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.

Monitoring the Antiplatelet Therapy Efficacy in Patients with Acute Ischemic Stroke.

BACKGROUND: Stroke is one of the leading causes of morbidity and mortality in populations of developed countries. Ischemic strokes account for 85 - 90% of all strokes, with the majority of strokes of non-cardioembolic pathogenesis. Platelet aggregation plays a key role in arterial thrombus formation. Therefore, effective antiplatelet therapy plays a key role in secondary prevention. Acetylsalicylic acid (ASA) is the main drug of choice, and clopidogrel therapy is another recommended treatment option. Monitoring of the efficacy of antiplatelet therapy has been intensively studied in patients with coronary artery disease in the context of coronary stent implantation. It is not yet part of the routine procedure in patients with stroke [1-3]. METHODS: This study investigated the efficacy of antiplatelet therapy with ASA and clopidogrel using optical and impedance aggregometry in 42 consecutive patients with acute ischemic stroke. Patients were treated with throm-bolysis at baseline and platelet function was examined 24 hours after administration, focusing on the occurrence of platelet hyperaggregability and assessing the efficacy of any chronically administered antiplatelet therapy. Subsequently, patients were administered a loading dose of ASA or clopidogrel followed by a check of its efficacy 24 hours after administration. In the following days, the maintenance dose of the drug was continued and regular laboratory monitoring of treatment efficacy was performed every 24 hours. RESULTS: In patients with atherothrombotic stroke indicated for antiplatelet therapy, monitoring of residual platelet activity allowed for detection of potentially atrisk patients. These were 35% (9% borderline ineffective) of patients taking ASA and 55% (18% borderline ineffective) of patients treated with clopidogrel. The dose was adjusted, increased the administered treatment and no recurrence of stroke was observed in this study group at 1-year follow-up. CONCLUSIONS: Personalized antiplatelet therapy based on platelet function tests appears to be a useful method to reduce the risk of recurrent vascular events.

Antiphospholipid antibody-mediated NK cell cytotoxicity.

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia that is characterised by thrombosis and obstetric complications in the presence of antiphospholipid antibodies (aPL). Pregnancy complications remain a challenging problem for patients with APS, especially during the first trimester. Although natural killer (NK) cells constitute up to 70% of decidual lymphocytes during the first trimester, their contribution to early pregnancy loss in APS is largely unknown. We aimed to analyse whether aPL are able to recruit antibody-dependent cellular cytotoxicity (ADCC) of NK cells, with special emphasis on the differences in the effects of aPL containing anti-ß2GPI domain 1 (anti-ß2GPI-D1) antibodies (aPL+/D1+) and those that do not (aPL+/D1-). Our findings revealed a differential distribution of NK subsets in the presence of different aPL. Namely, aPL+/D1- IgGs increased CD56dim/CD16dim cells, while aPL+/D1 + IgGs increased the number of CD56bright/CD16dim cells. ADCC NK cell cytotoxicity was found to be higher in the presence of aPL+/D1- IgGs, as defined by an increased target cell death, degranulation and increased expression of CD11b, CD69 and NKG2D. Overall, our evidence showed that aPL are able to recruit ADCC, suggesting NK cells as candidate cells for APS-related obstetric complications.

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.

Detection of Unknown and Rare Pathogenic Variants in Antithrombin, Protein C and Protein S Deficiency Using High-Throughput Targeted Sequencing.

The deficiency of natural anticoagulants­antithrombin (AT), protein C (PC), and protein S (PS)­is a highly predisposing factor for thrombosis, which is still underdiagnosed at the genetic level. We aimed to establish and evaluate an optimal diagnostic approach based on a high-throughput sequencing platform suitable for testing a small number of genes. A fast, flexible, and efficient method involving automated amplicon library preparation and target sequencing on the Ion Torrent platform was optimized. The cohort consisted of a group of 31 unrelated patients selected for sequencing due to repeatedly low levels of one of the anticoagulant proteins (11 AT-deficient, 13 PC-deficient, and 7 PS-deficient patients). The overall mutation detection rate was 67.7%, highest in PC deficiency (76.9%), and six variants were newly detected­SERPINC1 c.398A > T (p.Gln133Leu), PROC c.450C > A (p.Tyr150Ter), c.715G > C (p.Gly239Arg) and c.866C > G (p.Pro289Arg), and PROS1 c.1468delA (p.Ile490fs) and c.1931T > A (p.Ile644Asn). Our data are consistent with those of previous studies, which mostly used time-consuming Sanger sequencing for genotyping, and the indication criteria for molecular genetic testing were adapted to this process in the past. Our promising results allow for a wider application of the described methodology in clinical practice, which will enable a suitable expansion of the group of indicated patients to include individuals with severe clinical findings of thrombosis at a young age. Moreover, this approach is flexible and applicable to other oligogenic panels.

Evaluation of the Determination of Dabigatran, Rivaroxaban, and Apixaban in Lupus Anticoagulant-Positive Patients.

BACKGROUND: The effect of direct oral anticoagulants (DOAC) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa, is a well-known problem and can cause both false positive and negative results. In particular, the situation in patients who develop lupus anticoagulant (LA) antibodies is highly complex. To evaluate the effectiveness of DOAC therapy in lupus-positive patients, 31 samples were enrolled in this retrospective study. All patient samples were spiked with three types of DOAC (dabigatran, DABI; rivaroxaban, RIVA; and apixaban, API) in a concentration that significantly influenced the screening test for LA and thus can mask the presence of LA. Subsequently, the DOAC was always unbound by the DOAC-Stop procedure. DOAC levels before and after binding were determined by functional assays, followed by liquid chromatography coupled with mass spectrometry (LC-MS) analysis. METHODS: The determination of DOAC levels was performed by direct thrombin assay and determination of anti-Xa activity with specific calibration as functional tests for DABI and xabans (API and RIVA). To determine concentration levels of API, DABI, and RIVA, our in-house LC-MS method was used. RESULTS: The results of LA-positive samples show significant differences between functional tests and the LC-MS method both before and after DOAC binding. CONCLUSIONS: The acute findings of the presence of LA-type antibodies fundamentally affects the determination of DOAC by functional tests, and in this case, it is necessary to use LC-MS analysis to determine the true value. If patients treated with DOAC develop LA of medium and higher titers, we do not recommend checking DOAC levels with functional tests.

Hypertriglyceridemic Waist in Patients with Type 2 Diabetes: Its Relationship to Selected Markers of Vascular Damage.

Background: To evaluate the association between hypertriglyceridemic waist (HTGW), a promising marker of visceral adiposity and cardiovascular (CV) risk, and different indicators of vascular damage in type 2 diabetes (T2D) patients. Methods: This case-control study included 161 patients with T2D (91 males, 70 females) and 40 healthy controls (24 males, 16 females). HTWG was defined as waist circumference >90 cm in men or >85 cm in women and triglyceride concentrations >2 mmol/L. In addition to anthropometric and metabolic parameters, markers of endothelial dysfunction, namely von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1), were assessed. Arterial stiffness parameters were examined using the SphygmoCor system. Results: Individuals with T2D and HTGW showed the highest elevation of PAI-1 levels and significantly increased vWF levels compared with healthy controls. No significant differences in arterial stiffness markers were observed between T2D individuals. Age and, for several markers, systolic and/or diastolic blood pressure were identified as the main predictors for arterial stiffness, whereas PAI-1 and vWF levels were predicted by metabolic parameters. Conclusions: HTGW represents increased CV risk in T2D patients, mainly due to endothelial damage. The presence of HTGW had no significant effect on arterial stiffness compared with other T2D individuals.

A synergy of liquid chromatography with high-resolution mass spectrometry and coagulation test for determination of direct oral anticoagulants for clinical and toxicological purposes.

Direct oral anticoagulants are an alternative to anticoagulants based on vitamin K antagonists. Monitoring of direct oral anticoagulant concentration levels is necessary in specific cases (e.g. in emergency conditions, for determination of the cause of bleeding, adverse effects, risk of drug-direct oral anticoagulants interaction); therefore, a sensitive and specific method is needed. A methanol protein precipitation method followed by liquid chromatography with high-resolution mass spectrometry was developed for simultaneous separation and determination of apixaban, betrixaban, edoxaban, dabigatran, rivaroxaban and ximelagatran. The proposed method was fully validated in terms of linearity, the limits of detection and quantification, intra- and inter-day trueness and precision, recovery, matrix effect, process efficiency and stability. The method shows a strong correlation (Pearson's correlation coefficients > 0.92) with coagulation assays of apixaban, dabigatran and rivaroxaban (dilute thrombin time for gatrans and anti Xa factor (anti-Xa) activity for xabans). In addition, the developed method was applied for the identification and determination of apixaban and dabigatran in post-mortem serum samples. The developed method is a good alternative to coagulation tests which may show various interferences.

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

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

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.

17ß-Estradiol Promotes Proinflammatory and Procoagulatory Phenotype of Innate Immune Cells in the Presence of Antiphospholipid Antibodies.

Antiphospholipid syndrome (APS) is the most common cause of acquired thrombophilia and recurrent spontaneous miscarriages associated with extended persistence of antiphospholipid antibodies (aPL). How circulating aPL and high-17ß-estradiol (E2) environment contribute to the pregnancy complications in APS is poorly defined. Therefore, we aimed to analyse whether E2 could be responsible for the immune cell hyperactivation in aPL- positive (lupus anticoagulant, anti-cardiolipin, anti-ß2-glycoprotein) in women. For this, peripheral blood mononuclear cells (PBMCs) from 14 aPL- positive and 13 aPL- negative women were cultured in the presence or absence of E2, LPS or E2+LPS and cell immunophenotype and cytokine release were analysed. In the aPL+ group, E2 presence markedly increased the percentage of NK cells positive for CD69 (p < 0.05), monocytes positive for tissue factor (TF, CD142) (p < 0.05), and B cells expressing PD-L1 (p < 0.05), as well as the elevated production of IL-1ß comparing to aPL- women (p < 0.01). Regardless of aPL positivity, E2 augmented the procoagulatory response elicited by LPS in monocytes. Our findings show the ability of E2 to promote proinflammatory and procoagulatory phenotype of innate immune cells in individuals with aPL positivity. Our data highlights the significant impact of female hormones on the activation of immune cells in the presence of aPL.

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.

Monitoring of fibrinolytic system activity with plasminogen, D-dimers and FDP in primary total knee arthroplasty (TKA) after topical, intravenous or combined administration of tranexamic acid.

AIM: We assessed various ways of tranexamic acid (TXA) administration on the fibrinolytic system. Blood loss, transfusions, drainage and haematoma were secondary outcomes. METHODS: In this prospective study, we examined 100 patients undergoing primary total knee arthroplasty (TKA) between June and November 2018. Patients were randomly assigned to 4 groups according to the following TXA regimens: 1) loading dose 15 mg TXA/kg single intravenous administration applied at initiation of anesthesia (IV1); 2) loading dose 15 mg TXA/kg + additional dose 15 mg TXA/kg 6 h after the first application of TXA (IV2); 3) IV1 regime in combination with a local wash of 2 g of TXA in 50 mL of saline (COMB); 4) topical administration of 2 g of TXA in 50 mL of saline (TOP). RESULTS: Systemic fibrinolysis interference was insignificant in all of the regimens; we did not detect significant differences between IV1, IV2 and COMB in the monitored parameters within the elapsed time after the TKA; IV regimes had the lowest total drainage blood loss; the lowest blood loss was associated with the IV1 and IV2 regimens (IV1, IV2 < COMB < TOP); the lowest incidence of haematomas was in patients treated with TXA topically (i.e., in COMB + TOP). CONCLUSION: The largest antifibrinolytic effect was associated with intravenous administration of TXA. In terms of blood loss, intravenously administered TXA can interfere with the processes associated with the formation of the fibrin plug more efficiently than the simple washing of wound surfaces with TXA.

Searching for genetic variants associated with thrombophilia.

Thrombotic states are inherited or acquired predisposition for thrombosis in the human vascular system. Nowadays Leiden mutation and mutation in prothrombin G20210A contributing to congenital thrombophilia are routinely tested. These mutations have a high prevalence in the population. Congenital deficiencies of protein S, protein C and antithrombin III are rare thrombophilia with lower population frequency, but higher risk of thromboembolic event. The genetic causes are mutations in the genes, which encode these proteins. The choice of proper molecular genetic testing depends on the difference in the detection of well-known single nucleotide polymorphism or unknown/rare variant. For the detection of causative variant FV Leiden and prothrombin G20210A are mostly used PCR-RFLP, reverse Strip Assay®, allele-specific PCR, TaqMan real-time PCR and SNaPshot®. Precise patient selection should precede the genetic testing of rare variants in anticoagulant proteins. It is appropriate to use methodology of massive parallel sequencing supplemented by a methodology for the detection of larger gene rearrangements - MLPA. We are successfully employing this approach in our institute. This methodology is faster with larger analytic capacity compared to commonly used direct sequencing by Sanger method.

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.

Possibility of Coagulation System Activation Determination with Tissue Factor in Pregnancy Complications.

BACKGROUND: In this part of the study, where we determined the causes of preeclampsia and other obstetric complications, we focused on the role of tissue factor (TF) in the activation of these pathophysiological processes. Recent findings attribute a significant part of the activation of coagulation creation of autoantibodies. Once this mechanism is activated, the antibodies induce expression of tissue factor (TF, CD142) on monocytes and vascular endothelial cells. METHODS: We have proposed a monitor activation model of the coagulation system in preeclampsia and other pregnancy complications using TF expression on monocytes by flow cytometry and simultaneous determination the TF-induced thrombin generation in plasma. To determine expression of tissue factor (CD142) on monocytes, we proposed a method of multicolor flow cytometry using anti CD45 PerCP, anti CD14 APC, anti CD16b FITC, and anti CD142 PE antibodies and the corresponding isotype controls. RESULTS: We verified the model on patients with severe antiphospholipid syndrome, which is a high expression of antibodies, in particular against beta-2GPI. CONCLUSIONS: We demonstrated complete inhibition of TF expression on monocytes and a significant reduction of thrombin generation in plasma.

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.

Markers of endothelial activation in preeclampsia.

BACKGROUND: The study aimed at finding a laboratory approach to detect endothelial damage in normal pregnancy as well as in pregnancy complicated by preeclampsia using selected markers of endothelial activation. MATERIALS: A total of 403 healthy pregnant women without a history of deep vein thrombosis and/or hypertension were prospectively studied. From all women, venous blood was collected before the end of the 1st trimester, between weeks 24 and 28 of gestation, and in the 3rd trimester (weeks 34-36). Assays of tissue plasminogen activator, plasminogen activator inhibitor-1, von Willebrand factor activity and antigen, thrombomodulin, endothelial protein C receptor, and endothelial microparticles activated by TF were performed. RESULTS: When comparing women who developed preeclampsia during pregnancy (the average levels were 23.41 µg/L, 34.33 µg/L, and 53.56 µg/L in the 1st, 2nd, and 3rd trimesters, respectively) with healthy pregnant women (the average levels were 19.05 µg/L, 28.47 µg/L, and 39.86 µg/L in the 1st, 2nd, and 3rd trimesters, respectively) significant differences in the levels of thrombomodulin were found in all three trimesters. By contrast, no statistically significant differences in the levels of vWF (both antigen and activity), t-PA, EPCR, EMPs, MMP-2, MMP-9, and TIMP-9 were found in any trimesters in the same group. CONCLUSIONS: Pregnancy and preeclampsia strongly influence the levels of studied markers. The findings of this work confirm the possible predictive potential of thrombomodulin and PA-1.

The role of tissue factor in normal pregnancy and in the development of preeclampsia: A review.

BACKGROUND: Tissue factor (TF) is a key element for normal gestation, especially in the first trimester. TF levels are hence raised in pregnancy, producing an adaptive hypercoagulable state. Potentiated hypercoagulability however, is associated with disorders of pregnancy such as pre-eclampsia but the results of TF and its inhibitor, tissue factor pathway inhibitor (TFPI), measurement, in pre eclampsic women are ambiguous and the data conflicting. This review covers the current knowledge status of the role of TF assessment in pregnancy with a focus on its diagnostic utility. METHODS: A review of the literature using the following key words: tissue factor, thrombosis, inflammation, pregnancy, preeclampsia. RESULTS: The published literature shows raised and unchanged TF levels in various studies of pre-eclampsia along with equally conflicting data for TFPI. The various study designs and methods used in these studies makes valid comparison difficult. Meta analysis of 34 randomized trials showed that low-dose aspirin in early phases of gravidity (starting from the 16th week or earlier) significantly reduces the incidence of preeclampsia. CONCLUSIONS: Overall, the results of the literature search together with knowledge of the structure and biological effects of TF, suggest that measuring the level of plasma TF/TFPI is not ideal for determining the actual levels of TF in the uteroplacental circulation. The current view that endothelial dysfunction is the trigger for preeclampsia, suggests that aspirin may be an effective prophylaxis. Further research will be necessary: measuring the expression of tissue factor on monocytes using flowcytometry and comparing the development of this expression during normal pregnancy and pregnancy complicated by preeclampsia, for example. Another possibility is immunohistochemical determination of the level of TF expression directly in placental tissue.