Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban.

The effect of direct oral anticoagulants (DOACs) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa (FXa), is a well-known problem and can cause both false positive and negative results. Therefore, the correct interpretation of tests performed in patients receiving DOACs is necessary to avoid misclassification and subsequent clinical consequences. However, even with significant experience, there are situations where it is not possible to assess the influence of some methods. Particularly important is the situation in the diagnosis of lupus anticoagulants using the dilute Russell viper venom timetest, which is based on direct FXa activation. A very promising solution to this situation is offered by the DOAC laboratory balancing procedure DOAC-Stop. For evaluating the effectiveness of this procedure, 60 (20 apixaban, 20 dabigatran, and 20 rivaroxaban) patients treated with DOACs were enrolled. All patient samples were analyzed for the presence of individual DOAC types and subsequently subjected to the DOAC-Stop procedure.We evaluated its effectiveness by our own high-performance liquid chromatography-coupled tandem mass spectrometrymethod, which simultaneously sets all high-sensitivity DOACs. Unlike coagulation tests based on the determination of the residual effects of DOACs on target enzymes, which is complicated by extensive interindividual variation, this methodology is highly specific and sensitive.The DOAC-Stop procedure eliminated dabigatran from 99.5%, rivaroxaban from 97.9%, and apixaban from 97.1% of participants in our group. Residual amounts did not exceed 2.7 ng/mL for dabigatran, 10.9 ng/mL for rivaroxaban, or 13.03 ng/mL for apixaban, which are safe values that do not affect either screening or special coagulation tests.

Thrombin Generation Testing in Patients with Myelofibrosis.

BACKGROUND: Primary myelofibrosis (PMF) is a chronic clonal myeloid disorder. Together with essential thrombocythemia (ET) and polycythemia vera (PV), it belongs to a group of Philadelphia chromosome-negative myeloproliferative neoplasms. Thrombotic events are serious complications negatively influencing the quality and length of these patients' lives. The confirmed risk factors for venous thromboembolism are age over 60 years, a positive history of thromboembolism, presence of common cardiovascular risks, JAK2 V617F mutation and, according to some authors, leukocytosis. Various opinions on the role of thrombocythemia have been published. The present study was undertaken to evaluate the benefit of thrombin generation test and its potential use in predicting the risk of thrombosis in MF patients. METHODS: The analysis included plasma samples obtained from 36 patients diagnosed with MF in our center from 2004 to 2016 (JAK2 V617-positive 53%; CALR-positive 31%; MPL-positive 14%; triple negative 2%) and a control group comprising 20 healthy volunteer blood donors. Thrombin generation was measured in platelet-rich plasma using the TECHNOTHROMBIN® TGA kit (Technoclone, Austria) and the fully automated system Ceveron® Alpha (Technoclone). The results were correlated with clinical and laboratory parameters of the patients. RESULTS: There were differences in thrombin generation as expressed by endogenous thrombin potential (ETP) between patients and healthy controls, with ETP being lower in the patient group (p = 0.0003). Analysis confirmed a significant correlation between thrombin generation and platelet counts, with higher thrombin generation in patients with thrombocythemia > 400 x 109/L (p = 0.04). ETP values were consistently higher in earlier disease stages and lower in CALR-mutated myelofibrosis. CONCLUSIONS: In MF patients, thrombin generation is mainly influenced by platelet counts and, to a lesser extent, by mutation status, activity, and progression of the disease. Thrombin generation test results have confirmed that thrombocythemia is a potential risk factor for thrombotic complications.

Validation of a New Panel of Automated Chemiluminescence Assays for Anticardiolipin Antibodies in the Screening for Antiphospholipid Syndrome.

BACKGROUND: Antibodies anticardiolipin (aCL) and anti-ß2-glycoprotein I (aß2GPI) are two of three laboratory criteria of antiphospholipid syndrome (APS). All of assays of antiphospholipid antibodies (aPL), coagulation assays as well as ELISAs, show methodological shortcomings, that affect their sensitivity and specificity. Therefore, we decided to validate these parameters for a new chemiluminescent examination (CLIA). METHODS: aCL and aß2GPI antibodies were measured by ELISAs (AIDA, Bad Kreuznach, Germany) and aß2GPI with CLIA kits (Werfen, Barcelona, Spain). RESULTS: When we evaluated both assays, the coefficient of variation for CLIA was slightly lower (9.04 - 12.74%) than for ELISA (11.05 - 15.3%) and the LOD was 0.2 U/L. The dilution series showed significant linearity for all CLIA methods, aCL IgG, aCL IgM, aß2GPI IgG, and aß2GPI IgM (0 - 3000 U/L), and method comparison studies revealed good agreement with the currently used ELISA (Kappa values ranging 0.534 - 0.936) without determination of aß2GPI IgG. The determination aß2GPI IgG by CLIA method shows higher positivity in 31 samples. These new aCL IgG, aCL IgM, aß2GPI IgG, and aß2GPI IgM tests have excellent analytical characteristics and allow fully automated and simultaneous analysis on an analyzer. CONCLUSIONS: Chemiluminescent determination of an automated analyzer can improve the fundamental parameters of tests such as reproducibility between laboratories.

Hypertriglyceridemic waist - a simple clinical tool to detect cardiometabolic risk: comparison with harmonized definition of metabolic syndrome.

With the increasing prevalence of obesity and especially abdominal obesity, a simple clinical tool is needed that identifies the cardiometabolic risk for cardiovascular disease and type 2 diabetes. The aim of our study was to evaluate a broad spectrum of metabolic variables and IMT in subjects with and without hypertriglyceridemic waist (HTGW) and compare it with the harmonized definition of metabolic syndrome (MS) with both a higher (MS-I) and lower waist circumference (MS-II) for Europids. We enrolled 607 asymptomatic dyslipidemic subjects (295 men and 312 women) into our cross-sectional study. The subjects with HTGW had an atherogenic lipid profile (significantly higher triglycerides, AIP, non-HDL-C, lower HDL-C and ApoA-1, and the women also higher TC and ApoB), increased markers of insulin resistance (insulin, HOMA, C-peptide, proinsulin), inflammation (hsCRP), thrombosis (fibrinogen, PAI-1), SBP and DBP, and lower adiponectin (p<0.05-0.001 for all). These risk factors were entirely similar in HTGW, MS-I and MS-II. Age-adjusted IMT was significantly higher only in the women with HTGW but this significance disappeared after further adjustment for TC, SBP, and smoking. Our results support the routine use of HTGW as a simple and inexpensive screening tool to detect subjects at increased cardiometabolic risk in clinical practice.

Detection of clopidogrel resistance using ADP induced aggregometry with specific inhibitor PGE1.

BACKROUND: Antiaggregation therapy is still the most frequently used approach to prevent thrombotic events in cardiovascular diseases. It has a good clinical effect but increasing evidence shows high residual platelet aggregation activity in a number of patients. Laboratory methods only allow us to detect clopidogrel "non-responders" or "low responders". Recent methods are based on monitoring residual platelet aggregation activity (aggregation methods) or detecting the number of free epitopes for binding a specific monoclonal antibody such as vasodilator-stimulated phosphoprotein phosphorylation (VASP). METHODS: The aims of our study were comparison light transmission aggregometry (LTA) and multiple electrode platelet aggregometry (MEA) with induction by ADP in concentrations of 20 micromol/L with or without prostaglandin E1 (PGE1) for monitoring clopidogrel resistance. RESULTS: In the group of 84 patients with cardiovascular disease (CAD) studied, an impaired individual response to clopidogrel therapy was found 11.9% and 10.7% of the patients using MEA and LTA, respectively, induced by ADP with PGE1. The LTA and MEA methods with induction by ADP with PGE1 and without PGE1 were statistically compared using Spearman's nonparametric correlation analysis. Both methods with using PGE, showed a positive significant correlation (p = 0.003) in contrast with the results without PGE1 with a no significant correlation (p = 0.732). CONCLUSIONS: The sensitivity for detecting clopidogrel resistance correlates well with other data in the literature suggesting that there are 5%-30% clopidogrel low-responders depending on the type of platelet function assay used and the criteria for defining a low-responder [16-18]. These results favor implementation of the ADP test with PGE1 by MEA specifically for identification of low-responders to clopidogrel.

The advantages and limitations of impedance aggregometry in detection of 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). Current methods HIPA and SRA are time-consuming and difficult if HIT is clinically suspected; therefore, numerous new methods have recently been developed. METHODS: To determine HIT, impedance aggregometry using the Multiplate analyzer (MEA) as heparin-induced aggregation techniques and the Technozym HIT Ig 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. RESULTS: We examined 190 patients at clinically intermediate and higher risk of HIT according to the 4T score. All samples were examined by the ELISA test and MEA, with positive samples being further confirmed by high-concentration heparin. The methodology was modified with respect to the dilution for high positive samples and assessment has been extended to an index of inhibition. CONCLUSIONS: In the studied group, we demonstrated that MEA has sufficient sensitivity and higher specificity. In the group of patients, 10.0% showed positive results by MEA as compared with 7.3% determined by ELISA. Unlike the ELISA methods of the same quality, MEA is more suitable for detecting platelet-activating HIT antibodies in practice.

Evaluation of total adiponectin, adipocyte fatty acid binding protein and fibroblast growth factor 21 levels in individuals with metabolic syndrome.

Although many studies have investigated the relationships of several adipokines to metabolic syndrome (MetS), the interrelationships of adiponectin (ADP), adipocyte fatty acid binding protein (A-FABP) and fibroblast growth factor 21 (FGF 21) have not been described in detail. We examined 209 asymptomatic dyslipidemic patients divided into MetS+ (n=73) and MetS- (n=136) groups. The aim of study was to evaluate the relationships between observed adipokines, to compare the levels of total ADP, A-FABP and FGF 21 in individuals with and without MetS, and to elucidate the relationships of individual adipokines to lipid parameters, markers of insulin resistance and endothelial hemostatic markers in these groups. In MetS+ group, we found the independent positive association ADP with A-FABP (beta=0.4888, p=0.0382), A-FABP with FGF 21 (beta=0.3811, p=0.0002) and von Willebrand factor (beta=0.4502, p=0.0013), and FGF 21 with A-FABP (beta=0.4422, p=0.0002). Our study has confirmed the well-established risk profile of subjects with MetS, although clinically asymptomatic. MetS+ patients had also lower levels of ADP and higher levels of A-FABP and FGF 21. Our study evaluated the interrelationships of ADP, A-FABP and FGF 21 in asymptomatic dyslipidemic subjects with diagnosis of MetS. Especially strong association between A-FABP and FGF 21 needs to be clarified in further studies.

[Occurence, etiology and clinical significance of trombocytopenia in pregnancy]. / Incidence, etiologie a klinický význam trombocytopenie v gravidite

OBJECTIVE: The principal objective of the study is to compare results from the experimental group of pregnant women suffering from thrombocytopenia in pregnancy with results from the control group of pregnant women with normal physiologic blood platelet count. SETTING: Department of Obstetrics and Gynaecology of the Tomas Bata Regional Hospital Zlín, Obstetrics and Gynaecology Clinic, Haematology and Oncology Clinic of the Palacky University Teaching Hospital and Medical School in Olomouc, Obstetrics and Gynaecology Clinic of the Ostrava Teaching Hospital. METHODOLOGY: A group of 200 pregnant women suffering from thrombocytopenia underwent thorough medical tests. The level of platelets, presence of anti-platelets agents, liver function (LFT), anti-phospholipid antibodies, complete blood count with differential, specific antibodies for hepatitis B and C, Lyme borreliosis and cytomegalovirus were determined from venous blood using the EIA, ELISA methods. RESULTS: Medical articles and books about thrombocytopenia divide the causes for thrombocytopenia as follows: 79.5% benign gestational thrombocytopenia, 16% preeclampsia, 2.5% HELLP syndrome, 1% immune thrombocytopenia, 1% HVC. The number of women who developed physiological anaemia in pregnancy and were overweight is identical in the experimental group of pregnant women suffering from thrombocytopenia and in the control group of pregnant women with normal physiologic blood platelet count, and the proportion of the different age groups in the two groups of pregnant women is also identical. CONCLUSION: 32% of pregnancies in the experimental group ended in a caesarean section, of which 13.5% in a group of 127 pregnant women suffering from mild thrombocytopenia, 17.5% in a group of 71 pregnant women suffering from moderate thrombocytopenia and 1% in a group of 2 pregnant women suffering from severe thrombocytopenia. 20.5% pregnancies in the control group ended in caesarean section.

[Preeclampsia and thrombin generation test]. / Preeklampsie a trombin generacní test.

OBJECTIVE: Acquiring new information to allow prediction of the development of diseases associated with impaired coagulation. Design effective preventive measures most serious diseases (TEN) in the fields of gynecology and obstetrics. For pregnant women with preeclampsia, hypertension compared with women with normal pregnancies could lead to increased thrombin generation due to the synergistic effect of thrombotic risk factors. Based on the results and found statistically significant differences between the groups among pregnant can select for a higher risk of developing deep vein thrombosis. This risk group could then greatly benefit from more stringent follow-up and possible preventive treatment prophylactic doses of LMWH in reducing maternal and perinatal morbidity and mortality. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology, University Hospital Olomouc. METHODS: In early pregnancy - during pregnancy standard samples (up to the end of the first trimester) patients venous blood was sampled and they completed information questionnaire. A second sampling was carried out between 24 to 28 week, the third sample and between 36th to 40th week. Obtained blood samples were subsequently processed in the coagulation laboratory Hemato-Oncology Clinic and Olomouc. The blood samples were investigated protein C and S, antithrombin, FVIII level, FII, Leiden, and plasma endothelial microparticles, and lupus anticoagulant and APC resistance standardized methodologies. Thrombin generation was determined thrombin generation test. Thrombin generation was measured fully automatically using a kit (Technothrombin TGA, Technoclone, Vienna, Austria) and analyzer Ceveron Alpha (Technoclone, Vienna, Austria) with fully automatic analysis software. As the main parameter is evaluated by the maximum thrombin generation, at the same time, however, was also detected in the total amount of thrombin and the time until the beginning of the formation of thrombin. RESULTS: In the period 2008-2011 were analyzed blood samples of 303 healthy pregnant women. 215 women, ie 71% were nuliparas, 60 women, ie 19.8% were primiparas, 28 women, 9,2% were secundiparas. The average age of pregnant women was 28.6 years(± 3.8 years). The average maternal weight at the beginning of pregnancy was 63.6 kg (± 7.8 kg). Of the 303 women in 18 (6%) developed slight to moderate degree of preeclampsia or HELLP syndrome with varying severity of clinical manifestations. 20 mothers (6.6%) gave birth prematurely terminated before 37 week of pregnancy. 3 pregnancies (0.9%) were discontinued due to genetic indication for fetal birth defect. The complete study protocol (sampling in all three trimesters) thus completed 280 pregnancies. Of the three evaluated, parameter Lag time, ETP and peak we observed significant differences when comparing physiological pregnancies and pregnancies with preeclampsia (Table 3 and Figure 5-7), the statistical level of p < 0.01. In pregnancies with chronic hypertension, these differences were not significant. Comparison of 18 pregnancies, in which the III. trimester developed preeclampsia with other pregnant with physiological pregnancy did not show statistically significant differences in I. and II. trimester. The results suggest the activation of coagulation through the late stages of pregnancy. Results are influenced by strong clinical variability of disease. In severe and early preeclampsia this activation and significant differences begin much earlier. CONCLUSION: We demonstrated significantly higher activation of thrombin generation in women with preeclampsia [10]. Changes in preeclampsia are characterized by increased generation of thrombin in plasma. This fact may explain the partial success of the clinical use of aspirin in preeclampsia. In the third trimester, during the manifestation of the disease, patients with preeclampsia have significantly higher ETP compared to patients with a normal pregnancies. Pregnant women with chronic hypertension also show a slight increase in the activation of thrombin. However, these results are not statistically significant. Examination of coagulation in the first and second trimester in women who later developed preeclampsia, showed no statistically significant differences and thus can not be used in this case as predictive, but only as a diagnostic test.

[Endothelial dysfunction in pregnancies with chronic hypertension]. / Dysfunkce endotelu u tehotných s chronickou formou hypertenze.

AIM OF THE STUDY: To establish endothelial activation markers which could uncover endothelial damage during hysiological pregnancy and pregnacies with chronic hypertension. SETTINGS: Department of Hemato-oncology, Obstetrics and Gynecology and Department of Medical Genetics and Fetal Medicine Medical Faculty of Palacký University Olomouc, Department of Obstetrics and Gynecology Medical Faculty Ostrava. METHODS: We examined 298 pregnant women with a physiological pregnancy. Venous blood samples were collected from the women in both arms at the beginning of the pregnancy, a second sample was collected in the interval 24-28 weeks gestation, the third sample was colected about the 36 weeks of gestation. Parameters were examined using methods: t-PA - ELISA, PAI-1 - ELISA, vWF - EIA, ePCR - ELISA, MMP-2,9 - ELISA with fluorogenic detection, TIMP-2 - ELISA, endothelial microparticles - flow cytometry. RESULTS: In accordance with the literature, we have observed in our study significantly increased endothelial activatition in hypertensive pregnancies compared with women with physiological pregnancy, as evidenced by significant increases in vWF activity and antigen, thrombomodulin and PAI-1 in all trimesters. In the other investigated parameters statistically significant changes were not observed. CONCLUSION: We have found significant signs of endothelial dysfunction in the group of women with pre-existing hypertension, a wide range of parameters examined markers indicating an significantly increased endothelial activation pregnant women with pre-existing hypertension, confirming the need for strict follow-up of pregnant women with hypertensive disease.

[The occurence of genetic trombophilic markers in patients evaluated for infertility]. / Výskyt vybraných geneticky podmínených trombofilních markeru u pacientek podstupujících lécbu neplodnosti.

OBJECTIVE: To assess and compare the frequency of selected gene mutations of thrombophilic markers (FV Leiden, FII prothrombin G20210A and MTHFR C677T) in patients with primary and secondary infertility. DESIGN: Retrospective study. SETTING: Institute of normal anatomy, Faculty of Medicine and Dentistry, Palacky University Olomouc. METHODS: The study included 92 patients with primary infertility and 89 patients with secondary infertility. Indications for examination of these mutations were following: a positive family or personal history, a positive obstetrical history or a repeated failure of assisted reproduction treatment. RESULTS: According to our anticipation, women with the secondary infertility were significantly older(p < 0.0005) than those with primary infertility. No mutations of genes of examined thrombophilic markers (FV, FII and MTHFR), either alone or in combination, were found in only 8.7 % patients with primary infertility and in 5.6 % patients with secondary infertility. Significantly higher frequency of factor Leiden(p < 0.02) was observed in women with secondary infertility. There were no significant differences in the frequency of detected mutations of the remaining factors. CONCLUSION: Based on our findings we suggest that the assessment of selected gene mutations of thrombophilic markers should be a part of the diagnostic algorithm in patients with positive history for thrombophilic disorders.

The assessment of aspirin resistance by using light transmission and multiple electrode aggregometry.

INTRODUCTION: The issues related to aspirin [acetylsalicylic acid (ASA)] resistance are still under debate. Depending on the method of assessment and studied patients, the prevalence of ASA resistance is rather heterogeneous, ranging from 5% to 45%. The method most commonly used for assessing platelet function is their aggregation. ASA irreversibly inhibits cyclooxygenase-1 (COX-1) by acetylation. METHODS: This study aimed to compare light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) for the measurement of ASA resistance, using arachidonic acid as an inducer of the reaction. RESULTS: The study comprised 101 patients with stable ischemic heart disease taking a daily dose of 100 mg of ASA. The rates of ASA resistance were 22.22% and 21.21% as detected by LTA and MEA, respectively. The two methods were statistically compared using Spearman's nonparametric correlation analysis, with a positive significant correlation (P=0.01) and medium positive dependence between the methods (r=0.0539). CONCLUSION: If ASA resistance is detected by laboratory tests, replacement of ASA or its combination with other antiplatelet drugs as well as increased dosage may be considered.

[Monitoring of endothelial activation markers during physiological pregnancy]. / Monitorování hladin markeru aktivace endotelu behem fyziologické gravidity.

AIM OF THE STUDY: To establish endothelial activation markers which could uncover endothelial damage during physiological pregnancy. TYPE OF STUDY: Prospective study. METHOD: We examined 403 pregnant women with a physiological pregnancy. Venous blood samples were collected from the women at the beginning of the pregnancy, a second sample was collected in the interval 24-28 weeks gestation. Parameters were examined using methods: t-PA--ELISA, PAI-1--ELISA, vWF Ag--EIA ePCR--ELISA, MMP-2,9--ELISA with fluorogenic detection, TIMP-2--ELISA, endothelial microparticles - flow cytometry. RESULTS: The level of vWF antigen increased during the entire course of pregnancy (in the I. trimester the average level was 152.32%, in the II. and III. trimester 173.34% and 216.20% respectively). At the same time, vWf activity also increased (I. trimester average level 130.20%, II. and III. trimester 150.09% and 181.91% respectively). The level of thrombomodulin significantly increased during pregnancy (I. trimester average level 19.05 ng/ml, II. and III. trimester 28.47 ng/ml and 39.86 ng/ml respectively). The level of soluble form of EPCR increased during pregnancy (I. trimester average level 201.76 ng/ml, II. and III. trimester 274.68 ng/ml and 324.07 ng/ml respectively). The level of PAI-1 increased during the entire course of pregnancy (I. trimester average level 36.14 ng/ml, II. and III. trimester 50.07 ng/ml and 60.12 ng/ml respectively). The level of t- PA did not change significantly during the course of pregnancy (I. trimester average level 2.48 ng/ml, II. and III. trimester 2.97 and 3.34 ng/ml respectively). The levels of MMP-2 (I. trimester average level 9043.76 RFU, II. and III. trimester 9315.38 and 8800.27 RFU respectively), MMP-9 (I. trimester average level 8371.90, II. and III. trimester 8290.81 and 7470.50 respectively), TIMP-2 (I. trimester average level 92.5 ng/ml, II. and III. trimester 98.5 and 96.5 ng/ml respectively) or endothelial microparticles (I. trimester average level 3838.38 particles/microl, II. and III. trimester 3836.59 and 3650.59 particles/microl respectively) did not change significantly throughout the individual trimesters. CONCLUSION: We confirmed the hypothesis regarding the significant influence pregnancy has on changes in levels of these markers.

[Antithrombotic therapy in pregnancy]. / Antitrombotická terapie v gravidite.

Despite the fact of low prevalence of maternal death, deep venous thrombosis remains one of the most serious complication in pregnancy and puerperium. Virchows triad--vascular stasis, hypercoagulability, and vascular trauma plays the main role in the pathogenesis of deep vein thrombosis in pregnancy. Low molecular weight heparins and unfractionated heparins are the best treatment option. The aim of the treatment is to be effective in extension of thrombus and prevention of the postthrombotic syndrome and pulmonary embolism. Management of pregnant women with increased risk of venous thromboembolism can be stratified by determining whether the prior episode VTE was unprovoked or associated with a transient risk factor and the presence or absence of an inherited thrombophilia.

Immunohistochemical markers of proliferation and vascularisation in preneoplastic bronchial lesions and invasive non-small cell lung cancer.

Autofluorescence bronchoscopy (AFB) has been shown to be sensitive to detect preneoplastic lesions in central lung airways system. In early stages of carcinogenesis, up-regulation of cyclooxygenase (COX)-2, Ki67 and/or increased angiogenesis may play a role by promoting the proliferation of tumoral cells and their resistance to apoptosis, as well as angiogenesis, tumor cell invasion and setting up of the metastatic process. The present study compared the expression of proliferative (COX-2, Ki67 and PCNA) and angiogenic markers (CD34 and NG2) between preneoplastic bronchial squamous dysplasia lesions and invasive squamous cell carcinoma. Biopsies obtained during AFB [preneoplastic lesions: low-grade (lesions up to moderate dysplasia), n=13; high-grade lesions (severe dysplasia), n=12] and surgical specimens (resections of bronchogenic carcinoma, n=11) were stained with COX-2, Ki67, PCNA, CD34 and NG2 monoclonal antibodies. Microvessel density (MVD) was analysed based on anti-CD34 immunostaining. Lesions were positive for COX-2 in 12 out of 25 preneoplastic lesions, and in 10 out of 11 invasive carcinomas (p=0.025). In preneoplastic lesions, the mean percentage of Ki67 positive cells was lower compared to invasive carcinomas (37.4+/-5.8 versus 58.6+/-8.4%, p=0.043). In addition, significant differences in MVD were observed between preneoplastic and NSCLC specimen [35.3 (25.9, 61.9) versus 22.1 (20.1, 32.6), p=0.016]. No differences were observed in the mean percentage of PCNA or NG2 positive cells between preneoplastic lesions and invasive carcinomas. Findings of the present study indicate that increases in COX-2 and Ki67 expression may be associated with the development of bronchogenic carcinomas and possibly with acquisition of an invasive phenotype. In contrast, increased CD34 expression in preneoplastic lesions suggests that increased MVD may represent an early marker of lung carcinogenesis.

[Molecular methods in thrombophilic states diagnostics]. / Molekulární metody v diagnostice trombofilních stavu.

Molecular genetic methods passed into the field of investigation of thrombophilic states in 90th years of last century, along with the first discoveries of coagulation inhibitors (AT III, protein C and protein S). They have acquired a widespread use above all with the detection of the molecular basis of activated protein C (APC) resistance in 1994 by prof. Bertina. At the present time, a wide range of molecular genetic markers, linked with a clearly documented increased risk of thrombophilia are adapted. They include mutations of factor V Leiden 506R/Q, of protrombin 20210G/A, MTHFR 677C/T in homozygous form, mutation of PAI-1 4G/5G, mutations of different coagulation inhibitors and finally a range of polymorphisms with still not precisely defined increased risk for thrombophilia (F XIII Val34leu, platelets glycopeproteins, endothelial protein C receptor and trombomodulin). From the methodological viewpoint, all these techniques are based on the principle polymerase chain reaction (PCR). In the last period of time, however there was a rapid evolution, allowing a significant improvement in their laboriousness. Nowadays, splitting with the aid of restriction endonucleases, real time PCR or allel specific primers for PCR. The second, where molecular genetic methods are currently under use, is pathophysiological investigation of the single coagulation processes. Here, in a fact, most significant progress has been in the field of APC resistance made elucidation. Although still in the 90th years of the past century the genetical cause of these coagulation disturbance was unequivocally documented its clinically heterozygous appears not yet fully understood at the moment. Similarly, in prothrombin mutation, only the latest investigations have outlined the probable mechanism of expression. Concerning the future evolution of molecular genetic methods, there can be observed a clear cut tendency to better understanding the pathophysiologic cause of thrombophilia in comparison with the searching for new coagulation defects which consecutively bear lesser a relative risk of thrombosis.

[Disturbances of the plasma coagulation defects in retinal venous occlusions]. / Poruchy hemokoagulacní kaskády u retinálních venózních okluzí.

Occlusions of retinal veins (central and branch) represent multifactorialy-conditioned disease involving presumably older patients, in whom the changes of retinal vessels caused by hypertension and atherosclerosis present the most important pathophysiological factors for development of this disease. In last years, the intensive scientific research is focused to the explanation of the role of the defects of the coagulations cascade. Especially in younger patients, the most often mentioned defect of the coagulations cascade is called as APC-resistance. Up to 95% of all patients with APC-resistance are carriers of so called Leiden mutation. The aim of our study was to establish the prevalence of the ACP-resistance in 92 patients with central or branch occlusion of the retinal vein verified by means of angiography treated at the Department of Ophthalmology, Faculty Hospital, Olomouc, Czech Republic, EU, during the period 1999-2005. The control group consisted of 40 patients without any vascular, eye-related disease. In the group of patients with occlusion of the retinal vein, the prevalence of the APC-resistance was 10.9% and in the control group 5%. In the group of patients 55 years old and younger the prevalence of the ACP-resistance was 14.3%, in patients older than 55 years it was 5.6%. According to the relatively small groups of patients, the established difference did not reach the level of statistical evidence. Results of our study confirmed the conclusions of previously published papers that the prevalence of the APC-resistance is not significantly higher in patients with retinal vein occlusion according to the prevalence in controls.

[Coagulation factor VIII in the early postpartum period]. / Hladina faktoru VIII v casném poporodním období.

OBJECTIVE: Pregnancy is accompanied by changes in the coagulation and fibrinolytic systems. There is a marked increase in some of the coagulation factors, particularly fibrinogen and factor VIII. A high plasma levels of coagulation factor VIII is an important risk factor for thrombotic complications during pregnancy and puerperium. The aim of the study was to determine changes of the VIII:C in the early postpartum period. SETTING: Dept. of Obstetrics and Gynaecology, Medical Faculty of Palacký University, Olomouc. DESIGN: A longitudinal prospective study of 197 healthy women. Primi or multigravidas whose pervious pregnancies had been uncomplicated, aged 18-41 years. All of the deliveries were spontaneous and vaginal. First samples were taken between 24-72 hours postpartum. Women whose factor VIII plasma levels were higher than 150 (percentage of standard) were tested again after 6 weeks. Factor VIII:C was investigated by the one-step coagulation method. Statistical evaluation was done by Statsoft, Inc. (2001) Statistika Cz (Software system data analysis), version 6. RESULTS: Pregnancy is associated with increased levels of VIII:C. Mean value was 194.09 percentage of standards. 119 (60.4 %) of the tested women had VIII:C higher than 150%. The post-puerperal tests were done in 59 women and showed values similar to those from formerly published data in age-matched non-pregnant group. Mean value was 139.76%. CONCLUSION: Normal pregnancy is connected with increased levels of factor VIII. However elevated plasma levels of VIII:C is not associated with poor pregnancy outcome. The highest level of the clotting factor VIII was associated with patient's blood group A. Post-puerperal data showed distinct decrease of factor VIII. There is a necessity to rule out thrombophilia, in the case of the outlasting elevation of the factor VIII.

[Endothelial injury and activation of the coagulation cascade during radiofrequency catheter ablation]. / Endoteliální poskození a aktivace koagulacní kaskády behem radiofrekvencní katétrové ablace.

GOAL OF STUDY: To identify the extent of systemic activation of the coagulation cascade and to evaluate thrombogenic effect of the radiofrequency catheter ablation. METHODS AND RESULTS: Markers of activation of the coagulation cascade (D-dimers [DD]), markers of activation of the fibrinolytic system (tissue plasminogen activator [t-PA] and its inhibitor [PAI-1]), and markers of endothelial damage (von Willebrand factor [vWf]) were monitored in 50 patients undergoing catheter ablation. Levels of these substances were identified in time T0--at the beginning of the examination, T1--after finishing diagnostic part of the electrophysiological study, T2--after finishing all applications of radiofrequency energy, and T3--24 hours after T2. Levels of vWf were significantly elevated in time T1 compared to values in T0 (p < 0.001) and were further elevating after finishing the procedure in time T2 (p < 0.05). Levels of t-PA were also elevated in time T1, however after application of the radiofrequency energy, further increase in T2 was nonsignificant. Concentrations of PAI-1 were in time T2 significantly lower compared to T1 values (p < 0.001). Levels of DD were significantly elevated during entire procedure and elevated levels persisted even 24 hours later (p < 0.001). Levels of vWf a t-PA in time T2 correlated with total time of application of radiofrequency energy. Significantly higher activation of the coagulation cascade was identified, in patients undergoing isolation of pulmonary veins compared to patients undergoing catheter ablation of other arrhythmias. In the subgroup of patients treated with anticoagulation before the intervention elevation of DD levels in times T1 and T2 was lower compared to patients who did not undergo any treatment (p < 0.05). CONCLUSION: The radiofrequency catheter ablations activate the coagulation cascade. Moreover, application of the radio frequency energy increases systemic thrombogenic state and this effect "depends on the dose". A risk group make patients undergoing catheter isolation of pulmonary veins.

Detection of early atherosclerosis using the ultrasound parameter of the intima-media thickness of the common carotid artery in families with familial combined hyperlipidemia.

AIM: The aim of the present study was to quantify intima-media thickness (IMT) of the common carotid artery (CCA) in clinically asymptomatic members of familial combined hyperlipidemia (FCHL) families and to evaluate its association with lipids, apoproteins, blood pressure, surrogate markers of insulin resistance, fibrinogen and hs-CRP. METHODS: The group under study consisted of 82 individuals from 29 FCHL families (47 hyperlipidemic [HL] and 35 normolipidemic [NL]). They were compared with the age and sex adjusted control groups of healthy subjects (HL-c, n=20 and NL-c, n=20). IMT was measured by ultrasound at a far wall of both common carotid arteries. RESULTS: Hyperlipidemic subjects had increased IMT compared with healthy controls (0.695+/-0.118 vs 0.599+/-0.074 mm), with an age and sex corrected difference of 86 mm (p<0.001). No difference in IMT was recorded in NL FCHL members in comparison with their healthy controls. In HL subjects, significantly positive univariate correlations were observed between IMT and age, total cholesterol, LDL-cholesterol, non-HDL-cholesterol, apolipoprotein B, SBP, DBP, BMI, waist, fasting glycemia, C-peptide and proinsulin, whereas in NL subjects IMT correlated only with age. Multivariate regression analysis in FCHL subjects (HL+NL) revealed that age (p<0.001), sex (p<0.001), non-HDL-cholesterol (p<0.01) and BMI (p<0.05) were significant and independent predictors of IMT. CONCLUSIONS: The increase of IMT CCA in hyperlipidemic still clinically asymptomatic FCHL subjects corresponds to acceleration of the clinically ''silent'' atherosclerosis by about 8-14 years and is in agreement with their increased risk of atherosclerosis.