as a secondary malignancy in a young patient after successful treatment of acute promyelocytic leukemia.

BACKGROUND: Acute promyelocytic leukemia (APL) is a relatively rare subtype of acute myeloid leukemia. It has become the best curable subtype of acute leukemias in adults due to the inclusion of all-trans-retinoic acid (ATRA) in the treatment. Despite the efficacy of ATRA, chemotherapy must be added in APL patients in order to maintain durable complete remission. However, chemotherapy administration is inevitably related to many complications, including the risk of secondary malignancies. T-lymphoblastic lymphoma (T-LBL) is an infrequent disease that belongs to the group of highly aggressive lymphomas. CASE REPORT: The authors describe the case of a 25-year-old woman who was treated for APL in 2002 and developed precursor T-LBL 5 years later. CONCLUSION: Several cases of secondary acute lymphoblastic leukemias in 'cured' APL patients have been described, but probably no patient with secondary precursor T-LBL. Secondary malignancy has become one of the topics discussed (not only) in APL patients. It is apparently related to the excellent treatment outcomes and long-term survival. Better tailored treatment based on relevant prognostic factors allowing chemotherapy reduction or omission in some patients is needed.

Molecular pathophysiology of thrombotic states and their impact to laboratory diagnostics.

BACKGROUND: Molecular genetic methods were implemented in the detection of thrombophilic disorders in the 1990's with the discovery of coagulation inhibitors antithrombin III (AT III), protein C (PC) and S (PS). The discovery of the molecular cause of activated protein C (APC) resistance by Bertina in 1994 greatly expanded their utilization. METHODS AND RESULTS: Currently, a broad group of molecular genetic markers with a clearly demonstrated risk of thrombophilia are used--mutation of FV Leiden 506R/Q, mutation of prothrombin (F II) 20210G/A, mutation of methylenetetrahydrofolate reductase (MTHFR) 677C/T in homozygous form, mutation of plasminogen activator inhibitor (PAI-1) 4G/5G, mutations of single coagulation inhibitors as well as a number of polymorphisms with controversial thrombophilic risk such as F XIII Val34Leu, platelet glycoproteins, endothelial protein C receptor and thrombomodulin. Another area utilizing molecular genetic methods is research of the pathophysiology of individual coagulation processes. To date, the greatest advances in regard to APC resistance have been achieved here. Although the molecular cause of APC resistance was clearly demonstrated in the 1990's, its clinical variability has not yet been fully explained. The same is true for the second most widespread mutation, prothrombin gene mutation, where only the latest research has hinted at a possible mechanism of expression of the genetic changes in the actual coagulation process. CONCLUSIONS: The future of molecular genetic methods is in achieving a complex understanding of the pathophysiology of thrombophilia and not only in its utilization as a method for detecting many polymorphisms with a very low risk of thrombosis.