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.

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.

[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.

[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.

[Occurrence of gene mutations in factor V Leiden, prothrombin and methylenetetrahydrofolate reductase in patients with pre-eclampsia]. / Výskyt mutací genu faktoru V. Leiden, protrombinu a metylentetrahydrofolátreduktázy u pacientek s preeklampsií.

OBJECTIVE: The aim of the study was to detect the incidence of thrombophilia FV Leiden, prothrombin G20210A, MTHFR C677T, resistance to activated protein C, and deficiency of protein C and S in pregnant or puerperal women with preeclampsia and control group. DESIGN: Case-control study. SETTING: Department of Obstetrics and Gynecology, Department of Hemato-oncology, Medical Faculty, Palacký University, Olomouc. METHODS: The group of 38 women with confirmed diagnosis of preeclampsia were examined for resistance to activated C protein. (Coatest, APC resistence Chromogenix), levels of C and S protein. For the detection of FV:Q506, prothrombin G20210A and MTHFR C677T allels was done by PCR test. The control group had 50 women, chosen randomly. The statistical evaluation was performed by the STATISTICA program. For the analyzing of the continuous variables the Student's T-test was used. Values below p < 0.01 were considered to be statistically significant. RESULTS: Women who suffered from preeclampsia came to mean age of 32.8 +/- 4.3 (years), mean gestational age 33.6 +/- 2.8 (weeks), mean systolic pressure 163 +/- 21 mmHg, diastolic pressure 108 +/- 8 mmHg. All of the results were rendered to be statistically significant in comparison the control group. On the other hand, the results in observed haematologic parameters were not significant (Preeclampsia/controls). FV Leiden--heterozygous subjects 4/3, prothrombin G20210A 1/0, C677T heterozygous subjects 12P10, C677T 2/0, protein C and S deficiency 2(0. Resistance to activated C protein was found in 14 (37%) patients, in comparison to 6 (12%) in the control group (p = 0.0073). CONCLUSION: We didn't find any difference in the prevalence of genetic mutations in patients with preeclampsia compared to the control group. The prevalence of APC resistance was statistically higher in preeclamptic patients compared to the control group.

[Activated protein C resistance and deep venous thrombosis in pregnancy]. / Rezistence na aktivovaný protein C a hluboká zilní trombóza v tehotenství.

OBJECTIVE: The aim of the study was to detect the incidence of resistance to activated protein C in pregnant or puerperal women with confirmed diagnosis of deep venous thrombosis or pulmonary embolism. DESIGN: Case-control study. SETTING: Dept. of Obstetrics and Gynaecology, Dept. of Haemato-oncology, Medical Faculty, Palacky University, Olomouc. METHODS: The group of 33 women with confirmed deep venous thrombosis were examined for resistance to activated C protein. The levels of C and S proteins, antithrombin III., prothrombin, heparin II cofactor and plasminogen were measured. Patient with APC ratio below 1.86, were tested by PCR analysis for the detection of the FV:Q506 allele. The control group had 51 pregnant women, chosen randomly. The statistical evaluation was performed by STATISTICA program. For the analyzing of the continuous variables the Students' T-test was used. For the categorical variables the chi 2 test was used (for comparison of 2 relative values). Values below P < 0.01 were considered to be statistically significant. RESULTS: Women who have shown to suffer from deep venous thrombosis (DVT) came to the mean age of 31 +/- 3.1 years, weight 75.3 +/- 7.9 kg and BMI 28.7 +/- 4.3. All of the results above were rendered to be statistically significant. 54.5% of thrombosis occurred in the 3rd trimester, 61% were localised to the left iliofemoral vein. APC resistance appeared in 17 (51%) women with DVT, in comparison to 5 (9.8%) women in the control group (i.e. statistically significant). Factor V appeared in II (33.3%) women, in comparison to 3 (5.9%) women in the control group. CONCLUSION: APC resistance and factor V. Leiden represent as important factors in the aetiology of deep venous thrombosis in pregnancy.

[Mesenteric localization of thrombosis in the antiphospholipid syndrome]. / Mezenteriální lokalizace trombózy pri antifosfolipidovém syndromu.

The authors describe a thrombophil condition manifested at an atypical site--partial thrombosis of the sup. mesenteric vein. The diagnosis was made during surgery followed by haemocoagulation examination which repeatedly revealed positive lupus anticoagulans (LA). Early diagnosis of the acute state with initiation of anticoagulant treatment and solution of the diagnostic conclusion--antiphospholipid syndrome--make an optimal therapeutic procedure possible.

[Clinical aspects of Chlamydial infection]. / Klinické aspekty chlamydiových infekcí.

Chlamydiae are one of the most widespread pathogens of man, though up to 90% of infected people have few or no symptoms. This bacterial pathogen has been shown to cause respiratory track infections, pelvic inflammatory disease and articular diseases. We explored group of 20 patients with the elevated anti-chlamydial antibody titres and clinical significance of infection. All patients were also taking a tree-week course of antibiotics therapy.

L-asparaginase--a reassessment.

The authors review some historical data connected with the discovery of the enzyme l-asparaginase and then summarize the basic informations concerning the mechanism of action, clinical application, toxicity and future prospectives of this substance. From the clinical viewpoint, a broader use of l-asparaginase prepared from Erwinia carotovora instead of Escherichia coli seems to be warranted.

Bleeding complications in chronic lymphocytic leukaemia.

In a series of 164 patients, a retrospective study has been carried out on the incidence, frequency and character of bleeding episodes occurring in chronic lymphocytic leukaemia. The authors stress the relatively high frequency of haemorrhagic complications which were causing death in a significant proportion of cases (5.5%). Thrombocytopenia was the main precipitating factor of bleeding on most occasions.

Clinicopathological aspects of Richter's syndrome.

Richter's syndrome represents an acute transformation of chronic lymphocytic leukaemia or other type of low-grade malignant non-Hodgkin lymphoma to highly malignant anaplastic large-cell lymphoma. The prognosis of this complication is highly unfavourable. The authors report on eight cases of Richter's syndrome observed in a series of 198 patients with the clinical diagnosis of chronic lymphocytic leukaemia and evaluate some clinicopathological and immunological peculiarities of this disease.