Euroforgen-NoE collaborative exercise on LRmix to demonstrate standardization of the interpretation of complex DNA profiles.

There has been very little work published on the variation of reporting practices of mixtures between laboratories, but it has been previously demonstrated that there is little consistency. This is because there is no current uniformity of practice, so different laboratories will operate using different rules. The interpretation of mixtures is not solely a matter of using some software to provide 'an answer'. An assessment of a case will usually begin with a consideration of the circumstances of a crime. Assumptions made about the numbers of contributors follow from an examination of the electropherogram(s)--and these may differ between the prosecution and the defence hypotheses. There may be a necessity to evaluate several sets of hypotheses for any given case if the circumstances are uncertain. Once the hypotheses are formulated, the mathematical analysis is complex and can only be accomplished by the use of specialist software. In order to obtain meaningful results, it is essential that scientists are trained, not only in the use of the software, but also in the methodology to understand the likelihood ratio concept that is used. The Euroforgen-NoE initiative has developed a training course that utilizes the LRmix program to carry out the calculations. This software encompasses the recommendations of the ISFG DNA commissions on mixture interpretation and is able to interpret samples that may come from two or more contributors and may also be partial profiles. Recently, eighteen different laboratories were trained in the methodology. Afterwards they were asked to independently analyze two different cases with partial mixture DNA evidence and to write a statement court-report. We show that by introducing a structured training programme, it is possible to demonstrate, for the first time, that a high degree of standardization, leading to uniformity of results can be achieved by participating laboratories.

Beneficial effect of ultra-low-dose aspirin in platelet activity alterations and haemorrhage observed in experimental portal hypertension.

Ultra-low-dose aspirin has shown a prothrombotic effect in the laser-induced thrombosis model. Several studies of our laboratory have shown a positive effect in rats with two different experimental models of portal hypertension: portal vein ligation, a model with an almost normal liver, and 30 days of bile duct ligation, a model with cirrhosis and presence of ascitis. In both models of portal hypertensive rats, bleeding time was prolonged and thrombi formation, in a laser-induced model of thrombi production, decreased. The hypotheses of the presented studies were that ultra-low-dose aspirin could decrease the bleeding complications in these models and that the mechanism for these effects could act thorough the COX pathway. In different studies, ultra-low dose of aspirin normalized the induced hemorrhage time, thrombi production, and platelet-endothelial cell interaction. The possible beneficial role of these doses of aspirin and mechanism of COX 2 inhibition are discussed.

2006 GEP-ISFG collaborative exercise on mtDNA: reflections about interpretation, artefacts, and DNA mixtures.

We report the results of the seventh edition of the GEP-ISFG mitochondrial DNA (mtDNA) collaborative exercise. The samples submitted to the participant laboratories were blood stains from a maternity case and simulated forensic samples, including a case of mixture. The success rate for the blood stains was moderate ( approximately 77%); even though four inexperienced laboratories concentrated about one-third of the total errors. A similar success was obtained for the analysis of mixed samples (78.8% for a hair-saliva mixture and 69.2% for a saliva-saliva mixture). Two laboratories also dissected the haplotypes contributing to the saliva-saliva mixture. Most of the errors were due to reading problems and misinterpretation of electropherograms, demonstrating once more that the lack of a solid devised experimental approach is the main cause of error in mtDNA testing.

Effects of external electrical stimulation on laser-beam-induced experimental thrombosis.

External electrical stimulation (EES) has demonstrated venous antithrombotic properties. The aim of this investigation was to study its antithrombotic properties using unfractionated heparin (UFH) as a reference in arterial and venous mesenteric microvessels. For this purpose a rat model of laser-beam-induced thrombosis was used. Four groups of rats were investigated (n = 7): control, UFH (5 mg /kg), UFH (2.5 mg/kg) and electrical stimulation [e-vascular, E = 10, alternate polarity, in two protocols: (i) 30 min before and during thrombosis induction and (ii) only 30 min before thrombosis induction as preventive stimulation]. This effect was tested in arterial and venous microvessels in the four groups using the continuous protocol and only in arterial microvessels using the preventive protocol. Three parameters were studied: the number of laser beams needed to induce platelet thrombus formation, the number of emboli and the duration of embolization. Induced hemorrhagic time (IHT), platelet aggregation induced by ADP and coagulation tests (activated partial thromboplastin time, aPTT, prothrombin time, pTT, and fibrinogen level) were also determined. Continuous electrical stimulation decreased the number of emboli and the duration of embolization both in arterioles and in venules. It also significantly reduced the amplitude and velocity of the ex vivo platelet aggregation induced by ADP. Contrary to UFH, neither aPTT nor pTT were affected in platelet-poor plasma collected after the induction of thrombosis by laser beam, nor did it modify IHT. Preventive electrical stimulation produced similar results with fewer effects on induced venous thrombosis. These data suggest that EES has a potent antithrombotic effect on arterial and venous thrombosis without any hemorrhagic adverse effects, making it a very promising tool in the prevention and treatment of venous and arterial thromboembolic complications.

Increase of arterial thrombosis parameters in chronic Helicobacter pylori infection in mice.

An animal model was developed to study arterial thrombosis and determine if animals infected with Helicobacter pylori behave differently after induction of direct damage to blood vessels. Twenty-one C56/BL6 mice inoculated with the "Sydney strain" of H. pylori and 19 uninfected animals were kept for 1 year before testing. Vascular lesions were induced to mesenteric arterioles (15-25 microm diameter) by Argon laser. The dynamic course of thrombus formation was continuously monitored by a video camera for 10 min. Three parameters were assessed: (1) the number of laser pulses required to induce thrombus formation, (2) the number of platelet emboli removed by the blood flow and, (3) the duration of embolization. Additionally, blood was tested for platelet aggregation, fibrinogen, and cell count. Of the parameters measured, statistical differences between infected and uninfected mice concerned the number of emboli formed (6.00+/-2.18 infected vs. 3.89+/-1.37 non-infected, p=.0006) and the duration of embolization (2.41+/-0.73 min infected vs. 1.47+/-0.61 min non-infected p>.0001). A significant difference was also found in the fibrinogen levels between infected and uninfected mice. Chronic infection of mice with H. pylori leads to increased platelet embolization after damage to arterioles. These results are in favor of the possible involvement of H. pylori infection in the acute phase of coronary heart disease (CHD).

Time related neutralization of two doses acetyl salicylic acid.

Aspirin has a well established role in the prevention of arterial thrombosis. Discussion on the efficacy and safety of aspirin in the treatment and prophylaxis of thrombosis has become an important issue. In fact, hemorrhage complications are often associated with its use. On the other hand, previous studies showed unexpected thrombotic potencies associated with the presence of this drug at ultra low doses (ULD) in the circulation. In this study, we aimed to evaluate the effect of aspirin at ULD, injected 1, 2, or 3 hours after the administration of aspirin at 100 mg/kg, on hemostasis and bleeding in rats. We used an experimental model of thrombosis induced by laser beams to evaluate these effects. Platelet aggregation was determined by Cardinal and Flower method. Results from this investigation demonstrate that the neutralizing effect of aspirin at ULD did not operate significantly 1 hour after the injection of aspirin at 100 mg/kg. This effect was observed 2 and 3 hours after. The use of aspirin at ULD to neutralize the side effects of aspirin at high doses will reduce the hemorrhagic risk during extra corporeal circulation. The therapeutic benefit and safety of aspirin therapy in the treatment of cardiovascular diseases can be obtained.

Thrombogenic potential of contrast media in an experimental model of laser-induced thrombosis.

Controversy still exists about the pro- or antithrombotic side effects of contrast media used in daily medical practice. Recent reports have shown that various contrast media, including ionic compounds, have deleterious prothrombotic actions. A new evaluation of these adverse side effects is reported here, with the study of the dose-effect relationship. Two ionic (ioxaglate and diatrizoate) and two non-ionic contrast media (iopamidol and iohexol) were studied. Experiments were done on 22 groups of 5 Wistar male rats each, using a Laser Argon-induced thrombosis model in mesenteric microvessels. Three parameters were studied: the number of laser beams needed to induce platelet thrombus formation, the number of emboli, and the duration of embolization. Platelet count and platelet aggregation also were determined. Iopamidol and iohexol induced a significant rise in both the number of emboli and the duration of embolization in mesenteric microvessels at doses up to 1 mL/kg. Ioxaglate and diatrizoate also significantly increased these parameters at doses up to 2 mL/kg. All the products tested decreased platelet count, inducing a -17 to -30% variation from control values. Diatrizoate and ioxaglate inhibited platelet aggregation, while iopamidol and Iohexol behaved as activators. All non-ionic, and to a lesser extent, all ionic contrast media demonstrated prothrombotic properties.

Effects of acetyl salicylic acid therapy on an experimental thrombosis induced by laser beam.

Aspirin inhibits the synthesis of both platelet and vascular arachidonic acid metabolism which have opposite effects on platelet functions. The rationale for its clinical use as an antithrombotic drug has therefore been questioned. Therefore, we investigated the effects of acetylsalicylic acid (ASA) at 100 mg/kg on an experimental thrombosis induced by laser beams using different groups of rats that were previously treated with the same dose (100 mg/kg), according to the delay between the first and second injections. A partial occlusion was induced by laser beams in the rat mesenteric microvessels (15-25 m). The thrombus formed within seconds after the laser lesion; both it and the embolization which began within minutes after, were continuously accounted. Experiments were done on 11 groups of 5 animals each: 45 rats received a first injection of ASA at j(0) and a second injection 30 minutes before thrombosis induction at j(0)+x (x=2, 4, 6, 8, 9, 10, 12, 14 and 16 days). Different groups are defined according to the x value. The rats receiving NaCl 0.9% or a single injection of ASA at 100 mg/kg 30 minutes before thrombosis induction were used as control (Group I) and reference group (Group II) respectively. In this study, ASA treatment showed two types of results. The administration of ASA (100 mg/kg) 30 minutes before laser-induced thrombosis prevented thrombus formation. In the same way, ASA injected to rats already treated with the same dose 2 or 4 day later also demonstrated a potent antithrombotic effect. The same trends were observed with animals receiving the second injection (100 mg ASA) at j(0+8), j(0+12), j(0+14), and j(0+16). However, when injected to rats at j(0+6) and at j(0+10), ASA did not shown any effects on thrombus formation compared to the control (p>/=0.05). The same phases of ASA action were observed on the induced hemorrhagic time. The antithrombotic effects of the later second injection of ASA (100 mg/kg) were neutralized in rats previously receiving the same dose of this drug. This phenomenon seems to be periodic and is of great importance for the observance of ASA treatment.

Aspirin and fraxiparine in the prevention of laser induced thrombosis in the presence of iodinated contrast media.

The purpose of this study was to investigate the thromboembolic properties of ionic and nonionic contrast media in rats pretreated with aspirin and/or fraxiparine using an experimental model of laser induced thrombosis in the mesenteric microvessels of 17 groups of five male Wistar rats each. Two ionic (ioxaglate and diatrizoate) and two nonionic contrast media (iopamidol and iohexol), alone or associated with antithrombotic drugs (aspirin and/or fraxiparine) were studied. To evaluate the effects of these substances in this model, the number of laser beams needed to induce platelet thrombus formation, the number of emboli detached from the thrombus and the duration of embolization were quantified. Platelet aggregation induced by ADP, induced hemorrhagic time (IHT) and haemoglobin loss level were also determined. Both contrast media injected at 3 ml/kg caused a significant increase in the number of emboli and the duration of embolization (p<0.05). Pretreatment with aspirin and/or fraxiparine in the presence of ionic contrast media showed antithrombotic activities equal to those obtained when they were tested alone (p<0.05), while in the presence of nonionic contrast media, these drugs only neutralised the prothrombotic effects. There were no differences with the NaCl treated group (p>0.05). The ionic contrast media, and to a lesser extent the nonionic contrast medium: iohexol, inhibited platelet aggregation, while iopamidol behaved as an activator. The antithrombotic drugs tested in this study prevent the prothrombotic activities of contrast media therefore suggesting their use before radiographic procedures.

Combination of two doses of acetyl salicylic acid: experimental study of arterial thrombosis.

The antithrombotic effect of high dose acetylsalicylic acid is well known, and recently, in vitro studies hinted the potent thrombotic effect of ultra-low dose of acetylsalicylic acid (<1mg/day) showing a significant decrease in bleeding time. In this study, we investigated the effect of a combination between a high and an ultra-low dosage (100 mg/kg+ 10(-30) mg/kg) on an arterial thrombosis induced by a laser beam. We used an intravital microscopic technique, allowing to evaluate (anti)-thromboembolic events at previously determined locations of microvasculature. Thrombus formation was induced by argon-laser shot. The instrumental test setup was completed with a video system, to select mesenteric arterioles with the same diameter (between 15 and 25 microm). The changes in platelet aggregability were determined by Cardinal and Flower method, and the concentration of acetylsalicylic acid in the plasma was measured by high pressure liquid chromatography. Antithrombotic effect of high dose (100 mg/kg) acetylsalicylic acid was confirmed in all results obtained. Asa injected at ultra-low dose (10(-30) mg/kg) had a potent thrombotic properties and decreased significantly the bleeding time. The subcutaneous administration of the combination of the two doses permitted to come back to the control values, and the bleeding time was shortened compared to control group.

Fibrinogen as a factor of thrombosis: experimental study.

Epidemiological, clinical, and experimental studies have clearly demonstrated the strong association between baseline fibrinogen level and risk of thromboembolic complications. The pathogenesis of postoperative or post-traumatic thrombosis in man is associated with fibrinogen level in plasma. The purpose of this study was to evaluate the effects of fibrinogen administration on thrombus formation at different dosages. To investigate these effects, we used an experimental model of induced thrombosis in rat microcirculation. This model allows single endothelial cell destruction by laser injuries, thus leading to thrombus formation. Fibrinogen was injected intravenously via penis vein and tested at various dosages (50, 100, and 200 mg/kg), 60 minutes after injection on arterial thrombosis induction and 120 minutes after injection on venous thrombosis induction. Results showed that the administration of fibrinogen increases the number of emboli, the duration of embolization, the amplitude, and the velocity of the ex-vivo platelet aggregation induced by ADP (p < 0.05). A positive correlation between the percent of fibrinogen increase in plasma and the enhancement of thromboembolic risk in the experimented animals was observed.

Thromboembolic complications several days after a single-dose administration of aspirin.

The antithrombotic properties of acetyl salicylic acid (ASA) used at current doses are largely demonstrated. However, our previous study showed unexpected thrombotic potencies associated with the use of this drug. In this study we investigate the effect of aspirin on an experimental thrombosis induced by laser beams, according to its in vivo plasma concentration. Experiments were done on nine groups of seven Wistar male rats. The groups are defined by the delay between aspirin administration time and the laser-induced thrombosis time. Results from this study showed an enhancement of thromboembolic complications when thrombosis was induced 8 or 10 days after aspirin administration; the number of emboli and the duration of embolization are increased, compared to the control group. The prothrombotic properties of ASA demonstrated in this study, might limit its therapeutic benefit and might explain thromboembolic complications observed in some ASA-treated patients. These results also suggest a biological monitoring several days after aspirin administration to patients.

[Recent developments of genetic markers in legal practice]. / Données récentes sur les empreintes génétiques en pratique judiciaire.

The human identification in forensic science is currently based upon the study of highly polymorphic systems with mendelian transmission: the STRs (Short Tandem Repeats). These genetic markers are three to seven bases repetitive sequences spread all over the genome. They are detected by using the Polymerase Chain Reaction (PCR). This analysis method makes it possible to amplify several different loci from very low quantities of genomic DNA (Desoxyribonucleic Acid) in very short periods of time without using any radioactive substances. Interpretation of the results is simple because the amplified fragments are of a known size. The alleles known in each system with discrete values allow the calibration of the revealing gel and the populations studies; moreover, they may be entered into a database. The fragility of this test implies specific precautions with the Laboratory's organization in order to avoid any kind of contamination. Both the establishment of a quality assurance responding to the ISO 9002 standard together with internal quality controls would ensure that these tests are reliable and repeatable in the opinion of the Courts. Only authorized laboratories may perform DNA testing on the occasion of legal proceedings.

Evaluation of plasma levels of tumour necrosis factor alpha and interleukin-6 as rejection markers in a cohort of 142 heart-grafted patients followed by endomyocardial biopsy.

The rejection reaction after cell or organ transplantation has to be detected as early as possible in order to conduct optimal immunosuppressive treatment. Among the numerous events leading to rejection, cytokine production, especially of tumour necrosis factor alpha, is particularly important. Interleukin-6 and tumour necrosis factor alpha were investigated in 142 heart-grafted patients in order to define an early peripheral non-invasive marker of an acute rejection that could fit well with myocardial biopsy results. Cytokines were immunoenzymatically measured in blood specimens collected on the day of the endomyocardial biopsy. The values were compared to the grade of heart graft rejection established according to pathological criteria. Plasma interleukin-6 and especially tumour necrosis factor alpha determined on the day of the rejection diagnosis were significantly increased in the patient sample with moderate or severe rejection when compared with mean values of interleukin-6 and tumour necrosis factor alpha in the patient sample without rejection or with mild rejection (P = 0.04 and 0.001 respectively). Because high levels of tumour necrosis factor alpha may appear before histological signs, this biological marker could be useful in the follow-up of heart-grafted patients.

A review of the collaborative exercises on DNA typing of the Spanish and Portuguese ISFH Working Group. International Society for Forensic Haemogenetics.

Since 1992 the Spanish and Portuguese Working Group (GEP) of the International Society for Forensic Haemogenetics (ISFH) has been organizing collaborative exercises on DNA profiling with the aim of making progress on standardization and discussing technical and statistical problems in DNA analysis. A total of four exercises (GEP-92 to GEP-95) have been carried out until now. A consequence of these exercises was the creation of a quality control programme in Spain and Portugal in 1995 which was carried out simultaneously with the GEP-95 exercise. The number of participating laboratories increased from 10 in the first exercise (GEP-92) to 19 in the last exercise (GEP-95). Despite this increasing number of participating laboratories, results remained satisfactory. In the last exercises, all the laboratories used PCR-based DNA polymorphisms with an increasing number of markers obtaining good results. SLPs were used by only 30% of laboratories in the last two exercises but the results indicated a good level of expertise in most of these laboratories. The reasons for these successful results are the common use of the EDNAP protocol for SLP analysis and commercially available kits or common sequenced allelic ladders for PCR-based DNA polymorphisms.

Action of neurotransmitters: acetylcholine, serotonin, and adrenaline in an experimental arterial thrombosis induced by oxygen free radicals.

It is well known that high stress and particularly an enhancement of plasma catecholamines and myocardial infarction have a close relation. In addition, adrenaline is presented as a prothrombogenic agent in vivo. The role of the other agents such as serotonin or acetylcholine, in the development of arterial thrombosis is somewhat uncertain, although, the role of each of them is often considered at the level of vascular regulation only. Therefore, the present study was designed to investigate the effects of three neurotransmitters on experimental arterial thrombosis model induced by generation of free radicals. The results demonstrate that intravenously injection of adrenaline or serotonin (1 ng/kg) stimulated arterial thrombosis formation, whereas injection of high dose of acetylcholine (5 mg/kg) slackened the thrombosis formation.

Action of neurotransmitters: acetylcholine, adrenaline and serotonin on arterial thrombosis induced by a laser beam.

The releasing of catecholamines is increased in stress situation which promotes the formation of circulating platelet aggregates, and could participate in the arterial thrombosis formation in coronary diseases. The purpose of this study is to evaluate the thrombogenic action of some neurotransmitters, and their participation through the vessel's vasomotoricity, in the growth of an arteriolar thrombosis. Endothelial cells destruction, induced by a laser beam in mesenteric arteriole of the rat were observed to determine changes in thrombus growth, through the embolization and variation of vessel diameter. It is desirable to get insight into the interrelation of thrombus formation and local vasomotoricity in the presence of acetylcholine, adrenaline and serotonin. The administration of acetylcholine (5mg/kg) increases the number of emboli which detached from thrombus, and decreases the thrombus area. Therefore, acetylcholine induces a variation of the vessel's diameter, a vasodilation in the intact vessel and a vasoconstriction when the endothelium is removed. Two vasoconstrictor agents are used: adrenaline and serotonin which increase the number of laser injuries required to induce thrombus formation, decrease the number of emboli and the duration of embolization (p < 0.05). They cause a potent vasoconstriction. These neurotransmitters seem to be involved in the arterial thrombosis induced by laser beam, promoting or not the platelet aggregation, and modulating the vascular tone by the endothelium.

New class of heparin derivatives with a potent antithrombotic effect and a very limited hemorrhagic activity.

Eight heparin derivatives (HD1 to HD8) were prepared by mixing various doses of protamine with a fixed amount of heparin. After centrifugation and elimination of the formed complex, the supernatant was lyophilized and titrated. A dose of 5 mg (dry weight)/kg of these heparin derivatives was injected subcutaneously to rats in classical thrombosis model induced by stasis. Antithrombotic, hemorrhagic and anticoagulant activities are investigated and compared to those of Unfractionated Heparin (UFH) and LMWH (Enoxaparin). Seven rats in each group were studied. Significant antithrombic effect was exhibited by HD1, HD2, HD3, and HD4 which decreased progressively. Only HD1, HD2, and HD3 augmented hemorrhagic activity but to a lower degree than UFH and LMWH. No change was observed by coagulation assays; Activated Partial Thromboplastin Time (APTT) and Diluted Thrombin Time (dTT) and there was no effect on platelet aggregation except for UFH. These heparin derivatives might present advantages over UFH and LMWH in the treatment of thrombosis.

Importance of photo activation of rose bengal for platelet activation in experimental models of photochemically induced thrombosis.

Generation of oxygen free radicals in the lumen of a vessel leads to NO inactivation, modification of lipid components of platelets and endothelial cell membranes, and platelet activation. On this basis, many experimental models of thrombosis have been developed, where the formation of a platelet rich thrombus follows the illumination of a vessel with an appropriately filtered light after intravenous administration of Rose Bengal or another sensitizing dye. However the detailed mechanism of thrombus formation remains poorly known. This work appreciates the contribution of platelet activation directly induced by oxygen free radicals in formation of the platelet rich thrombus, from a study of human platelet aggregation in presence of photo-activated Rose Bengal. The results demonstrate that direct activation of platelets by free radicals generated by Rose Bengal is of low or no importance in formation of the thrombus. Therefore, the main trigger of platelet aggregation and thrombosis in these models is primary endothelial cells injury.