Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms.

To better understand envenoming and to facilitate the development of new therapies for snakebite victims, rapid, sensitive, and robust methods for assessing the toxicity of individual venom proteins are required. Metalloproteinases comprise a major protein family responsible for many aspects of venom-induced haemotoxicity including coagulopathy, one of the most devastating effects of snake envenomation, and is characterized by fibrinogen depletion. Snake venoms are also known to contain anti-fibrinolytic agents with therapeutic potential, which makes them a good source of new plasmin inhibitors. The protease plasmin degrades fibrin clots, and changes in its activity can lead to life-threatening levels of fibrinolysis. Here, we present a methodology for the screening of plasmin inhibitors in snake venoms and the simultaneous assessment of general venom protease activity. Venom is first chromatographically separated followed by column effluent collection onto a 384-well plate using nanofractionation. Via a post-column split, mass spectrometry (MS) analysis of the effluent is performed in parallel. The nanofractionated venoms are exposed to a plasmin bioassay, and the resulting bioassay activity chromatograms are correlated to the MS data. To study observed proteolytic activity of venoms in more detail, venom fractions were exposed to variants of the plasmin bioassay in which the assay mixture was enriched with zinc or calcium ions, or the chelating agents EDTA or 1,10-phenanthroline were added. The plasmin activity screening system was applied to snake venoms and successfully detected compounds exhibiting antiplasmin (anti-fibrinolytic) activities in the venom of Daboia russelii, and metal-dependent proteases in the venom of Crotalus basiliscus. Graphical abstract ᅟ.

Skin permeation and retention of topical bead formulation containing tranexamic acid.

The objective of this study is to develop a topical bead formulation of tranexamic acid (TA) which can be used concomitantly with laser treatment. The bead formulation of TA (TAB) was successfully prepared by fluidized bed drying method. Physicochemical properties of the TAB were evaluated in terms of chemical stability of TA and differential scanning calorimetry. TA in the bead was stable up to six months at 25°C and existed as amorphous state. In vitro skin permeation and in vivo skin retention of TA in the beads were significantly higher compared to a commercial product. When the bead was dissolved into distilled water and applied concomitantly with laser treatment, the amount of TA retained in the skin in the in vivo study was inversely proportional to the energy levels of laser treatment, indicating absorption into subcutaneous tissue and drainage to systemic circulation. Therefore, when laser treatment is used concomitantly with TAB, energy level should be very carefully monitored to avoid possible adverse events associated with systemic side effects of TA.

In vitro effects of an extracorporeal membrane oxygenation circuit on the sequestration of ϵ-aminocaproic acid.

OBJECTIVE: To assess the in vitro effects of drug sequestration in extracorporeal membrane oxygenation (ECMO) on ϵ-aminocaproic acid (EACA) concentrations. METHODS AND DESIGN: This in vitro study will determine changes in EACA concentration over time in ECMO circuits. A pediatric dose of 2,500 mg was administered to whole expired blood in the simulated pediatric ECMO circuit. Blood samples were collected at 0, 30, 60, 360 and 1440-minute intervals after initial administration equilibration from three different sites of the circuit: pre-oxygenator (PRE), post-oxygenator (POST) and PVC tubing (PVC) to determine the predominant site of drug loss. The circuit was maintained for two consecutive days with a re-dose at 24 hours to establish a comparison between unsaturated (New) and saturated (Old) oxygenator membranes. Comparisons between sample sites, sample times and New versus Old membranes were statistically analyzed by a linear mixed-effects model with significance defined as a p-value <0.05. RESULTS: There were no significant differences in EACA concentration with respect to sample site, with PRE and POST samples demonstrating respective mean differences of 0.30 mg/ml and 0.34 mg/ml as compared to PVC, resulting in non-significant p-values of 0.373 [95% CI (-0.37, 0.98)] and 0.324 [95% CI (-0.34, 1.01)], respectively. The comparison of New vs. Old ECMO circuits resulted in non-significant changes from baseline, with a mean difference of 0.50 mg/ml, 95% CI (-0.65, 1.65), p=0.315. CONCLUSION: The findings of this study did not show any significant changes in drug concentration that can be attributed to sequestration within the ECMO circuit. Mean concentrations between ECMO circuit sample sites did not differ significantly. Comparison between New and Old circuits also did not differ significantly in the change from baseline concentration over time. Sequestration within ECMO circuits appears not to be a considerable factor for EACA administration.

Analysis of Prethrombotic States in Patients with Malignant Tumors.

BACKGROUND: This study aimed to investigate the incidence and risk factors for a prethrombotic state in patients with malignant tumors. MATERIALS AND METHODS: Plasma d-dimer (D-D) in patients with malignant tumors was measured. Abnormal rates of D-D and possible risk factors like gender, age, type of tumor, and staging of tumor were analyzed. RESULTS: Of 1,453 patients, 629 demonstrated plasma D-D abnormality (43.3%). The D-D abnormal rate of male patients (n=851, 43.5%) was not statistically significantly different from that for female patients (n=602, 43.0%) (p>0.05). D-D abnormal rate increased with age and was statistically significant among different age groups (p<0.05). Regarding staging of tumor, D-D abnormal rate in patients with phase I was 2.0%, 6.2% in phase II, 47.6% in phase III and 83.1% in phase IV, with statistically significant differences between phase III and II, as well as phase III and IV (p<0.01). CONCLUSIONS: A prethrombotic state was closely related to malignancy of tumors. The risk factors for a prethrombotic state include age and tumor stage.

Variation of D-dimer values as assessment of pulmonary thromboembolism during adulticide treatment of heartworm disease in dogs.

Dirofilaria immitis adult worms lodged in the pulmonary arteries are the causative agent of heartworm disease, characterized by endarteritis and obstruction of the blood flow. Dying worms form embolic fragments which trigger thrombosis. Thus, pulmonary thromboembolism is an inevitable consequence of successful adulticide therapy. Clinical signs of pulmonary thromboembolisms are highly variable and non-specific, and its diagnosis is often difficult to obtain. The D-dimer assay is considered the marker of choice for dogs with a suspicion of pulmonary thromboembolism. The aim of the present study was to evaluate the variation of D-dimer and platelets in 15 heartworm-infected dogs during the adulticide treatment with ivermectin, doxycycline and melarsomine. Nine dogs were considered to have a low parasite burden and 6 had a high burden. Measurements were carried out on days 0, 60, and afterwards weekly on days 67, 75, 82, 91, 98, 106, 113, 120, and finally on day 271 after treatment. 40% of dogs showed pathological concentrations of D-dimer and 40% showed slight or mild thrombocytopenia on day 0. The levels of D-dimer decreased by day 60, probably due to the joint action of the ivermectin, doxycycline and exercise restriction. All dogs with high parasite burden showed elevated values of D-dimer on several occasions during the treatment, reaching the highest values the first and second week after the injections of melarsomine. Only 33.3% of the dogs with low parasite burden showed elevated D-dimer levels at some point during the treatment, and all of them showed undetectable levels from the third week after the first injection of melarsomine. On day 271, all dogs showed undetectable levels of D-dimer. There was no correlation between thrombocytopenia and levels of D-dimer during the treatment. The evaluation of serum D-dimer appears to be a supportive test in the assessment and monitoring of pulmonary thromboembolism in dogs with heartworm disease during the adulticide treatment.

[Emergency department point of care testing: what are the benefits and for which patients?]. / Laboratoire délocalisé aux urgences: quels bénéfices pour quels patients?

Laboratory tests contribute to patient length of stay in the emergency department. Therefore, rapid tests performed at the bedside (POCT or point of care testing) are attractive because they allow the emergency physician to obtain immediate biological, diagnostic and/or prognostic data. Userfriendly and with validated analytical performance, POCT have the potential to reduce laboratory time, patient length of stay and time to treatment or disposition. The expected benefit from POCT implementation will depend on the type of patients involved (inpatient or outpatient), their clinical condition and their overall care. Furthermore, logistical and economic implications should also be taken into account.

[The unresolved issue of false-positive D-dimer results in the diagnostic workup of pulmonary embolism]. / Diagnostic d'embolie pulmonaire: problématique résiduelle des D-dimères faussement positifs.

The unresolved issue of false-positive D-dimer results in the diagnostic workup of pulmonary embolism Pulmonary embolism (PE) remains a difficult diagnosis as it lacks specific symptoms and clinical signs. After the determination of the pretest PE probability by a validated clinical score, D-dimers (DD) is the initial blood test in the majority of patients whose probability is low or intermediate. The low specificity of DD results in a high number of false-positives that then require thoracic angio-CT. A new clinical decision rule, called the Pulmonary Embolism Rule-out criteria (PERC), identifies patients at such low risk that PE can be safely ruled-out without a DD test. Its safety has been confirmed in US emergency departments, but retrospective European studies showed that it would lead to 5-7% of undiagnosed PE. Alternative strategies are needed to reduce the proportion of false-positive DD results.

[Changes of haemostatic parameters in patients with acute cardioembolic stroke].

Haemostatic parameters: platelet aggregation, hematocrit, fibrinogen, antithrombin III, fibrinolytic activity and euglobulin lysis time, plasminogen, plasminogen activator and anti-activator activity, thrombin- antithrombin and plasmin-antiplasmin complexes and serum von Willebrand factor were studied at the most acute stage of cardioembolic stroke (1-7 days after development of neurological symptoms). State of the vascular wall was assessed using the "cuff"-test. The activation of hemostasis in patients with cardioembolic stroke was characterized by the hypercoagulation and decrease of fibrinolysis and athrombogenic potential of vessel wall properties (antiaggregation, fibrinolytic and anticoagulative). In conclusion, haemostatic disbalance and decreasing of athrombotic potential of the vessel wall may be the indicator of embolism in patients with cardioembolic stroke.

Computerized alert reduced D-dimer testing in the elderly.

OBJECTIVE: To assess the effect of a targeted age-specific computerized alert to reduce D-dimer testing in elderly patients. STUDY DESIGN: A single-crossover cluster randomized trial of computerized alerts during physician order entry involving 8 ambulatory care clinics in a group-model integrated care delivery system. METHODS: The rate of completed D-dimer tests per 1000 patient visits, ratio of completed venous ultrasonography to completed D-dimer tests, and rate of completed venous ultrasonography per 1000 patient visits. RESULTS: The rate of completed D-dimer tests per 1000 visits among patients 65 years and older in intervention clinics decreased from 5.02 to 1.52 (95% confidence interval [CI], -4.20 to -2.80; P <.001), which persisted throughout the study period. The rate of completed D-dimer tests per 1000 visits among patients 65 years and older in control clinics decreased from 3.14 to 2.11 (95% CI, -1.66 to -0.04; P <.001 for interaction). After activation of the alert in control clinics, the rate of completed D-dimer tests per 1000 visits among patients 65 years and older decreased from 2.11 to 0.81 (95% Cl, -1.79 to -0.80; P <.001). After activation of the alert in each clinic group, the ratios of completed venous ultrasonography to completed D-dimer tests increased from 1.17 to 4.05 (95% CI, 2.52-3.22) and from 2.25 to 7.29 (95% CI, 3.74-6.35) in intervention clinics and control clinics, respectively (P <.001 for both). CONCLUSION: An electronic age-specific alert targeted to a specific condition reduced D-dimer testing in this elderly population of outpatients and demonstrated a persistent effect.

[Clinical and radiological findings in patients diagnosed pulmonary thromboembolism by pulmonary computerized tomography angiography]. / Pulmoner bilgisayarli tomografi anjiyografi ile pulmoner tromboemboli tanisi konulan hastalarda klinik ve radyolojik bulgular.

Pulmonary computed tomography angiography (PCTA) is the initial imaging test for the diagnosis of pulmonary thromboembolism (PTE). In the study, it was aimed to determine the clinical, radiological findings in patients diagnosed PTE by PCTA, to investigate the relationship between the thrombus localisation and the clinical, laboratory parameters. 172 patients diagnosed PTE by PCTA between 2004 and 2007 were included in the study. The clinical, laboratory parameters, thrombus localisation were evaluated. Mean age (female/male: 99/73) was 58.27 +/- 15.11, mean Wells score was 2.99 +/- 2.40. 39.5% (n= 68) of patients had low risk, 50.6% (n= 87) intermediate, 9.9% (n= 17) high risk. The most common comorbidities were cardiovascular diseases (n= 46, 26.7%), COPD (n= 26,15%). Recent operation history (n= 47, 27.3%), immobilisation (n= 37, 21.5%) were the most frequent risk factors. Dyspnea (89%), chest pain (59.9%) were the most common complaints. Deep venous thrombosis was detected by Doppler USG in 56.4% of patients. The most common site of thrombus was the right lower lobe artery (44.2%). In 30% of patients, the most proximal level of thrombus was the main pulmonary arteries (MPA). Mean age of patients with MPA thrombus (61.96 +/- 14.47), was higher than patients with distal thrombus (56.62 +/- 15.16, p= 0.03). Patients with the recent operation history (41% vs. 21%, p= 0.009), cancer (24% vs. 2.5%, p< 0.001) had higher rates of MPA thrombus. In patients presented with syncope,16.9% of them had a MPA thrombus compared to others having 3.3% rate of other thrombus localisations (p= 0.004). Mean Wells score in patients with MPA thrombus was higher compared to others (3.59 +/- 2.38/2.72 +/- 2.37, p= 0.02), however it didn't differ the extent of proximal thrombus between low, intermediate and high risk patients. The mean level of D-dimer was not different between patients with MPA thrombus and the others. D-dimer level was significantly higher in patients with thrombus localized at truncus pulmonalis (1357 microg/mL vs. 724 microg/mL). There was no significant difference between Doppler USG positive and negative patients for DVT. In conclusion, it was determined that the thrombus was at MPA in one third of the patients, a significant relationship between the presence of the recent operation, cancer history and syncope with MPA thrombus. In patients with a thrombus at truncus pulmonalis, D-dimer levels were higher.

Spectrophotometric determination of tranexamic acid using vanillin.

A new spectrophotometric method has been examined for the determination of the tranexamic acid (TA) by derivatization with vanillin (VAN). The molar absorptivity of TA was calculated 25,160 L x mol(-1) x cm(-1) at lambdamax 354 nm and obeyed the Beer's law within 0.5-2.5 microg x mL(-1). The color reaction was highly stable and did not show any change in absorbance up to 24 h. The method was applied for the analysis of TA from capsules, injections and tooth pastes. The amounts of TA found in capsules, injections and tooth pastes of various pharmaceutical companies were observed with 249.0-250.9 mg/capsule, 249.3-250.7 mg/injection and 0.048%-0.049% in tooth pastes with relative standard deviation (RSD) 0.2%-5.0% (n = 3).

The use of D-dimer testing and Wells score in patients with high probability for acute pulmonary embolism.

RATIONALE, AIMS AND OBJECTIVE: To investigate if a combination of Wells pre-test probability score and D-dimer testing could be used as a safe base for making clinical decisions on further investigations for patients with intermediate to high risks of pulmonary embolism (PE). METHODS: One hundred and twenty patients with signs or symptoms of acute PE were investigated with pulmonary angiography (PA) or contrast enhanced computed tomography of the pulmonary arteries (CTPA), D-dimer testing (Tinaquant) and clinical scoring using the Wells pre-test probability score during their first 48 hours at the hospital. Patients were recruited consecutively from emergency departments at two teaching hospitals. RESULTS: The cut-off value of 0.5 mg L(-1) in D-dimer analysis is proved adequate with a negative predictive value (NPV) of 92% in this group of patients with intermediate to high risks. The combination of D-dimer testing and Wells score increases the NPV to 94%. The specificities of both tests were low. CONCLUSION: D-dimer and Wells pre-test probability scores are safe to rule out acute PE even in patients with at least an intermediate risk of PE, but the specificity is low. D-dimer testing had a higher NPV than Wells score and the combination improved the algorithm further. The cut-off level for a high risk of PE measured with the Wells score was four and it seems reasonable to use that cut-off level in future algorithms. In addition, both PA and CTPA can present false positive and negative results difficult to interpret.

Melatonin administration increased plasminogen activator activity in ram spermatozoa.

The aim of the present study was to investigate the effect of melatonin on plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in ram spermatozoa and seminal plasma, in correlation with changes in blood testosterone. Melatonin implants (18 mg) were placed subcutaneously in sixteen Chios rams in autumn and spring. Semen samples for spectrophotometrical assays were collected 36 h before the implantation of melatonin and thereafter once a week, for 17 weeks. Blood samples for testosterone assay (RIA) were collected 8h before implantation (one sample/30 min x 7.5 h) and thereafter every 15 days for 105 days after implantation. For each ram, six parameters of testosterone were estimated: mean value, basal level, number of peaks, peak amplitude, peak duration and mean testosterone concentration during peaks. Melatonin implantation during autumn induced an increase in PAA and t-PAI in spermatozoa; melatonin implantation in spring induced an additional increase in u-PAI and PI; no change in PAA, PAI or PI was found in seminal plasma, during autumn or spring. The melatonin-induced increase of PAA, PAI and PI in spermatozoa was in positive correlation with the increase of testosterone mean value, basal level and number of peaks; the increase of testosterone parameters was greater in autumn compared to spring. Changes of PAA, PAI and PI of spermatozoa, under the influence of melatonin, might indicate changes in the fertilizing ability of spermatozoa, since plasminogen activators and their inhibitors are present on the plasma and the outer acrosomal membrane of spermatozoa and are released during the acrosome reaction.

[Diagnosis of pulmonary embolism]. / Diagnostic de l'embolie pulmonaire.

The diagnosis of pulmonary embolism (PE) is challenging because the symptoms are unspecific and an objective confirmation of the disease is required. Clinical symptoms and physical examination can be used to estimate the clinical probability of the disease used to interpret the results of the diagnostic tests. In patients with low or moderate clinical probability most quantitative D-dimer tests allow to exclude PE safely. A positive proximal venous ultrasound enables to confirm PE but the sensitivity is too low to exclude the diagnosis. In patients with a positive D-dimer test, multidetector spiral CT allows to confirm or exclude PE with confidence in most of the cases. Lung scanning is still useful in patients with renal impairment or allergy to contrast medium.

The role of CT pulmonary angiography in patients with suspected pulmonary embolism admitted to general medicine.

BACKGROUND: CT pulmonary angiography (CTPA), D-dimer testing, and pre-test probability scoring have greatly improved the ability to manage patients with suspected pulmonary embolism. International guidelines suggest combining these investigations for the best yield. We have been investigating the use of CTPA in patients with suspected pulmonary embolism (PE) admitted to the Department of General Medicine at Christchurch Hospital, New Zealand. METHODS: A retrospective audit of 100 patients with suspected pulmonary embolism who had a CTPA performed between October 2003 and April 2004. RESULTS: CTPA was positive for PE in 31% of admissions. The pre-test probability was documented in only 4% of admissions. All patients with PE had a significantly elevated D-dimer (> 499 ng/mL). Wells score calculated by the investigators showed 59 (59%) to have a low, 33 (33%) a moderate, and 8 (8%) a high risk for PE. Of these, PE was diagnosed in 9 (15%), 15 (45%), and 7 (88%) respectively; 93% of patients had a blood gas performed, yet only 77% had the D-dimer measured. No patient with a measured and negative D-dimer had a diagnosis of a PE; 32 CTPAs were performed on 32 patients out of hours. CONCLUSION: There was a very low uptake of the formal use of pre-test probability scores by medical registrars. This audit confirms that, in patients with low or moderate risk of PE and a negative D-dimer, an alternative diagnosis should be considered. The management of suspected venous thromboembolism (VTE) could be improved; it is likely that after hours CTPA could be reduced.

The emergency department utility of Simplify D-dimer to exclude pulmonary embolism in patients with pleuritic chest pain.

STUDY OBJECTIVE: Pleuritic chest pain is a common presenting complaint in the emergency department (ED) and a symptom of pulmonary embolism. Patients with pleuritic chest pain would benefit from a simple and rapid way of screening for pulmonary embolism. The aim of this study is to assess the utility of Simplify D-dimer as a rule-out tool for pulmonary embolism in ED patients with pleuritic chest pain. METHODS: This was a prospective diagnostic study in a large city-center ED. Four hundred twenty-five patients with pleuritic chest pain were prospectively recruited between February 2002 and June 2003. Simplify D-dimer testing was performed on each patient in the ED. All patients followed an independent reference standard diagnostic algorithm for pulmonary embolism. Each patient was followed up clinically for 3 months. RESULTS: The calculated sensitivity of Simplify D-dimer for pulmonary embolism was 81.8% (95% confidence interval [CI] 61.4% to 92.7%), and specificity was 74.2% (95% CI 69.6% to 78.4%). The negative predictive value was 98.6% (95% CI 96.6% to 99.6%), positive predictive value 15.0% (95% CI 9.1% to 22.7%), negative likelihood ratio 0.25 (95% CI 0.10 to 0.52) and positive likelihood ratio 3.17 (95% CI 2.30 to 3.97). The study cohort pretest probability was 5.3%. A negative Simplify result reduced the posttest probability to 1.3% (95% CI 0.5% to 3.4%). CONCLUSION: The Simplify D-dimer is not sufficiently sensitive to exclude the diagnosis of pulmonary embolism in all patients presenting to the ED with pleuritic chest pain.